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VirD - A Virion Display Array For Profiling Functional Membrane Proteins

To facilitate high-throughput biochemical analyses of membrane proteins, we have developed a novel display technology in a microarray format. Both a single-pass (CD4) and a multiple-pass (GPR77) human transmembrane proteins were engineered to be displayed in the membrane envelop of herpes simplex virions. These viruses produce large spherical virions displaying multiple copies of envelop proteins. Our aim was to engineer this virus to express these human proteins during the virus productive cycle and incorporate the human proteins into the virion during the assembly process. Another strategy presented includes engineering a fusion of glycoprotein C (gC), a major constituent of herpes simplex virus type 1 (HSV-1) virions, by hijacking the cis-acting signals to direct incorporation of the chimeric protein into the virion. The expression of the human proteins in infected cells, at the cell surface and in purified virions, is in the correct transmembrane orientation and the proteins are biochemically functional. Purified virions printed on glass slides form a high-density Virion Display (VirD) Array and the displayed proteins were demonstrated to retain their native conformations and interactions on the VirD Array judging by similar assays, such as antibody staining, as well as lectin and ligand binding. This method can be readily scaled or tailored for different modalities including to a high-content, high-throughput platform for screening ligands and drugs of human membrane proteins.

vird_-_a_virion_display_array_for_profiling_functional_membrane_proteins

Introduction<!>Cells and Viruses<!>Antibodies<!>Plasmids<!>Marker-rescue/marker-transfer assays<!>Red-ET recombination<!>Transfection of Bacmid DNA to re-constitute infectious virus<!>Western blot analysis<!>Immunofluorescence and Confocal Analysis<!>Virion preparation<!>Flow-Cytometry<!>Enzyme-linked immunosorbent assay (ELISA)<!>VirD Array fabrication<!>Antibody assays on VirD Arrays<!>Ligand binding assay on VirD Array<!>Lectin binding assays on VirD Arrays<!>Results and Discussion<!>Conclusions

<p>Approximately one-third of the human proteome is comprised of membrane proteins that belong to protein families with a variety of biochemical activities, such as transporters, channels, receptors, recognition molecules, and adhesion molecules. Membrane proteins are critically important molecules for cell survival, maintenance of cell homeostasis, cell signaling, immune surveillance, molecular transport, and cell-cell communication. This class of proteins represents up to 70% of therapeutic targets for all prescribed drugs. Therefore, development of a high- throughput platform that enables profiling membrane proteins in an active conformation for their biochemical activities will have an important impact on drug discovery by streamlining small molecule screening methods. However, membrane proteins, especially those containing multi- pass transmembrane (TM) domains, are notoriously difficult to study because they have to be embedded in a membrane to maintain a native conformation and many require proper posttranslational modifications (PTMs), such as glycosylation, which occurs during transport in the cellular secretory pathway. Although membrane protein microarrays have been produced, biochemical purification using detergents limits the throughput and subsequent manipulation of such arrays1, 2.</p><p>To develop a new, high-throughput platform that displays human membrane proteins in their native conformation, we made use of herpesvirus virons as a vehicle to fabricate a Virion Display microarray, dubbed VirD Array. Herpesviruses, such as herpes simplex virus type 1 (HSV-1), produce large membrane-enveloped virons that contain high copies of the viral glycoproteins, such as the three major glycoproteins gB, gC, and gD, that are distributed regularly in the circular virion structure. The DNA genome of this virus can be genetically manipulated to express foreign proteins in their native configuration or as fusions with the viral proteins. Our singular goal was to develop a method to efficiently incorporate human membrane proteins in HSV-1 virions and then to discover whether these proteins were functional in the viral membrane. We explored two strategies for the VirD array: 1) Clone a full-length human ORF at the gB locus and express the gene under control of the strong gB promoter; 2) Fuse a human ORF to the TM and cytoplasmic domains of gC and express this chimeric gene at the gC locus under the control of the gC promoter (Fig. 1). Previous studies have also utilized a gC chimera approach (hepatitis C virus glycoprotein E2) as well as expression from the gC locus (CD4) to incorporate foreign proteins in HSV-1 virions3, 4. Important for these strategies to work is the observation that the absence of either gB or gC does not affect the ability of the virus to assemble mature enveloped virions in infected cells5–7.</p><p>To test the feasibility of the two approaches, we chose CD4 as a classical type I membrane protein with a single TM domain and GPR77 (a.k.a. C5L2) as a representative of the multi- spanning (seven TM regions), G-protein coupled receptor (GPCR) membrane protein. CD4 is a well-characterized membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigens and is also a receptor for the human immunodeficiency virus8. GPR77 is involved in the complement system of the innate immune response with a canonical ligand identified (i.e., complement component C5a)9. Our goal was to examine the expression and incorporation of these human membrane proteins into HSV-1 virions using the two strategies outlined above and to determine whether these human membrane proteins are maintained in their native form in purified virions immobilized on a glass surface at high density.</p><!><p>Vero cells, transformed Vero cell lines and human foreskin fibroblasts (HFT) were grown in minimum essential medium - alpha medium supplemented with 10% fetal calf serum (Gibco-Invitrogen) and passaged as described in Desai et al. (1998)10. HFT is an immortalized cell line that is transduced with a retrovirus expressing human telomerase11. D6 (UL27 transformed) was used as the host cell for growth of the recombinant viruses that expressed genes from the gB locus5. The A1.1 (UL27 and UL28 transformed) cell line12 was used for the marker-rescue/marker-transfer method to introduce the human genes cloned into the gB loci13 and was a kind gift from Fred Homa of the University of Pittsburgh. Stocks of the parental wild-type virus strain KOS (HSV-1) and the mutant and recombinant viruses were prepared as previously described10. All tissue culture and virus manipulation was performed in a Biosafety Level 2 facility. Infected cells and media containing infectious material was treated with 10% sodium hypochlorite. Other material utilized during the handling of virus infected cells was subjected to steam sterilization in an autoclave.</p><!><p>Antibodies reactive to human CD4 and GPR77 were purchased from Santa Cruz Biotechnology and Sigma-Aldrich, respectively. PE-conjugated anti-CD4 antibody used for flow cytometry analysis was obtained from BD Biosciences. The V5 monoclonal antibody was purchased from Invitrogen Life Technologies. Anti-HSV-1 gB antibody clone B6 and anti-gC antibodies were a kind gift from Joseph Glorioso (University of Pittsburgh). Anti-HSV-1 gD antibody DL6 was a generous gift from David Johnson (OHSC) and Gary Cohen and Roz Eisenberg (Penn University). The VP5 antibody LP12 was kindly provided by Tony Minson (University of Cambridge, UK).</p><!><p>Plasmid pKΔ4B was derived by Cai et al.14 following engineering of linker-insertion mutants in the glycoprotein B gene. DNA sequences encoding amino acids 43 through 711 of gB were deleted and a BglII restriction site added to maintain the protein reading frame14. pKΔ4B was digested with Xho1 and BglII, treated with antartic phosphatase (NEB) and ligated with an Xho1-BglII PCR fragment amplified from pKΔ4B which deletes all gB amino acids from 1–43 (gBΔSS) but retains the gB promoter sequences (Table 1). This plasmid was designated pKgBΔSS. The sequence of gB amino acids spanning 711 to 796 were deleted from pKgBΔSS by cassette PCR mutagenesis. The PCR fragment was cloned as a BglII-BamH1 into pKgBΔSS and the resulting plasmid was labeled pKgBPR. The human CD4 and GPR77 sequences were amplified from the plasmids from the Ultimate ORF collection (Life Technologies). The sequence encoding the V5 epitope was included in the reverse primer (Table 1). The final gB promoter driven gene plasmids were labeled pKgB:CD4 and pKgB:GPCR77. Sequence analysis of the different plasmids was done prior to introduction into the virus genome. Plasmids were linearized with BamH1 for homologous recombination.</p><!><p>The marker rescue of UL28 and marker transfer of the gB:human ORF gene was accomplished using the method described in Desai et al.13. A1.1 cell monolayers (1×106) in 60 mm petri dishes were co-transfected with 25 µl of infected cell DNA (KΔ4BX) and 0.1–0.05 µg linearized plasmid DNA using the calcium phosphate precipitation method. When plaques began to appear (72 h after transfection) the cell monolayers were harvested, freeze/thawed once, sonicated and total virus progeny titered. The recombinant virus was isolated by single plaque purification on D6 cells. Additional plaque purification was carried out by limiting dilution on the D6 cell line.</p><!><p>The KOS BAC37 genome15 was transferred into TOP10 cells (Stratagene) for this method. KOS BAC37 was kindly provided by David Leib, Dartmouth University, NH. The procedure to engineer gC chimera fusions into the virus genome used the Gene Bridges Red-ET method and the protocols provided16. The kanamycin cassette surrounded by gC homologous sequences was amplified using gC-Kan-F and gC-Kan-R primers and pRPSL-neo as a template. This kanamycin gene was introduced into KOS BAC37 replacing the gC gene. Colonies that grew on kanamycin plates were screened for streptomycin sensitivity before the next step. The CD4-gC and GPR77-gC fusion genes were made using Overlap PCR methods using the primers listed in Table 1. The CD4-gC and GPR77-gC chimera fusions were amplified using the RedET primers listed in Table 1 and were used to replace the kanamycin gene. Successful isolates carrying the correct chimeric genes were identified by PCR assays and the inserted gene in the BAC genome was sequenced prior to reconstitution of infectious virus.</p><!><p>The KOS Bacmids carrying the glycoprotein C chimera gene fusions were prepared using the PureLink nucleic acid purification kit (Life Technologies). The Bacmid DNA was transfected into Vero cells (5 X 105) in 12 well trays using Lipofectamine 2000 reagent (Life Technologies). Plaques generally began to appear after 3 days and this infected cell lysate was used to amplify and prepare working stocks of each of the gC chimera viruses.</p><!><p>Infected cell extracts were resolved by SDS-PAGE in MES buffer and transferred to iBlot membranes (Life Technologies) using an iBlot apparatus (Life Technologies) according to the manufacturer's protocol. The transferred membranes were blocked with blocking buffer (TBS with 5% non-fat milk) at room temperature for an hour with gentle shaking, and then incubated with primary antibodies (1:5000 dilution in blocking buffer) at room temperature for an hour with gentle shaking. The membranes were washed for 5 min with TBS+0.1% Tween20 (TBST) buffer for 3 times with shaking. HRP-conjugated anti-mouse antibodies (GE Healthcare) were incubated on the membranes at 5,000-fold dilution in blocking buffer for an hour with gentle shaking. The membranes were washed for 5 min with TBST buffer for 3 times with shaking and incubated with ECL Plus Western Blotting detection reagents (GE Healthcare) for 5 min before signals were visualized by ImageQuant LAS 4000 imaging system (GE Healthcare).</p><!><p>HFT cells in LabTek (#1 borosilicate glass) four well chamber slides (6×105 cells) were infected at an multiplicity of infection (MOI) of 10 plaque forming units (PFU) per cell. Infected cells were washed 2X with DPBS (Dulbecco's phosphate buffered saline), fixed with 4% paraformaldehyde in DPBS for 25 min; washed 2X with DPBS and permeabilized with 0.25% triton X-100 in DPBS for 30 min. After permeabilization, the cells were washed 2X with 3% BSA in DPBS and non-specific reactivity was blocked for 30 min in the same buffer. For cell surface labeling the detergent permeabilizaton step was omitted and the cells incubated with blocking buffer. Primary antibody was diluted in 3% BSA/DPBS and 250 µl added to each chamber well for 60 min (room temperature). Subsequently the cells were washed 3X with 3% BSA/DPBS and then incubated with Cy3-labeled secondary antibody (Jackson Laboratories) for 45 min (room temperature). The cells were then washed 3X with 3% BSA/DPBS and then incubated in Fluormount G (EMS) prior to imaging. The stained infected cells were analyzed in a Zeiss LSM 510 confocal microscope. Most images were collected with a pinhole set at 1 Airy unit.</p><!><p>Extracellular virions were prepared from HFT cells. Generally 8.6 X 106 cells in 100 mm petri dishes were infected at an MOI of 10 PFU/cell. The culture medium was harvested at 72 h post-infection, clarified by centrifugation at 3500 rpm for 30 min at 4°C. The supernatant was layered on a 20% sucrose cushion (W/V in growth medium) and centrifuged in a Beckman SW41 (39 K for 30 min) or SW32 (24 K for 60 min). The virion pellet was resuspended in PBS at 4°C overnight and then used for subsequent analyses. For VirD Array printing the virion preparations were resuspended in PBS plus 35% glycerol. The titers of KOS (wild-type) virions, which are infectious, was monitored by plaque assays during the different purification and manipulation procedures to ensure biological integrity of the virions.</p><!><p>Extracellular virions (150 µl volumes) were incubated with PE-conjugated flow antibodies (20 µl) and incubated at room temperature (tube rocker) for one hour in the dark. The virions (volume adjusted to 500 µl with PBS) were then sedimented through 20% sucrose cushion (250 µl) in an Eppendorf tube at 16000 g for 60 min. The supernatant was discarded and the virus pellet resuspended in 200 µl PBS. The labeled virions were analyzed in a BD FACSARIA II instrument using the DIVA software (version 6.1.3).</p><!><p>Serial dilutions of virions were incubated in Nunc MaxiSorp flat-bottom 96 well white plates. The sealed plates were incubated at 4°C for 2 days. The wells were washed with PBS + 0.02% Tween-20 (PBS+T20) 3 X for 5 min each time on a platform shaker and then blocked with 2% BSA in PBS+T20 for 60 min at room temperature. Primary antibody dilutions were made in blocking buffer generally 1:2000 to 1:250 and incubated for 60 min. Secondary HRP conjugated mouse antibody was used at a 1:1000 concentration. The plates were washed 3X with PBS+T20 for 5 min each wash after both primary and secondary antibody binding. The reaction was quantitated using SuperSignal ELISA Pico (Pierce) chemiluminescent substrate according to the manufacturer's procedure and the plate read in a Glomax luminometer to determine relative light units (425 nm).</p><!><p>Purified virions were arrayed in a 384-well plate and spotted on FAST slides (Whatman) in a 4x4 pattern along with BSA as a negative control. The printed arrays were stored at −80°C.</p><!><p>VirD Arrays were blocked with blocking buffer (TBS with 3%BSA) at room temperature for an hour with gentle shaking, then incubated with primary antibodies (1:1000 dilution in blocking buffer) at room temperature for an hour with gentle shaking. The arrays were washed for 5 min with TBS+0.1% Tween20 (TBST) buffer for 3 times with shaking. To visualize the presence of human or viral proteins, Cy5-labeled anti-mouse antibodies (The Jackson Laboratory) were incubated on the arrays at 1,000-fold dilution in blocking buffer. The arrays were washed for 5 min with TBST buffer for 3 times with shaking, briefly rinsed with water, and dried by spinning. The slides were finally scanned with a GenePix 4000B scanner (MDS Analytical Technologies).</p><!><p>The VirD Array was blocked in TBST with 1% BSA for 1 h at room temperature with gentle shaking. Complement anaphylatoxins C3a (Alpha Diagnostic Intl), C4a (MyBioSource), and C5a (Abcam) were individually labeled with Cy5 NHS Easter (GE Healthcare) and incubated on the VirD Array at 1 μM in ligand binding buffer (1 mM MgCl2, 2 mM CalCl2, 0.2% BSA, and 25 mM HEPES, pH 7.4) at room temperature for 1 h with gentle shaking. The array was washed for 5 min in ice-cold washing buffer (0.5 M NaCl in 10 mM HEPES, pH 7.4) for 3 times with shaking, dried by spinning, and scanned as described above.</p><!><p>VirD Arrays were blocked in PBS with 1% BSA for 1 h with gentle shaking. Lectins (EY Laboratories) were labeled with Cy5 NHS Easter (GE Healthcare) and incubated on the VirD Array at 1 μg/ml in PBS with 0.5 mM CaCl2 and 1% BSA at room temperature for 1 h with gentle shaking. The array was washed for 5 min in PBST for 3 times with shaking, dried by spinning, and scanned as described above.</p><!><p>Recombinant methods were used to generate four viruses (see Experimental Section for details). Viruses, gB:CD4 and gB:GPR77, express the full-length human membrane proteins at the gB locus under the control of native gB promoter. We also incorporated a V5 epitope tag at the C-termini of both proteins for biochemical detection purposes. Viruses labeled CD4-gC and GPR77-gC, express human membrane proteins fused to the gC C-terminal domain (i.e., 481 to 511 aa), which contains the TM and a short cytosolic domain. Like the human genes cloned at the gB locus, the gC chimeras were cloned at the gC locus under the control of native gC promoter (Fig.1).</p><p>The precise mechanism by which many of the HSV-1 glycoproteins are incorporated into mature virions is still not determined. The absence of either one of the major glycoproteins, gB, gC, gD or gH-gL, does not appear to affect the incorporation of the others even though these proteins function as a complex during virus entry and egress17. Glycoprotein M may play a role in the incorporation of gC in certain cell types18. The TM domain may have a role in this process as well as the cytosolic tails of these glycoproteins which could mediate virion incorporation by interaction with the underlying tegument structure19. We first attempted to make chimeras of the human proteins with gB, but none of the fusion proteins were expressed at the cell surface and as a consequence these chimera proteins were not detected in our extracellular virus particle preparations similar to the observation demonstrated for the inner nuclear membrane protein UL3420. What became apparent was the abundant stable accumulation of the human polypeptide expressed from the gB promoter in infected cells. Thus, we generated an expression module to express the native human gene as a "viral gene" with the goal that the expressed proteins would become incorporated into mature virions during virus egress and maturation. Because there was evidence in the literature that fusion of foreign genes to gC were incorporated into the virion4, 21, this type of strategy was also developed to potentially increase the virion incorporation using cis-acting signals present in those sequences.</p><p>To examine whether CD4 and GPR77 were expressed and correctly processed through the secretory pathway, human fibroblasts infected with the recombinant and parental viruses were stained with antibodies against the ecto-domains of CD4, GPR77, and HSV-1 gD to visualize cell surface localization of these antigens (Fig. 2a). As expected, gD was detected on the surfaces of infected cells (Fig. 2a; insets). CD4 expressed either from the gB promoter or as a gC-chimeric protein was also detected on the cell surface as judged by the fluorescence signals. GPR77 was detected on the cell surface, but the distribution of the fluorescence was different depending on whether it was expressed from the gB promoter or as a gC-chimera. These results suggest that CD4 and GPR77, like gD, expressed off the HSV-1 genome were delivered to the surface of the plasma membrane via the canonical secretory pathway. KOS- and K082-infected cells did not react with the antibodies to CD4 and GPR77 (data not shown). The intracellular distribution of CD4 and GPR77 was examined by staining with V5 antibody following permeabilization of infected fibroblast cells (Fig. 2a; right panel). HSV-1 glycoproteins localize to nuclear, endoplasmic reticulum, Golgi and cell surface membranes during productive infection. The intracellular distribution of CD4, whether the cells were stained with anti-CD4 or anti-V5 antibodies, was similar to the intracellular distribution of gD (Fig. S1).</p><p>Further biochemical evidence for the expression of at least the V5-tagged human proteins was obtained using immunoblot analysis of total infected cell lysates (Fig. 2b; left panel). We also examined virion incorporation of these human membrane proteins. Wild type and gB null (K082) virions5, as well as gB:CD4, gB:GPR77 virions, were harvested from infected cell culture supernatants, clarified by low speed centrifugation and purified through a 20% sucrose cushion. These virions were analyzed using the same immunoblot methods with anti-V5 antibodies. Both gB:CD4 and gB:GPR77 virions showed strong anti-V5 reactivity at the expected molecular weights of CD4 and GRP77, while no detectable signals were observed in the other virions (Fig. 2b; right panel). Anti-gD antibodies were used as a loading control. We have also purified virus from infected cell culture supernatants using sucrose gradients and glycerol-tartrate cushions. These virions have similar virus polypeptide profiles to the particles purified using the sucrose cushion and all contained the human membrane protein, which was detected with the V5 antibody (data not shown). Together, these data confirmed that both CD4 and GPR77 were synthesized and incorporated into virions produced in infected human cells.</p><p>To demonstrate that human proteins could be displayed in the correct orientation after virion incorporation, we performed flow cytometry analysis of purified virions stained with PE-labeled antibodies that recognize the ecto-domain of CD4 (Fig. 3). An HSV-1 recombinant virus that incorporates the Venus fluorescent protein in the capsid was used to identify and gate purified virions (Experimental Section). K082 virions were used as a negative control for antibody binding specificity. Judging from the amounts of PE fluorescence detected within the gated virion populations, 85.5% of gB:CD4 virions were labeled with PE antibody and slightly more, 96.1% of CD4-gC virions were bound to PE antibody. These results were consistent with data from different virion preparations using similar experimental conditions. This observation was further confirmed using a standard ELISA analysis using chemilluminescent substrates for detection (Fig. S2). All virion preparations were stained with anti-gD antibodies as expected. Virions expressing CD4 or GPR77 were stained with the respective antibodies. There was little or no reactivity of the CD4 and GPR77 antibodies with the KOS or K082 virions. The signal observed with anti-gD antibodies was significantly higher because of the higher affinity of this monoclonal antibody for its antigen. Taken together, these results demonstrated that the human membrane proteins were incorporated in the correct transmembrane orientation because they reacted with antibodies that recognize the extracellular domains of these proteins. This observation suggests that the membrane proteins were embedded in the virion envelope in their native conformation.</p><p>To test whether these recombinant virions could be immobilized in a microarray format at high density, while maintaining their functional integrity, we spotted them on different glass surfaces at various titers. Using wild-type KOS virions that can be titered, we typically derived virions that were at a concentration of 4×108 PFU/µl. Of this we used 0.7 nl to spot the glass surface (3 times), delivering potentially ~106 PFUs to the surface. Using anti-gD antibodies, we determined that nitrocellulose-coated slides (i.e., FAST) provided the optimal detection as low as 500,000 virions (KOS plaque forming units) per spot and the anti-gD signals started to reach saturation after the titer was increased to >4,000,000 virions (Fig. 4). Therefore, we decided to construct the VirD Array with seven different virus preparations at a titer of 8,000,000 virions (KOS plaque forming units) per spot in a 4 × 4 format. It is likely that not all of the different virion preparations will be of the same concentration because of the differences in their genetic backgrounds.</p><p>To visualize and examine the integrity of immobilized virions on glass, the arrays were stained with anti-gD ecto-domain and anti-VP5 antibodies, the latter recognizes the major capsid protein, VP5. As expected, all seven virion sectors showed strong anti-gD signals but much lower anti- VP5 signals, indicating that the vast majority of the immobilized virions were intact (Fig. 5; left panel). This conclusion was further supported by the observation that the anti-gD signals were greatly decreased on the VirD Arrays after the virion envelopes were stripped with a mild detergent treatment using NP40 (Fig. 5; right panel). In contrast, strong anti-VP5 signals were seen in all seven virion sectors following this treatment. Moreover, staining the VirD Arrays with anti-gB and -gC antibodies confirmed the expected absence of gB and gC proteins in gB:CD4/GPR77 and CD4/GRP77-gC, respectively (Fig. 6).</p><p>Because glycosylation is important for human membrane protein activity, we employed fluorescently labeled lectins (i.e., SNA-II, PHA-L, CA, and WGA) to profile glycan structures using the VirD Arrays22, 23. Comparison of the lectin staining patterns between wild type, gB-KO (K082) and gC-KO (gCΔ39)7 virion sectors showed that gC is more heavily glycosylated than gB, because all four lectins showed much weaker binding signals to the gC-KO virion sector (Fig. 7). This finding correlates with data showing HSV-1 gC binds to peanut lectin and Helix pomatia lectin24. A more careful analysis of lectin CA staining pattern, which recognizes Galβ(1-4)GlcNAc, GalNAcα(1-4)GlcNAc, or NeuAcα(2-6)Galβ(1-4)GlcNAc, indicated that CD4 was probably modified by these carbohydrates. This is because the CD4-gC (i.e., gC-) virion sector showed significantly higher signals than both gC-KO and GPR77-gC (i.e., gC-) virion sectors. This observation is further supported by the same SNA-II (recognizing terminal Galβ, GalNAcβ, or NeuAcα(2-6)Galβ (1-4)GlcNAc) staining pattern because it is known that SNA-II should recognize the same glycan structures or partial glycan structures as CA does based on the database of lectin specificity at Consortium for Functional Glycomics web site (http://www.functionalglycomics.org) (Fig. 7). Interestingly, a very similar glycan structure NeuAcα(2-3)Galβ(1-4)GlcNAc was previously identified on mouse CD4 expressed in CHO cells using a mass spectrometry approach, indirectly supporting our observation here8. Thus, human CD4 is very likely to be glycosylated with NeuAcα(2-6)Galβ(1-4)GlcNAc. We, however, could not definitively determine any specific glycan structures associated with GPR77, probably due to a limited number of lectins used in this study. Regardless, the above results suggest that the virion-displayed human membrane proteins were glycosylated.</p><p>To determine whether human CD4 and GPR77 proteins displayed on the surface of virions immobilized on a glass surface were in the correct orientation, we stained the VirD Arrays with antibodies that each recognizes the ecto-domains of CD4 or GRP77 (Fig. 8). We observed strong and specific staining signals in the gB:CD4 and CD4-gC, gB:GPR77 and GRP77-gC virion sectors, respectively, indicating that these proteins are in the correct orientation and both membrane protein display strategies worked.</p><p>Finally, to demonstrate that the virion-displayed human membrane proteins were functional on the glass surface, we probed the VirD Array with a fluorescently labeled canonical ligand, complement anaphylatoxin C5a, of GPR77 with a KD of 2.5 nM (see Experimental Section for more details)9. As shown in the right panel of Figure 8b, Cy5-labled C5a showed relatively strong binding activity to the GPR77-gC virion sector on the array, albeit weaker binding signals were associated with the gB:GPR77 virion sector. No detectable fluorescence signal was observed in the other virion sectors, suggesting the interactions between C5a and GPR77 on the VirD Array was specific. The other two complement anaphylatoxins, C3a and C4a, were also tested on the VirD Arrays. Neither anaphylatoxin C3a nor C4a showed any significant binding signals to the two GPR77 virion sectors on the VirD Arrays (data not shown), further confirming the specific interactions between C5a and GPR77. Taken together, these results indicate that GPR77 was displayed correctly on the virions and maintained its functional conformation on the VirD Array.</p><!><p>Display of soluble peptides or protein in various formats has been effective using different carrier systems25. In this study, we demonstrate fabrication of a VirD Array that displays human membrane proteins on the envelopes of engineered HSV-1 virions immobilized at high density on solid glass surfaces. Using antibodies and lectins we showed that two human membrane proteins, CD4 and GPR77, are in the right orientation and potentially glycosylated. We further demonstrated that virion-displayed GPR77 was in its active conformation via a binding assay with its cognate ligand C5a. The VirD Array approach has several obvious advantages: 1) Displayed human membrane proteins are embedded in human cell membranes, a more physiologically relevant environment that can help maintain their native conformation; 2) As demonstrated with GRP77, membrane proteins with multiple TM domains are likely to be folded correctly in the virion envelopes; 3) Since the virus exploits the human secretory pathways, the displayed human proteins are likely to maintain their canonical PTMs as they are transported through the secretory pathways; this was demonstrated via lectin binding assays; and 4) The VirD Array is expected to be readily transformed to a high-content platform that can display virtually all of the human membrane proteins close to their native conformation on a single glass slide.</p><p>Although only two human membrane proteins were employed to establish the VirD Array technology in this study, we envision that all of the human membrane proteins can be included to construct a comprehensive human membrane protein VirD Array. Once such a high-throughput platform is established, it will allow us to perform high-throughput screens for novel drug target identification against membrane proteins, to identify ligands of various types of receptors, and to profile PTM of membrane proteins. For example, fluorescently labeled ligands can be used to probe the VirD Arrays in order to "deorphanize" the GPCRs. Using pathogen-encoded, secreted peptides as probes, the VirD Arrays will be capable of identifying pathogenic elicitor-host receptor interactions. A more sophisticated approach is, perhaps, to engineer a reporter system expressed in the tegument of virions on the VirD Arrays so that the state (e.g., open versus closed) of a given ion channel displayed on the virions can be examined in a high-throughput fashion. When this system is coupled with drug screens, we envision the VirD Array approach will have a significant impact on identifying drug targets for GPCRs and ion channels.</p>

PubMed Author Manuscript

Carbon nanodots as molecular scaffolds for development of antimicrobial agents

We report the potential of carbon nanodots (CNDs) as a molecular scaffold for enhancing the antimicrobial activities of small dendritic poly(amidoamines) (PAMAM). Carbon nanodots prepared from sago starch are readily functionalized with PAMAM by using N-ethyl-N\xe2\x80\xb2-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). Electron microscopy images of these polyaminated CNDs show that they are approximately 30\xe2\x80\x9360 nm in diameter. Infrared and fluorescence spectroscopy analyses of the water-soluble material established the presence of the polyamidoaminated moiety and the intrinsic fluorescence of the nanodots. The polyaminated nanodots (CND-PAM1 and CND-PAM2) exhibit in vitro antimicrobial properties, not only to nonmultidrug resistant bacteria but also to the corresponding Gram-negative multidrug bacteria. Their minimum inhibitory concentration (MIC) ranges from 8 to 64 \xc2\xb5g/mL, which is much lower than that of PAMAM G1 or the non-active PAMAM G0 and CNDs. Additionally, they show synergistic effect in combination with tetracycline or colistin. These preliminary results imply that CNDs can serve as a promising scaffold for facilitating the rational design of antimicrobial materials for combating the ever-increasing threat of antibiotic resistance. Moreover, their fluorescence could be pertinent to unraveling their mode of action for imaging or diagnostic applications.

carbon_nanodots_as_molecular_scaffolds_for_development_of_antimicrobial_agents

<p>The ever-increasing incidence of bacterial resistance to existing antibiotics has created a need to broaden the targets as well as to develop new antimicrobials and strategies to combat antibiotic resistant bacteria.1,2 Carbon nanodots (CNDs) are a fascinating new class of nanomaterials that are promising molecular templates for various different types of applications including imaging, sensing, drug delivery, photocatalysis, and more.3–6 They are readily prepared from starch and other carbonaceous sources7–9 and their low toxicity index promises numerous biomedical applications besides their fluorescent properties.10, 11</p><p>Carbon nanodots, like their nanotube congeners, offer reactive surface functional groups that can be oxidized by acid reflux to generate carboxylic acid containing dots.8, 12–14 Such surface decorated functional moieties on the carbon dots allowed for further passivation, with various compounds such as N-acetyl-cysteine, PEG1500N, and other polymers, to improve their fluorescence properties.15–17 Accordingly, CNDs could serve as a molecular scaffold for grafting small polycationic amines. The nanoscale carbon dots offer high surface areas suited for concentrating such cationic densities for enhanced antimicrobial activity. Structurally large polycationic compounds including poly-lysines, cationic amphipathic peptides, and large polyamine dendrimers have been reported to exert antimicrobial activities. They disrupt the integrity of bacterial membranes, which possess an overall net anionic charge, via favorable electrostatic and hydrophobic interactions18–20 Moreover, some of these polycationic compounds enhanced the uptake of small hydrophobic antibiotics into the bacterium, and consequently, presented synergistic effects. For example, an alpha-helical cationic peptide was reported to exert a potent synergistic effect with chloramphenicol against some types of bacteria.21</p><p>Poly(amidoamines) (PAMAM) dendrimers consist of an interior ethylene diamine core surrounded by successive branching layers (generations) that terminate with amino groups.20, 22 Although the higher generation PAMAM dendrimers (greater than generation three, G3) exhibit antibacterial properties, the flexible and open lower generation dendrimers lacks significant efficacy.20 Therefore, we explore carbon nanodots as a molecular scaffold for conjugating these lower generation PAMAM (G0 and G1) to concentrate their aminated cationic densities and hence, assess these conjugates for enhanced antimicrobial activity. Dendritic PAMAMs expressing primary amino groups are readily utilized for conjugation onto the surface carboxylated CNDs. Some common conjugation approaches in dendrimer engineering include their nucleophilic acyl substitution reaction with N-hydroxysuccinimide (NHS) activated carboxylic acids or N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydoxybenzotriazole (HOBt) coupling chemistry.23, 24</p><p>The preparation of conjugated CND and PAMAMs were realized using the previously reported surface oxidized CNDs,25 via an amidation reaction by EDC and NHS activations of the carboxyl carbon toward nucleophilic acyl substitution (CND-PAM1 and CND-PAM2, Scheme 1). The syntheses was achieved in one-step by either stirring at room temperature for 5–6 hrs or under microwave irradiation at 60 W for 10–15 min with a maximum temperature of 75°C (see Supplementary data). The water-soluble excess EDC, NHS, and salts were readily removed by dialysis and their separation was monitored by size exclusion HPLC. Both CND-PAM1 and CND-PAM2 were obtained as yellowish brown powder following lyophilization. The morphology of CND-PAM2 is slightly larger that CND-PAM1 and their diameter size ranges form 30–60 nm (Figure 1). Both conjugates were also characterized using infrared, fluorescence and ultraviolet spectroscopy as discussed below.</p><p>Figure 2a shows the infrared spectra of the polyamidoaminated CND (CND-PAM1), CND, and PAMAM G0 (PAM-1). CND-PAM1 shows characteristic absorption bands of surface functional groups, for example, there are stretching vibrations of N-H at 3100–3600 cm−1, C-H at 3000-2800 cm−1, and bending vibrations of CH2 1350–1460 cm−1. The amide I band (C=O stretching) occurs at 1616 cm−1 and the amide II band, resulting from the interaction of N-H bending and the C-N stretching of the C-N-H groups, is observed at 1541 cm−1, as previously reported.26</p><p>Similarly, CND-PAM2 exhibits absorption bands corresponding to the polyamidoaminated CND (CND-PAM2), CND, and PAMAM G1 (PAM-2). Both CND-PAM1 and CND-PAM2 show most of the IR absorption bands that are present in their corresponding PAMAM precursor and the O-H out-of-plane bending absorption band (650–520 cm−1), which is characteristic of the hydroxyl-rich starch derived CND precursor (Fig. 2a and 2b). The above-observed cues of functional groups present in the CND and PAMAM conjugates, in contrast to that recorded for the CND precursor; demonstrate that the PAMAM dendrimer are linked to the CND molecular framework.</p><p>Strong fluorescence emission was observed for the polyaminated CNDs at a range of 420–460 nm when the excitation wavelength is between 330 and 350 nm (Fig. 3). These results are consistent with those reported for the starting material25 except for a 10 nm red shift observed in the emission maximum peak for CND-PAM1 to 440 nm (Fig. 3a). Additionally, the fluorescence peak for CND-PAM1 is broader than that observed for CND-PAM2 (Fig. 3b). The latter could be attributed to a larger variation in the polyamination of CND by the structurally smaller PAMAM G0 compared to the larger PAMAM G1.</p><p>The dried powdery polyaminated CNDs were evaluated for antimicrobial activity by following the antimicrobial susceptibility testing standards. The precursors of the conjugates including CND, PAMAM G0, PAMAM G1, and equal mixtures of CND and PAMAMs were also examined for comparison. Table 1 shows the MIC values (µg/mL), the minimum concentration of the test compound necessary to inhibit detectable bacterial growth at 620 nm. The data shows promising MICs for both polyaminated CNDs, but no significant antimicrobial activities were observed for the controls and nonconjugated mixtures of CND and PAMAMs. Both polyaminated CNDs exhibited the same activity against representative Gram-negative (E. coli) and –positive (S. aureus) bacteria (Table 1). However, they show a four-fold selectivity for E. coli at an MIC of 8 µg/mL. Accordingly, they were subsequently tested against three prioritized Gram-negatives from the "ESKAPE" pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) that are categorized by the US Center for Disease and Control as urgent or serious because they are responsible for two thirds of all health care-associated infections.2</p><p>MIC results show that the antimicrobial activities of these surface polyaminated CND scaffold are effective against both antibiotic resistant (K. pneumonia 1706, P. aeruginosa 1744, and A. baumannii 1605) and non-resistant strains (K. pneumonia 13883, P. aeruginosa 27853). Remarkably, they are fourfold more effective in killing the resistant K. pneumonia strain than the corresponding non-resistant strain. This suggests that the resistant strain may be more vulnerable to these compounds regardless of the degree of polycationization. However, CND-PAM 1 is consistently twice as active as CND-PAM2 against the select Gram-negative bacteria suggesting that CND functionalized with smaller dendrimeric amines maybe more effective against Gram-negatives. Notably, each polyaminated CND type exhibits the same MIC value against P. aeruginosa 27853, P. aeruginosa 1744 (resistant strain), and A. baumannii 1605 (resistant strain), suggesting that a threshold in antimicrobial activity may have been reached by each polycationized CND.</p><p>In general, the antimicrobial activity and selectivity of CND-PAM1 and CND-PAM2 are similar (MIC values range from 8 to 32 µg/mL), and are comparable to those reported for outer-membrane cell wall disrupting amphipathic α-helical peptides through detergent-like action.27, 28 This suggests that CNDs could be useful as a molecular scaffold for grafting small polycationic amines to enhance their antimicrobial activities by increasing their cationic densities for optimal interactions with the polyanionic outer surface of the bacterial membrane. Such electrostatic perturbation could result in bacterial membrane-disruptive effects that could assist in the simultaneous uptake of small hydrophobic antibiotics and thereby impart synergistic antimicrobial effects. Accordingly, we evaluated them for in vitro synergism in combination with tetracycline, an antibiotic commonly used to treat pneumonia, acne, and urinary tract infections, and colistin, an antibiotic of last therapeutic option for infections caused by multidrug resistant (MDR) 'superbugs'. Checkerboard titration assay29 was conducted to measure the interaction between the conjugates and antibiotics in an in vitro bacterial culture to determine whether they acted in synergy to increase killing efficiency. This combination assay provides a numerical value calculated as a fractional inhibition concentration (FIC) index by measuring the effective MIC for the combined test compounds. Accordingly, an FIC index of 0.5 corresponds to a 4-fold decrease in the MIC of each test compound in combination constitutes synergism.</p><p>As shown in Figure 4, both CND-PAM1 and CND-PAM2 in combination with tetracycline or colistin exhibited some variations in partial synergistic antimicrobial activity (FIC > 0.5 and < 1). However, tetracycline and CND-PAM1 showed an additive effect (FIC = 1) against the resistant strain K. pneumonia, whereas colistin and CND-PAM2 exhibited a greater than four-fold increase in activity (FIC = 0.35) against A. baumannii (see Supplemental section). In general, the multi-drug resistant A. baumannii is more vulnerable to the combination treatment as observed by the associated lower FIC indexes (Fig. 4). The majority of these polyaminated CNDs exhibited partial synergism with tetracycline and colistin, independent of bacterial type. This is consistent with synergism displayed by polycationic peptides, which are attributed to their detergent-like mode of action on the bacterial membrane. Similarly, the polycationic CNDs could disrupt the integrity of bacterial membrane, resulting in enhanced antibiotic uptake and faster inhibition of bacterial growth.</p><p>In conclusion, we have reported the use of CNDs as an effective molecular scaffold for conjugating small dendritic poly(amidoamines)s to increase their antimicrobial efficacy. Moreover, these poly(amidoamines) functionalized CNDs in combination with tetracycline or colistin show improved antimicrobial activities. Overall, the results obtained from this study indicate that CNDs can serve as a promising molecular scaffold for the conjugation of dendritic polyamines that can be used as synergists or carriers for small hydrophobic antibiotics to enhance their uptake and hence increase antibacterial action. Moreover, the intrinsic fluorescence properties of CNDs could be useful for optimizing the rational design of targeted antimicrobial combination therapy with select antibiotics.</p>

PubMed Author Manuscript

pH-Triggered Assembly of Natural Melanin Nanoparticles for Enhanced PET Imaging

Natural melanin nanoplatforms have attracted attention in molecular imaging. Natural melanin can be made into small-sized nanoparticles, which penetrate tumor sites deeply, but unfortunately, the particles continue to backflow into the blood or are cleared into the surrounding tissues, leading to loss of retention within tumors. Here, we report a pH-triggered approach to aggregate natural melanin nanoparticles by introducing a hydrolysis-susceptible citraconic amide on the surface. Triggered by pH values lower than 7.0, such as the tumor acid environment, the citraconic amide moiety tended to hydrolyze abruptly, resulting in both positive and negative surface charges. The electrostatic attractions between nanoparticles drove nanoparticle aggregation, which increased accumulation in the tumor site because backflow was blocked by the increased size. Melanin nanoparticles have the natural ability to bind metal ions, which can be labeled with isotopes for nuclear medicine imaging. When the melanin nanoparticles were labeled by 68Ga, we observed that the pH-induced physical aggregation in tumor sites resulted in enhanced PET imaging. The pH-triggered assembly of natural melanin nanoparticles could be a practical strategy for efficient tumor targeted imaging.

ph-triggered_assembly_of_natural_melanin_nanoparticles_for_enhanced_pet_imaging

Introduction<!>Materials and Reagents<!>Cell Line and Animal<!>Preparation of PEG-Functionalized Melanin Nanoparticles (PEG-MNPs)<!>Preparation of pH-Sensitive Melanin Nanoparticles (pH-MNPs)<!>Characterization of Melanin Nanoparticles<!>68Ga2+ Radiolabeling<!>Cell Viability<!>Subcutaneous Tumor Models<!>Small Animal PET Imaging<!>Biodistribution Studies<!>Statistical Analysis<!>Preparation and Characterization of pH-MNPs<!><!>Preparation and Characterization of pH-MNPs<!><!>Preparation and Characterization of pH-MNPs<!><!>Preparation and Characterization of pH-MNPs<!><!>Radiolabeling With 68Ga and Stability in vitro<!><!>Biocompatibility of MNPs<!>Small Animal PET Imaging<!><!>Biodistribution Study<!><!>Conclusions<!>Data Availability Statement<!>Ethics Statement<!>Author Contributions<!>Conflict of Interest

<p>With the continuing development of nanotechnology, there is still strong demand for the design of new nanoparticles that have the properties of biocompatibility, long circulation time, low immune response, low toxicity, and biodegradability for biomedical applications (Jiao et al., 2018; Yang et al., 2019; Ou et al., 2020). Nature has inspired scientists to mimic precise dimensional biopolymer systems that play crucial roles in the physiology of many organisms and disease processes. Great efforts have been devoted to the modification of natural nanoparticles with high applicability potential (Cormode et al., 2010; Carrera et al., 2017; Aqil et al., 2019).</p><p>Among potential nanoparticles, melanin has attracted increasing attention because of its physicochemical properties. Melanin is an endogenous pigment that is distributed widely throughout human tissues and organs such as skin, mucous membranes, retinas, gallbladder, and ovaries, making it safe for in vivo application (Watts et al., 1981). Recent investigations demonstrated that melanin could serve as a photothermal agent (Liu Y. et al., 2013; Chu et al., 2016) and a photoacoustic probe (Ju et al., 2016; Liu et al., 2018) because of its strong near-infrared light absorption and high photothermal conversion efficiency. Moreover, melanin is an effective drug delivery system that can load chemotherapeutic drugs with aromatic structures via π-π stacking and/or hydrogen binding (Zhang et al., 2015), and the drug release can be stimulated by multiple methods, including near infrared light, pH, and reactive oxygen species (Araújo et al., 2014; Wang et al., 2016; Kim et al., 2017). As the structure of melanin includes abundant carboxyl groups, amino groups, and phenolic hydroxyl groups, it can serve as a natural multi-site metal chelating agent, making it capable of complexing many metal ions under mild conditions (Kim et al., 2012; Thaira et al., 2019). Many radionuclides are metallic elements, such as 64Cu, 89Zr, 68Ga, 177Lu, and 99mTc. Much effort is required to synthesize bifunctional chelators by labeling these radionuclides and optimizing the labeling conditions (Kang et al., 2015; Gai et al., 2016, 2018). Melanin may provide a facile strategy for labeling with radiometals. Cheng's group actively chelated melanin to 64Cu2+ and Fe3+ for PET and MRI imaging with high loading capacity and stability, indicating that melanin is a promising multimodality imaging nanoplatform (Fan et al., 2014; Hong et al., 2017).</p><p>Melanin can be made into nanoparticles with controllable sizes from a few nanometers to hundreds of nanometers (Ren et al., 2016; Amin et al., 2017; Lemaster et al., 2019). Studies have shown that small nanoparticles (<20 nm) can avoid macrophage recognition and penetrate tissues more deeply (Perrault et al., 2009; Liu C. et al., 2013). However, unfortunately, when small nanoparticles reach the tumor site, they continue to backflow into the bloodstream or are cleared into the surrounding tissues, decreasing retention within the tumor (Larsen et al., 2009; Zeng et al., 2016). Nanoparticles about 100 nm in size have been reported to have good retention but still high accumulation in the liver and pancreas before reaching the tumor, resulting in relatively low drug concentrations at the tumor site (Jain and Stylianopoulos, 2010; Albanese et al., 2012).</p><p>To overcome these limitations, we introduce a pH-triggered approach to aggregate small-sized melanin nanoparticles (pH-MNPs). The MNPs are redecorated with hydrolysis-susceptible citraconic amide, which can maintain a small size in the blood. When they reach the tumor site, spontaneous aggregation occurs in response to the tumor's acidic microenvironment. The aggregation of melanin nanoparticles cannot exceed the size of the blood vessels, and they become trapped in the extracellular matrix between cells because of their increased size, resulting in enhanced retention in the tumor site (Liu X. et al., 2013). In addition, the pH-melanin was labeled by 68Ga, and the in vivo PET imaging and biodistribution profiles of 68Ga-pH-MNPs were evaluated. We ascertained that the pH-triggered assembly of natural melanin nanoparticles could result in enhanced PET imaging, which could be a practical strategy for efficient tumor imaging.</p><!><p>Melanin was purchased from Sigma-Aldrich. Methoxy polyethylene glycol amine (mPEG2000-NH2) was purchased from the Shanghai Aladdin Biochemical Technology Co., Ltd.</p><!><p>H22 mouse hepatocarcinoma cells were purchased from the American Type Culture Collection (ATCC) and cultured in standard cell medium recommended by ATCC. Male BALB/c mice (6–8 weeks, 20–22 g) were provided by the animal center of Tongji Medical College (Wuhan, China). The mice were raised at an animal facility under special pathogen-free (SPF) conditions with a 12 h light/dark cycle and free access to food and water. The animal study was reviewed and approved by the Laboratory Animal Management Committee of Tongji Medical College of Huazhong University of Science and Technology.</p><!><p>Thirty mg of the melanin granule was dissolved in 10 ml of NaOH (0.1 N) and sonicated for 30 min with a bath type sonicator. Then, 90 mg of mPEG2000-NH2 (Mw = 2,000) aqueous solution was dropped into the above aqueous solution and stirred with a magnetic stirrer. After vigorous stirring for 12 h, the mixed solution was retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water.</p><!><p>Thirty mg of the melanin granule was dissolved in 10 ml of NaOH (0.1 N) and sonicated for 30 min with a bath type sonicator. Then, 90 mg of mPEG2000-NH2 (Mw = 2,000) and 270 μmol of ethylenediamine were added into the above aqueous solution and stirred with a magnetic stirrer. After vigorous stirring for 12 h, the mixed solution was retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water. Then, 200 μmol of citraconic anhydride was added into the obtained 10 ml of melanin aqueous solution (1 mg/ml of water) and the pH was adjusted to 9.0 with NaOH (0.1 N). After vigorous stirring for 12 h, mPEG and the citraconic amide modified MNPs were retrieved by centrifugation (MWCO-10,000, Millipore) at 4,000 rpm for 30 min and washed several times with deionized water.</p><!><p>The size and zeta potential of MNPs under pH 9, 7.4, and 6 were measured by a dynamic light scattering (DLS) instrument (Malvern instruments Ltd). The morphologies of MNPs were obtained under a transmission electronic microscope (TEM) at 100 kV.</p><!><p>68GaCl2 was washed from a 68Ge/68Ga radionuclide generator by 4 × 1 ml high purity hydrochloric acid (HCl, 0.05 M), and we took the one with the highest radioactivity. One ml of 68GaCl2 nearly 5 mCi in 0.05 M HCl was added into 200 μl PEG-MNPs or pH-MNPs (0.5 mg/ml of MNPs), then 0.25 M NaOAc was added dropwise to adjust the pH to 4, 5, 6, 7.4, respectively and incubated at room temperature for 30 min. The radiolabeled MNPs were purified by a PD-10 column (GE Healthcare) to remove the free 68Ga. The final product was washed out by PBS and passed through a 0.22 μm Millipore filter into a sterile vial for in vivo PET imaging. The radiolabeling yield was evaluated by dividing the radioactivity of the purified radiolabeled MNPs by the total radioactivity added. The stability of 68Ga-labeled MNPs was determined in vitro by incubating in saline or human plasma at a physiologic temperature for 3 h. An aliquot of 68Ga-labeled MNPs was removed at 1, 2, and 3 h intervals and the radiochemical purity was determined by ITLC (TLC scanner, BIOSCAN, USA). GF254 silica gel plates were used as the stationary phase and citrate buffer (0.1 M) was used as the mobile phase.</p><!><p>The in vitro cytotoxicity of MNPs was determined in H22 mouse hepatocarcinoma cells by the CCK-8 assay. H22 cells were cultured in DMEM (GIBCO, Carlsbad, CA, USA), supplemented with 10% fetal calf serum (FCS), 2 mmol/l glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells (5,000/well) were seeded in 96-well plates with 100 μL/well medium and incubated overnight with 10% fetal bovine serum DMEM medium at 37°C and in an atmosphere of 5% CO2. Cells were then cultured in the medium supplemented with different doses of PEG-MNPs and pH-MNPs. The final concentrations of MNPs in the culture medium were fixed at 100, 50, 25, 10, and 5 μg/ml, untreated cells were used as the control (with 100% cell viability), and the medium without cells was used as the blank. After treatment for 24 and 48 h, respectively, the medium was removed and DMEM medium containing 10% CCK-8 was added. After incubation for 30 min at 37°C, the absorbance at 450 nm was measured by using an automatic enzyme standard instrument (Bio-Rad iMark).</p><!><p>The H22 cells were maintained in the ascitic form by sequential passages into the peritoneal cavities of BALB/c mice, by weekly intraperitoneally (i.p.) transplanting 1 × 107 tumor cells in 0.2 ml. The ascites were collected, diluted with sterile saline, and the cell concentration was adjusted to 1 × 107/ml. The diluted solution (0.2 ml) was administered subcutaneously in the right shoulder of each mouse. When the tumors reached 0.5–0.8 cm in diameter, the tumor-bearing mice were subjected to in vivo PET imaging and biodistribution studies.</p><!><p>Small animal PET imaging of tumor-bearing mice was performed on a microPET-CT (TransPET Discoverist 180 system, Raycan Technology Co., Ltd, Suzhou, China). 68Ga-labeled PEG-MNPs and 68Ga-labeled pH-MNPs (180.0 ± 5.0 μCi) were injected via the tail vein, respectively (n = 4). At different times after injection (1, 2, and 3 h), mice bearing H22 tumors were anesthetized with 2% isoflurane in 100% oxygen for maintenance during imaging, and placed prone near the center of the FOV of the scanner. PET/CT images were obtained with the static mode for 10 min followed by a CT scan in the normal mode. The PET images were reconstructed using the three-dimensional (3D) ordered-subsets expectation maximization (OSEM) algorithm with a voxel size of 0.5 × 0.5 × 0.5 mm3. CT images were reconstructed using the FDK algorithm with 256 × 256 × 256 matrix. Images were displayed with software Carimas (Turku PET Center, Turku, Finland). No background correction was performed. The radioactivity uptake in the tumor and normal tissues were calculated using a region of interest (ROI) drawn over the whole organ region and expressed as a percentage of the injected radioactive dose per gram of tissue (% ID/g).</p><!><p>The biodistribution studies were performed in H22 tumor-bearing BALB/c mice (6–8 weeks), weighing 20–22 g, which were randomly divided into six groups (five mice per group). 68Ga-labeled PEG-MNPs and 68Ga-labeled pH-MNPs were intravenously injected through a tail vein and the mice were sacrificed at 1, 2, and 3 h intervals. The blood and organs of interest (e.g., brain, heart, lungs, liver, spleen, kidneys, stomach, small intestine, large intestine, muscle, bones, and tumor) were harvested, then weighed and measured using an automated gamma counter (2470 WIZARD, PerkinElmer, Norwalk CT, USA). The amount of radioactivity in each tissue sample was reported as the percentage of the injected dose per gram of tissue (%ID/g).</p><!><p>Quantitative data are expressed as means ± standard deviation (SD). Means were compared using Student's t-test (two-tailed) with a P-value <0.05 indicating significance.</p><!><p>The design and synthetic procedures of pH-MNPs are schematically illustrated in Figure 1. Firstly, the natural melanin was modified with mPEG-NH2 and ethylenediamine to provide many terminal amine groups on the surface. Then, the primary amine groups were reacted with citraconic anhydride to form amide bonds (Figure 1). The citraconic amide moiety on the surface is selectively hydrolysis-susceptible in mildly acidic environments. Under neutral and alkaline conditions, the citraconic amide bonds are stable and maintain negative charges. Triggered by pH values lower than 7.0, such as those present in tumor tissues that are often rendered acidic by hypoxia, the citraconic amide moiety tended to hydrolyze abruptly, resulting in both positive and negative surface charges as its terminal group changed from a carboxylate anion to a protonated amine group (Nam et al., 2009). The electrostatic attraction between nanoparticles drove nanoparticle aggregation (Supplementary Figure 1). The steric effect of mPEG may have hindered the electrostatic attraction, but the reduction of the surface modification of mPEG affected the water solubility of the MNPs. A ratio of ethylenediamine to mPEG of about 6 was reported to achieve a balance between steric hindrance and water solubility.</p><!><p>A schematic illustration of the preparation process of the pH-MNPs.</p><!><p>The product of each step of the synthesis was measured by the zeta potential and FT-IR spectra. In the FTIR spectrum of pristine melanin, the broad and strong bands in the 3,300 ~ 3,400 cm−1 region were due to the -OH and -NH stretching. The characteristic peaks at 1,600 cm−1 were attributed to the aromatic ring C=C, C=N bending, and C=O stretching in indole and indoline structures. The FT-IR spectra detected characteristic alkyl C-H bands around 2,910 cm−1 and C-O-C stretching bands from PEG at 1,100 cm−1 after the introduction of ethylenediamine and PEG on the surface (Figure 2). Although the FT-IR spectra did not provide any additional information about the pH-MNPs, the zeta potential described a considerable change in the surface charge at each step of the surface modification (Figure 3A). Melanin itself is a negatively charged polymer, and the surface potential after the introduction of PEG remained negative (−12.8 ± 1.3 mV). After a reaction with a large amount of ethylenediamine, the surface charge changed from negative to positive (6.7 ± 0.9 mV) because of the presence of the protonated amine. The surface charge then became negative again after a reaction with citraconic anhydride, indicating successful conjugation and conversion of the amine group to a carboxylate anion. Dynamic light scattering was employed to examine the size of the MNPs after surface modification. The hydrodynamic diameters of the PEG-MNPs, PEG-EDA-MNPs, and pH-MNPs were all ~12 nm, demonstrating no significant size differences between nanoparticle type (Figure 3B).</p><!><p>FT-IR spectra of pristine melanin, PEG-EDA-MNPs, and pH-MNPs.</p><p>Characteristics of MNPs. (A) Zeta potential of PEG-MNPs, PEG-EDA-MNPs, and pH-MNPs. (B) Hydrodynamic size of PEG-MNPs, PEG-EDA-MNPs, and pH-MNPs. Bars represent means ± SD (n = 3). All of the samples are adjusted to neutral pH value by buffer solution.</p><!><p>To characterize pH-induced aggregation behavior in solution, we compared the stability of pristine melanin, PEG-MNPs, PEG-EDA-MNPs, and pH-MNPs under different pH conditions. As shown in Figure 4, pristine melanin only dissolved in the alkaline solution, while the PEG-MNPs and PEG-EDA-MNPs maintained good solubility in acidic, neutral, and alkaline conditions. However, pH-MNPs exhibited specific aggregation in response to acidic conditions. At pH 9 and 7.4, the solution of pH-MNPs was clear and translucent, and at a mildly acidic pH 6, flocculation and precipitation occurred. All of the photos were taken after the samples had been standing at room temperature for ~12 h, and the pH-MNPs maintained a stable precipitation state, indicating that the aggregation was irreversible after complete hydrolysis. The hydrodynamic size and zeta potential of pH-MNPs at different pH values were measured by dynamic light scattering (DLS), with PEG-MNPs as the control group. As shown in Supplementary Figure 2, the size of pH-MNPs was found to be 3,316 ± 271 nm with a wide size distribution at pH 6, while the particles showed a small size and narrow size distribution at pH 7.4 and 9. In the control group, the size of PEG-MNPs did not change and maintained at 12.2 ± 1.3 nm under different pH values. Supplementary Figure 3 showed the zeta potentials of pH-MNPs and PEG-MNPs at different pH values. At pH value of 9, the zeta potentials of pH-MNPs was −12.6 ± 1.0 mV, and the value positively shifted to −9.3 ± 1.8 mV under neutral conditions. After exposure to an acidic environment (pH 6), the surface charge of pH-MNPs shifted to a positive value (4.9 ± 0.3 mV), indicating the citraconic amide moieties had been hydrolyzed into protonated amine groups. PEG-MNPs also showed a trend in that the zeta potential shifted positively as the pH value decreased. At pH 9, the zeta potential was −16.4 ± 0.7 mV, and it became −12.8 ± 1.3 mV at pH 7.4. After exposure to pH 6 buffer, the zeta potential positively shifted to −9.3 ± 0.3 mV, but remained negative. This result was due to the protonation of phenolic and amino groups of PEG-MNPs.</p><!><p>Stability of pristine melanin, PEG-MNPs, PEG-EDA-MNPs, and pH-MNPs under different pH conditions. Standing ~12 h, photos taken of all samples. The red arrow indicates precipitation at the bottom of the bottle.</p><!><p>Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were conducted to monitor the variation of particle size and morphology between different time points during the pH-triggering process of pH-MNPs. The TEM images in Figure 5A illustrate that the average size of the prepared pH-MNPs was nearly 10 nm with a narrow size distribution, which is consistent with the results obtained by DLS. Upon pH triggering, the agglomeration degree of pH-MNPs gradually grew, and the size to which the pH-MNPs aggregated became larger. After 10 min of exposure to an acidic environment (pH 6), the size increased to 100–160 nm with messy shapes observed by TEM, and DLS measurement showed two peaks with PDI 0.542, indicating a wide size distribution (Figure 5B). TEM measurement after 2 h of exposure confirmed the growth of some aggregates over time: the hydrodynamic size of pH-MNPs continually increased to the micron level in Figure 5C, whereas such pH-induced aggregation was not observed in PEG-MNPs (Supplementary Figure 4). These results strongly support that pH-MNPs had the ability to undergo pH-triggered aggregation. The aggregation of pH-MNPs began early (within 10 min), and flocculation occurred within 2 h. This rapid pH-response ability provides the possibility of subsequently 68Ga-labeling for PET imaging, which is desirable because the half-life of the 68Ga nuclide is only 67.7 min.</p><!><p>TEM images (left) and DLS images (right) of pH-MNPs in pH 6 buffer at different elapsed times of (A) 0, (B) 10, and (C) 120 min.</p><!><p>Melanin has the ability to coordinate with metal ions without an additional chelator because of its inherent structure. That enables us to prepare radiometal-labeled melanin nanoparticles for molecular imaging. Furthermore, melanin can bind metal ions at a wide pH range because of different chelating sites on the molecule function at different pH ranges. Under acidic conditions, the carboxyl groups are mainly involved in binding metal ions to form complexes, whereas under alkaline conditions, the phenolic hydroxyl groups play a major role (Sarna et al., 1980). In this research, we used 68Ga to radiolabel pH-MNPs without any linker at different pH values. The 68Ga-pH-MNPs exhibited high loading capacities at pH 4 and 5 with non-decay-corrected yields of 89.6 ± 6.2 and 87.5 ± 8.3%, respectively (Figure 6A). As the pH increased, the labeling yield gradually decreased, with only 52.3 ± 12.4% yield at pH 7. Considering the acid-triggered assembly of pH-MNPs, we still tried to use 68Ga for labeling under neutral conditions for subsequent in vivo studies, but the labeling yield was not very high. The 68Ga-pH-MNPs were prepared under the labeling conditions of pH 7, 37°C, and 30 min incubation. After purification using a PD-10 column, the radiochemical purity of the 68Ga-pH-MNPs was determined by ITLC. On the ITLC plate, 68Ga-pH-MNPs remained close to the origin (Rf = 0.12), and no free 68Ga was observed at the solvent front (radiochemical purity: >96%; Supplementary Figure 5). The stability assay of 68Ga-pH-MNPs in saline solution and human plasma showed that the radiochemical purity of 68Ga-pH-MNPs remained above 95% throughout the 3 h incubation period, indicating excellent stability in vitro (Figure 6B).</p><!><p>Characterization of radiolabeling with 68Ga. (A) Radiolabeling yield of 68Ga-pH-MNPs at different pH and (B) stability of 68Ga-labeled MNPs incubated in saline or human plasma for 1, 2, and 3 h.</p><!><p>To evaluate the in vitro cytotoxicity of the synthesized MNPs, CCK-8 assays were performed on H22 mouse hepatocarcinoma cells. For these assays, cultured cells were exposed to PEG-MNPs and pH-MNPs (5–100 μg/mL) for 24 and 48 h. The results showed that PEG-MNPs and pH-MNPs did not inhibit H22 cell viability at any concentration at either time point (Supplementary Figure 6), indicating that the synthesized MNPs have high biocompatibility in vitro.</p><!><p>For PET imaging, ~6.66 MBq (180 μCi) of 68Ga-pH-MNPs and 68Ga-PEG-MNPs were injected intravenously into H22 tumor-bearing mice. At different time points after injection (1, 2, and 3 h), tomographic images were acquired. Figure 7 shows representative decay-corrected whole-body images. A stronger PET signal in the tumor was observed for 68Ga-pH-MNPs than 68Ga-PEG-MNPs at all time points. The difference in tumor accumulation when 68Ga-PEG-MNPs are employed may be due to backflow into the bloodstream over time. In contrast, the pH-triggered aggregation of 68Ga-pH-MNPs, which can be trapped in tumor tissue, led to enhanced PET imaging. In addition to that within the tumor, moderate activity accumulation was found in the liver because nanoparticles are easily captured by the reticuloendothelial system. The heart was visible, perhaps because of the circulation of small-sized melanin nanoparticles in the blood. Quantitative analysis of three-dimensional regions of interest over multiple image slices revealed that the tumor uptake of 68Ga-pH-MNPs was up to 2.4 times higher than that of 68Ga-PEG-MNPs at 3 h post-injection (4.47 ± 0.73 vs. 1.87 ± 0.56% ID/g, respectively, p < 0.01; Supplementary Figure 7).</p><!><p>The overlaying of the PET and CT images of H22 tumors acquired at 1, 2, and 3 h after the intravenous injection of (A) 68Ga-pH-MNPs and (B) 68Ga-PEG-MNPs. Representative decay-corrected coronal and transaxial are displayed on the top and bottom respectively. The white arrow indicates tumor site.</p><!><p>The biodistribution results are shown in Figure 8. The radioactivity in blood gradually decreased over time, indicating that 68Ga-pH-MNPs were gradually cleared from circulation (Figure 8A). The liver showed the highest uptake among the tissues studied (7.47 ± 0.76% ID/g at 1 h), and then the level reduced gradually but was still prominent at 3 h post-injection (4.51 ± 0.72% ID/g). Relatively lower activity accumulation was observed in the spleen and kidney. The 68Ga-pH-MNPs was mainly cleared through the hepatobiliary system. The tumor uptake of 68Ga-pH-MNPs consistently increased, and the enhanced retention was maintained throughout all time points (2.54 ± 0.38, 3.35 ± 0.13, and 3.86 ± 0.25% ID/g at 1, 2, and 3 h p.i., respectively). In contrast, the tumor uptake of 68Ga-PEG-MNPs decreased from 2.14 ± 0.38% ID/g (1 h p.i.) to 1.34 ± 0.25% ID/g (3 h p.i.) (Figure 8B). The results were consistent with the PET images above. The tumor-to-muscle ratio of 68Ga-pH-melanin increased significantly from 4.74 ± 0.76 at 1 h p.i. to 29.30 ± 5.64 at 3 h p.i., a much greater increase than that of 68Ga-PEG-melanin (2.88 ± 0.74 at 3 h p.i.). However, the tumor-to-blood ratio was relatively low, probably because nanoparticles were still circulating in the blood (Supplementary Table 1). Therefore, the pH-MNPs can achieve enhanced tumor retention for PET imaging.</p><!><p>Biodistribution study of H22 tumor-bearing mice (n = 5) at different time points in (A) the 68Ga-PEG-MNPs group and (B) the 68Ga-pH-MNPs group.</p><!><p>In this work, we have successfully designed and prepared natural melanin nanoparticles that can form aggregates in response to pH changes. Under mildly acidic conditions, the pH-MNPs began to aggregate and became trapped by their increasing size, resulting in enhanced tumor retention. We also demonstrated that the pH-MNPs could be successfully radiolabeled with the 68Ga nuclide in a pH-neutral environment by simple mixing. The resultant 68Ga-pH-MNPs exhibited enhanced PET imaging, which could provide a promising strategy for molecular imaging and future clinical trials.</p><!><p>The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.</p><!><p>The animal study was reviewed and approved by Laboratory Animal Management Committee of Tongji Medical College of Huazhong University of Science and Technology.</p><!><p>QL conceived the idea and supervised the research work overall. HF and YG contributed to the experiment methods and data analysis. QL wrote the manuscript and drew all the figures. YG came up with ideas for the manuscript. XL contributed to the revision of the paper. 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>

PubMed Open Access

Nickel Catalyzed Electrochemical C(sp 2 )−C(sp 3 ) Cross-Coupling Reactions

HIGHLIGHTS Electrochemical C(sp 2 )−C(sp 3 ) coupling reactions are developed using bench stable, inexpensive substrates and Ni catalysts;  The electrochemical cross-coupling exhibits broad substrate scope and good yields;  The electrochemical cross-coupling are practical in making pharmaceutical candidates; Reaction scalability was demonstrated using flow cell synthesis. with a broad scope, good yields, and practical applications, which expands the synthetic toolbox to forge carbon-carbon bonds.Summary: Nickel (Ni) catalyzed carbon-carbon (C−C) cross-coupling reactions haves been considerably developed in last decades and has demonstrated unique reactivities compared to palladium. However, existing Ni catalyzed cross-coupling reactions, despite success in organic synthesis, are still subject to the use of air-sensitive nucleophiles (i.e. Grignard and organozinc reagents), or catalysts (i.e. Ni 0 pre-catalysts), significantly limiting their academic and industrial adoption. Herein, we report that, through electrochemical voltammetry screening and optimization, redox neutral C(sp 2 )-C(sp 3 ) cross-coupling reactions can be accomplished in an undivided cell configuration using bench-stable aryl halide or β-bromostyrene (electrophiles) and benzylic trifluoroborate (nucleophiles) reactants, non-precious, bench-stable catalysts consisting of NiCl2•glyme pre-catalyst and polypyridine ligands under ambient conditions. The broad reaction scope and good yields of the Ni-catalyzed electrochemical coupling reactions were confirmed by 50 examples of aryl/β-styrenyl chloride/bromide and benzylic trifluoroborates. Its potential applications were demonstrated by electrosynthesis and late-stage functionalization of pharmaceuticals, and natural amino acid modification. Furthermore, to testify practical industrial adoption, three electrochemical C−C cross-coupling reactions were demonstrated at gram-scale in a flow-cell electrolyzer. An array of chemical and electrochemical studies mechanistically indicates that the studied electrochemical C−C cross-coupling reactions proceed through an unconventional radical trans-metalation mechanism. The presented Ni-catalyzed electrochemicalC(sp 2 )-C(sp 3 ) cross-coupling paradigm is highly productive, easily operative, and atomically economic, and is expected to find wide-spread applications in organic synthesis.

nickel_catalyzed_electrochemical_c(sp_2_)−c(sp_3_)_cross-coupling_reactions

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

<p>The Bigger picture: Electrosynthesis has recently been recognized an enabling technology for organic synthesis. In principle, substrates or catalysts in electrosynthesis can be selectively anodically or catholically activated to participate desired reaction sequences in an electrolyzer.</p><p>The attractive synthetic merits of electrosynthesis include migrating the use of reactive (sometimes even dangerous) oxidants and reductants, enabling the access of highly reactive catalytic intermediates which are not easily handled in traditional thermal reactions, and thus representing a green, atomically economical synthetic strategy. Most of reported electrosynthesis methodologies were based on an anodic or cathodic process. However, paired redox neutral electrosynthesis merging simultaneous anodic and cathodic processes remains challenging. Herein, we report a redox neutral Ni-catalyzed electrochemical C(sp 2 )-C(sp 3 ) cross-coupling paradigm</p><!><p>In the past half-century, transition metal catalyzed carbon-carbon (C−C) cross-coupling reactions have gained significance advances regarding reaction scopes, selectivity, and catalytic mechanisms, and achieved tremendous success in organic synthesis of pharmaceutical molecules, agrochemicals, and organic materials. [1][2][3] Catalyzed C−C cross-coupling reactions have historically been dominated by Pd-based catalysts. [4][5] In addition to replacing the expensive, precious Pd metal, Ni metal is characteristic of more negative 2+/0 and 1+/0 redox potential than Pd 2+/0 to enable unique oxidative addition reactivities in activating C-X (X = Cl and Br) bonds 6 and has found increasing importance in C-C cross coupling reactions. [7][8] However, Ni catalyzed C−C crosscoupling reactions are still limited by a number of well-known synthetic limitations. Ni-based Kumda, Negish, and Suzuki, and reductive couplings are practically hampered by the use of either strong nucleophiles, sacrificed reductants, or sensitive Ni 0 pre-catalysts, e.g. widely used</p><p>Ni(COD)2 (where COD is 1,5-cyclooctodiene) and typically require rigid reaction conditions using inert atmosphere glovebox or Schenk-line techniques. It remains a long-standing challenge to develop Ni-catalyzed cross-coupling reactions using bench stable chemicals and easy-handling conditions for widespread academic and industrial adoption. 8 Efforts have been made to develop well-defined air stable Ni II and Ni 0 pre-catalysts [9][10] and encapsulated Ni 0 pre-catalysts. 11 However, these practices are still limited by the pre-formation of Ni pre-catalysts under rigid air-free conditions and the need of special stabilization ligands for most of them.</p><p>On the other side, literature has witnessed the powerful applications of electrochemistry in organic synthesis. [12][13][14][15] By precisely controlling redox potential in an electrolyzer cell, substrates or catalysts can be selectively anodically or catholically activated to participate desired reaction sequences. [12][13][14][15] Thereby, electrosynthesis not only migrates the use of reactive (even dangerous)</p><p>oxidants and reductants and enables the access of highly reactive catalytic intermediates which are not easily handled in traditional thermal reactions, representing a green, atomically economical synthetic strategy. In spite of being known for many decades, until very recently electrosynthesis has aroused recurred attention and is believed to impart profound impacts on organic syntheis. [12][13][14][15] For instance, anodic reactions including alcohol oxidation, 16 C−H functionalization, [17][18][19][20][21][22][23][24] alkene functionalization, [25][26] cyclization, [27][28] and C−O 29 and C-N [30][31] couplings, and cathodic reactions including arene or alkene hydrogenation, [32][33] and arylboronic acid hydroxylation 34 were demonstrated with good selectivity and yields. Ni-catalyzed cathodic reductive C−C homocouplings were first reported by Jennings and co-workers in 1976. Ni-catalyzed reductive cross-electrophile C−C couplings was pioneered by Jutand, Perchion and coworkers [35][36][37] and have been recently advanced by several groups, representing an attractive technology for C-C formation without using strong, reactive reductants as in traditional thermal reactions. [38][39][40][41][42] However, Nicatalyzed redox-neutral cross couplings remain very rare, 43 in which anodic oxidation of an nucleophile and cathodic reduction of an electrophile are coupled to forge the C-C bond formation while no sacrificed stoichiometric electron donor is required. It is also worth noting that more than 97.5% of ca. 900 electrosynthesis methodologies reported between 2000 and 2017 were based on an anodic or cathodic process. 13 The development of paired redox neutral electrosynthesis has been very challenging as merging an anodic redox reaction and a cathodic redox reaction is often plagued by side reactions of reactive intermediate in each redox reaction. 13 For example, homo-</p><!><p>Instead of randomly testing combinations of nucleophiles, electrophiles, and catalysts, we first set out to identify individual anodic and cathodic SET half-cell reactions for the proposed full-cell C−C coupling reactions using the electrochemical cyclic voltammetry (CV) method. For the cathodic half-cell reaction, we aimed to explore the SET reduction of Ni II -based catalysts to activate aryl and vinyl halide electrophiles by the Ni III/I(II/0) redox cycle to achieve R−Ni III(II) −X intermediate, which is mechanistically accessible in traditional Ni-based thermal couplings. 6 For the anodic half-reaction, nucleophiles including carboxylic acid 12 and organic trifluoroborate 44 are well documented as carbon radical precursors (Rʹ• in Figure 1) upon SET oxidation. It is noted that organic trifluoroborates have been used as versatile radical precursors for metal photoredox catalytic coupling reactions with aryl halides by Molander and coworkers. 45 Electrochemical screening of the proposed half-cell reactions was conducted through the cyclic voltammetry (CV) method using a three-electrode system. As shown in Figure 2B(i) (gray curve), in the presence of 3 equivalents 2,2'-bipyridine (2,2'-bpy) ligand, NiCl2•glyme displayed a reversible redox signal at E1/2 = -1.49 V (vs. Fc +/0 ), which corresponding to the Ni II/I redox couple.</p><p>Then, 10 equivalents of organic halides (R−X) were added to the electrolyte and CV curves were collected again. Among tested organic halides, C(sp 2 ) precursors (arly halide and alkenyl bromide)</p><p>or C(sp) precursors (alkynyl bromide) could be activated by the Ni I intermediate while C(sp 3 ) precursors were inactive. For example, when methyl 4-bromobenzoate was added (green trace in precursors (alkynyl bromide) and C(sp 3 ) sources (potassium benzyltrifluoroborate, phenylacetate,</p><p>LG We then optimized the NiCl2•glyme/polypyridine catalyst system using cyclic voltammetry with methyl 4-bromobenzoate as a model electrophile. As shown in Figure 2C(i), seven different polypyridine ligands including 4,4'-di-tert-butyl-2,2'-bipyridyl (dtbbpy), 6,6'-dimethyl-2,2'bipyridyl (dmbpy), 2,2'-bpy, dimethyl 2,2'-bipyridine-4,4'-dicarboxylate (dmcbpy), 1,10phenanthroline (1,10-Phen), 2,2'-biquinoline (biq), and terpyridine were screened to identify the most suitable ligand for the Ni-catalyst. Among all the ligands, dtbbpy prompted the strongest current intensity increase (green curve), indicating that Ni I (dtbbpy) + is the most reactive species to oxidative addition of the C-Br bond of methyl 4-bromobenzoate. Besides 2,2'-bpy and dtbbpy, 1,10-Phen also aroused strong current response (purple curve) and thus can also be a suitable ligand.</p><p>Terpyridine ligand displayed the lowest current response under the same conditions (Figure S5).</p><p>We further investigated the effect of Ni/ligand ratio on the reactivity of the Ni-catalyst. The CV curves of NiCl2•glyme with addition of various ratio of dtbbpy ligand showed continuous change (Figure S5). In the absence of the dtbbpy ligand, no reversible redox signal was observed. When 1 -3 equivalents of dtbbpy ligand was added, there were two set of quasi-reversible redox signals.</p><p>Further increase the ligand ratio to 5 equivalents, the redox signals overlapped to one set of fully reversible redox signal. It indicates that there is an equilibrium for Ni II complexes in the solution:</p><p>Ni II ↔ Ni II (dtbbpy) ↔ Ni II (dtbbpy)2 ↔ Ni II (dtbbpy)3, which is consistent with a previous UV-Vis study. 30 In the presence of methyl 4-bromobenzoate substrate, the addition of 1. S1). It was found that the yield for 1 was further improved to 93% using K2CO3</p><p>additive. The essentiality of NiCl2•glyme catalyst, dtbbpy ligand, and electrolysis was determined by control experiments (Table S1, SI). In addition, both reaction selectivity and rate were largely affected by current intensity. Lower selectivity was obtained under a higher or lower current intensity (64% under 1.0 mA, 77% under 5.0 mA current). Under 1.0 mA current electrolysis, the reaction was significantly decelerated as a reaction time of 48 h needs to fully convert the substrate.</p><p>Other solvents, such as THF, MeCN, CH2Cl2, MeOH, and DMSO were not effective to this reaction (only 0 -15% yield was observed, Table S2, SI). Moreover, similar as under thermal reaction conditions, 16 the reactivity and selectivity of this reaction is highly sensitive to the ligand structure (Table S3, SI). In particular, dtbbpy and 2,2'-bpy ligands exhibited the best efficiencies with isolated yields of 93% and 87%, respectively. 1,10-Phen and tridentate terpyridine (tpy) ligands gave moderate yields of 67% and 73%, respectively. It is noteworthy that the best selectivity between cross-coupling product 1 and homo-coupling product 1' was obtained by using the dtbbpy (95:5) and tpy (96:4) ligands, which tend to suppress the homo-coupling of strong electrophiles. However, other ligands (dmbpy, dmcbpy, and biq) were not effective. Moreover, no product was observed when a bidentate bis-phosphine ligand, 1,2-bis(diphenylphosphino)benzene (dppb) was used (Table S3). It was observed that the dppb ligand underwent oxidation near to the oxidation potential of benzyltrifluoroborate, which could destabilize the corresponding Ni catalyst (Figures S6 and S7).</p><p>After establishing optimal reaction conditions for yield and selectivity for the Ni-catalyzed electrochemical C(sp 2 )-C(sp 3 ) cross-coupling, we next tested the reaction scope on both aryl halide and benzylic trifluoroborate using the most efficient Ni/dtbbpy catalyst. As shown in Figure 3, a wide range of aryl chlorides including both electron-rich and electron-deficient arenes were suitable to this Ni-catalyzed electrosynthesis system (1 to 5). The electron-deficient aryl chlorides</p><p>(1 to 3, 74% to 86% yield) delivered better yield than the electron-rich ones (4 and 5, 46% and 31% yield). It is probably due to the low activity of electron-rich aryl chloride substrates with the Ni I intermediate. Aryl bromides displayed better efficiencies than the corresponding aryl chlorides, as 1 to 5 were isolated in 77% to 93% yield by using aryl bromide substrates. The reaction exhibited comparable efficiency upon scale-up, for example, 89% yield was obtained on a 2.5 mmol scale reaction of 1 (0.5 g). The substituent position of aryl bromide displayed a moderate effect to the reaction efficiency, as the para-, meta-and ortho-substituted methyl bromobenzoate delivered 93%, 71%, and 89% yield (1, 6 and 7), respectively. Aryl bromides with functional groups as diverse as ester (1, 6, 7, and 10), ketone (2), fluoride (3), methoxy group (9 and 10), amide (14 and 15), aldehyde (11), nitrile (12) and alkenyl (19) were effective in this reaction. Substrates possessing strongly electron-donating substituents such as t Bu and methoxy groups could also provide moderate to good yield (72% for 8 and 53% for 9). When 4-bromo-phenol was used as the electrophile as a control experiment for entry 9, no cross-coupling product was observed, which is attributed to the oxidation of the substrate itself at a less positive potential than the borate nucleophile. The observation emphasizes the protection of oxidization susceptible functional groups under the investigated electrochemical conditions. It is interesting that for the substrates possessing strong electron-withdrawing substituents such as aldehyde, acetyl, and cyano groups, best results were obtained by using 2,2'-bpy ligand (83% and 91% yield for 2 from chloride and bromide, respectively, 82% yield for 11, and 74% yield for 12). Furthermore, in the case of 4bromo(trifluoromethyl)benzene, homo-coupling product, 4,4'-bis-(trifluoromethyl)biphenyl (13'), was obtained as the only product when using dtbbpy and 2'2-bpy ligands. Interestingly, 21% yield of cross-coupling product 13 was obtained by using the tpy ligand, implying the Ni/tpy ligand combination is more compatible with electron deficient electrophiles to suppress homo-coupling. In addition to examine the substituent positions and functional groups of the aryl halide substrates, we also investigated the tolerance of this electrosynthesis system to common protecting groups which are widely used in organic synthesis, such as amide, tert-butyloxycarbonyl (Boc), benzyl ether (BnO), and acetal. All of these protecting groups were well tolerated, as evidenced by good isolation yield of 14 to 18 (67% to 86% yield). The π-conjugation extended aryl bromide substrates including 4-bromophenylethene, 3-bromofluorene, and 2-bromonaphthalene also smoothly proceeded this cross-coupling reaction with moderate to good yield (19 to 21, 43% to 84% yield). Moreover, a variety of aryl bromides consisting of nitrogen-containing heterocyclic groups including 6-bromoquinoline, 6-bromoisoquinoline, and Boc protected 6bromotetrahydroisoquinoline, and 5-bromoindole, which are prevalent building blocks in bioactive molecules, delivered moderate to good yield (22 to 25, 52% to 81% yield).</p><p>The substrate scope of benzylic trifluoroborate salts was also investigated. As shown in Figure 3, both electron-rich and electron-deficient benzylic trifluoroborates were approved efficient carbon radial precursors in this cross-coupling reaction (26 to 31, 74% to 95% yield). Functional groups, including esters, methoxy group, and trifluoromethyl group were tolerant to this Nicatalyzed electrosynthesis. The substituent positions displayed negligible effects to the reaction efficiency, as comparable yield was obtained for the para-, meta-, and ortho-substituted benzylic trifluoroborates (26 to 28, 77% to 82% yield). In the presence of two strong electron-donating methoxy (MeO-) groups, the highest yield, 95%, was gained for 29, which is interpreted as the favorable oxidation kinetics of the corresponding trifluoroborate substrate. The π-conjugation extended naphthalen-2-ylmethyl trifluoroborate is also highly productive in this electrochemical C(sp 2 )-C(sp 3 ) cross-coupling reaction, as 72% yield was obtained for 32. Beside the benzylic trifluoroborates, (benzyloxy)methyl trifluoroborate also manifested reasonable reactivity in this reaction with a yield of 47% (33). In the CV screening studies (Figure 2B), some other substrates also showed reactivity in the anodic half-reaction. For example, β-bromostyrene and methyl 3-bromopropiolate showed reactivity in the anodic half-cell reaction (Figure S4, SI). β-bromostyrene was briefly examined as an electrophile for the Heck-type like C(sp 2 )-C(sp 3 ) cross-coupling. In the reaction of βbromostyrene and potassium trifluoro(4-(methoxycarbonyl)benzyl)borate, 48% yield of product 34 and 47% yield of the homo-coupling product 34' were obtained by using dtbbpy as ligand.</p><p>According to entry 13, the tpy ligand exhibited the better selectivity to suppress the homo-coupling product. Then the coupling reaction using β-bromostyrene was optimized with the typ ligand. The improved yield and selectivity for the cross-coupling product 34 were obtained in the presence of the tpy ligand (83% yield, 90% selectivity) (Table S4, SI). As shown in Figure 3, both electronrich and electron-deficient benzylic trifluoroborates were efficient in this cross-coupling reaction (34 to 40, 63% to 92% yield). Functional groups including esters, methoxy group, and benzodioxol group were tolerant in this Ni-catalyzed electrochemical reaction. The π-conjugation extended naphthalen-2-ylmethyl trifluoroborate also provided good reactivity in this reaction, as 67% yield was obtained for 41. However, other anodic nucleophiles (3-bromopropiolate, phenylacetic acid, potassium pivalate, and potassium phenyltrifluoroborate) didn't provide satisfactory results (see Figure S9 and the SI for more discussions).</p><p>To demonstrate potential applications of this Ni-catalyzed electrochemical C(sp 2 )-C(sp 3 ) cross-coupling methodology, we first exploited the synthesis of pharmaceutical molecules containing the diphenylmethane structural component. Beclobrate analog (42, a hypolipidemic candidate 46 ) and Bifemelane (43, an antidepressant candidate 47 ) were synthesized with 74% and 56% overall yield, respectively (Figure 4A and 4B). We further utilized this methodology in latestage functionalization of pharmaceuticals which is a popular way for fast discovery of new drag candidates. Fenofibrate is a pharmaceutical molecule of the fibrate class and used to treat abnormal blood lipid levels. 48 As shown in Figure 4C, Fenofibrate was successfully converted to a series of brand-new compounds (44 to 48, 41% to 86% yield) in up to 2.5 mmol (0.93 g) scale form a regular vial electrolyzer cell. Another new Clofibrate derivative (a lipid-lowering agent) was synthesized using this electrochemical approach (49, 63% yield) (Figure 4D). In addition, the electrochemical C−C cross-coupling reaction was also effective in modification of brominated natural amino acids, e.g. phenylalanine, (Figure 4E) (50, 83% yield). To further testify the potential industrial adoption of the present electrochemical cross-coupling reaction, flow cell synthesis (Figure 4F and 4G) was demonstrated with compounds 1, 29 and 48 with a reaction scale greater than 2.0 g. It should be noted reaction solutions were only flushed with nitrogen gas in the flow cell synthesis without using rigid glovebox or Schlenk-line techniques. Under the flow-cell condition, all three compounds were obtained with good to excellent yields (86% for 1 at 3.0 g scale, 92% for 29 at 2.0 g scale, and 84% for 48 at 3.0 g scale).</p><p>To gain mechanistic understandings of this Ni-catalyzed electrochemical C(sp 2 )-C(sp 3 ) cross-coupling reaction, a radical-trapping experiment was conducted for the anodic half-reaction.</p><p>As shown in Figure 5A, controlled potential electrolysis (at 1.2 V, vs. Fc +/0 ) of the potassium trifluoro(4-(methoxycarbonyl)benzyl)borate and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) in a divided-cell produced radical coupling product 51 with 86% isolated yield, which confirms the formation of 4-(methoxycarbonyl)benzyl free radical in the anodic oxidation process. In addition, plots of overpotential over the logarithm of kinetic current and the corresponding fitted Tafel plots were constructed to determine charge transfer rate constants (k 0 ) of potassium benzyltrifluoroborate and phenylacetic acid in the presence of 2.5 equiv Cs2CO3 in the anodic oxidation process (Figure 5B and see the SI for detail). k 0 of potassium benzyltrifluoroborate and cesium phenylacetate were calculated as 5.56 x 10 -5 cm/s and 1.39 x 10 -5 cm/s, respectively. The higher charge transfer rate constant of potassium benzyltrifluoroborate indicates faster electrochemical reactivity to generate carbon radicals than cesium phenylacetate, which is consistent with the better efficiency of potassium benzyltrifluoroborate in the cross-coupling reaction than cesium phenylacetate (30% yield). It is believed that the quick formation of the Electrochemical studies were conducted to gain additional mechanistic insights for the cathodic process. As shown in Figure 5B, methyl 4-bromobenzoate substrate displayed irreversible redox signal with onset potential at -2.05 V (vs Fc +/0 ) and dtbbpy ligand delivered reversible redox signal with E1/2 = -2.70 V (vs. Fc +/0 ), respectively. The mixture of NiCl2•glyme and 1.5 equiv dtbbpy ligand exhibits three redox peaks at E1/2 = -1.74 V, -2.44 V, and -2.70 V (vs. Fc +/0 ), which corresponding to Ni II/I , Ni I/0 redox couples, and the free ligand. When the methyl 4-bromobenzoate substrate was added, significant increase of reductive current and disappearance of the return peak was observed for the Ni II/I redox couples. It indicates that the Ni I is the reactive species for the oxidative addition of aryl halide. In addition, CV curves of the reaction mixture displayed -1.60 V and 0.33 V (vs. Fc +/0 ) onset potentials for cathodic and anodic half-reactions, respectively (Figure S10). The potential of cathode was retained between -1.7 and -1.9 V (vs. Fc +/0 ) during the reaction (Figure S11), and the observation further indicates that the Ni I/0 redox couple is not involved in the cathodic process.</p><p>Based on the chemical and electrochemical studies, a possible reaction mechanism for this Ni-catalyzed electrochemical C(sp 2 )-C(sp 3 ) cross-coupling is proposed and illustrated in Figure 6. 45 which relies on an iridium photocatalyst to activate trifluoroborates and regenerate a Ni 0 catalyst, the key mechanistic difference of the present electrochemical coupling is that both the reactive carbon radical and Ni I intermediate are generated electrochemically. Without using the expensive iridium photocatalyst and the reactive Ni 0 catalyst, the present electrochemical cross coupling is more affordable, scalable, and practical. Molander's photocatalytic cross coupling is also capable of using alkyl trifluoroborate nucleophiles as radical precurors. 49 Nevertheless, this electrochemical cross-coupling protocol is not effective to handle reactive alkyl radicals. Through optimization of reaction conditions and catalysts, it is likely to expand the scope of nucleophiles to alkyl and phenyl trifluoroborates, and even carboxylic acids for electrochemical cross coupling.</p><!><p>In summary, a Ni-catalyzed electrochemical cross-coupling methodology was developed to forge the C(sp 2 )-C(sp 3 ) bond with broad substrate scope, excellent functional group tolerance, selectivity, and good yields. In addition, the cyclic voltammetry proved an effective and efficient way for the discovery, optimization, and mechanistic understanding of anodic and cathodic halfreactions and can be used as a go-to method for developing other useful electrosynthesis methodologies. Compared to traditional thermal Ni catalyzed cross-coupling reactions, the present electrochemical approach is advantageous as all reactants and catalysts are bench stable without using reactive oxidants/reductants and complex inert atmosphere techniques. As exemplified in gram-scale synthesis in the flow-cell synthesis and the late-stage functionalization of pharmaceuticals, this electrochemical C−C coupling methodology is expected to be widely applied to the construction of C(sp 2 )-C(sp 3 ) bonds in developing pharmaceutical molecules, agrochemicals, and organic materials. The Ni-catalyzed electrochemical C-C cross coupling reactions can be further advanced for broader substrates and extended to other types of coupling reactions. Moreover, the present new C-C bond formation paradigm (and also extended reactions) can offer rich opportunities to pursue fundamental mechanistic studies and thus lead to the discovery of new catalytic knowledge at the interface of synthetic chemistry and electrochemistry.</p>

ChemRxiv

IFT20 is critical for collagen biosynthesis in craniofacial bone formation

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies.

ift20_is_critical_for_collagen_biosynthesis_in_craniofacial_bone_formation

Introduction<!>Animals<!>Histology and immunohistochemistry<!>Micro-computed tomography (pCT) analysis<!>Cross-link analysis<!>Quantitative real-time RT-PCR<!>Western blotting analysis<!>Statistical analysis<!>Disruption of Ift20 results in craniofacial bone defects in the adults<!>IFT20 plays a role in lysine (Lys) PTMs of collagen in bone maturations<!>IFT20 modulates collagen biosynthesis via FKBP65 regulation

<p>A fundamental role of intraflagellar transport (IFT) is to assemble primary cilia(1, 2). Once overlooked as an evolutionary vestige, primary cilia are now considered to be a critical organelles indispensable for regulating tissue development and homeostasis(3–5). In humans, mutations in ciliary genes can affect development of the skeletal system(6–9). We and others demonstrated that the IFTs are critical for regulating skeletogenic cell proliferation, survival and differentiation(10–15). However, at present, little is known about their roles in the biosynthesis of the organic matrix critical for regulating bone mineralization.</p><p>Bone is mainly composed of two phases, an organic matrix, principally fibrillar type I collagen, and inorganic mineral crystals. The minerals are encased within and around collagen fibrils in a highly organized manner, indicating that collagen controls spatial aspects of mineralization(16, 17). To perform this structural function, not only the quantity of collagen but also its quality, as determined in part by its post-translational modifications (PTMs), is vitally important. In the past, we proposed that the final collagen PTM, covalent intermolecular cross-linking, plays a key role in spatially organizing the mineral deposition and growth in bone(18–22). Recent studies have also demonstrated that mutations in genes encoding the collagen PTM enzymes and associated ER chaperones results in various types of recessive osteogenesis imperfecta (OI)(23–25). Lys hydroxylation of collagen, catalyzed by lysyl hydroxylases 1-3 (LH1-3), is a critical collagen PTM to determine the fate of cross-linking chemistry(26–28). Among the LH isoforms, LH2 (mostly a longer isoform of LH2, i.e. LH2b), which is encoded by the PLOD2 gene, is the only LH that is capable of hydroxylating Lys in the telopeptides, thus, critical for the formation of stable Hylald-derived cross-links(29, 30). This LH2-catalyzed PTM is associated with a number of diseases including Bruck syndrome/OI(31, 32), fibrosis(33, 34) and cancer metastasis(35–38). Several groups including ours have demonstrated that LH2 expression directs the cross-linking pathway and regulates matrix mineralization in vitro(29, 39, 40). Interestingly, both hypo- and hyper-LH2 activities resulted in defective mineralization, indicating that a specific level of LH2-catalyzed telopeptidyl modification and resulting cross-linking are necessary for proper bone mineralization(35, 39). This is also well exemplified in that OI cases can be caused by loss-, and gain-of-function of LH2(31, 41, 42). A series of recent studies have revealed that LH1 and 2 activities are regulated by a number of endoplasmic reticulum (ER) chaperone-complexes. LH1 is regulated by cyclophilin B, Synaptonemal Complex 65 (SC65), and prolyl 3-hydroxylase 3 (P3H3) (22, 43, 44), while LH2 is regulated by FKBP65(45), HSP47 and Bip(41). These components may control LH activities positively or negatively, ultimately leading to a specific cross-linking pattern that is critical for proper mineralization. However, at present, to the best of our knowledge, there is no study on the association of collagen PTMs with skeletal ciliopathies.</p><p>The aim of this study is to investigate the role of IFT20 in collagen biosynthesis in bone development. Our study may shed light on the pathogenesis of not only for skeletal ciliopathies, but also for other skeletal disorders related to abnormal collagen biosynthesis, including OI.</p><!><p>The Animal Welfare Committee and the Institutional Animal Care and Use Committee of The University of Texas Medical School at Houston approved the experimental protocol. Ift20-floxed mice (#012565), Ocn-Cre mice (#019509), Wntl-Cre mice (#009107) and Rosa26 reporter mice (#007906) were obtained from the Jackson Laboratory.</p><!><p>Picrosirius red staining was performed using 1% picrosirius red solution (Sigma-Aldrich; 365548 and P6744). FKBP65 (Proteintech; 12172-1-AP, 1:200) and EGFP (abcam; ab13970, 1:1,000) antibodies were used for immunostaining. Images were captured with an Olympus FluoView 1000 confocal microscope.</p><!><p>The distal femoral metaphyses were scanned using a μCT system at 90kV of energy and 88μA of intensity (CosmoScanGX: Rigaku corporation, Tokyo Japan). One hundred slices of metaphyses under the growth plate, constituting 1.0 mm in length, were selected and reconstructed to produce 2D and 3D images (Analyze12.0: AnalyzeDirect Inc., Overland Park, KS).</p><!><p>Skull were harvested at E18.5 and p90, pulverized, demineralized with EDTA, reduced with standardized NaB3H4, acid hydrolyzed and subjected to amino acid and cross-link analyses as reported(46). The reducible cross-links, dehydro (deH)-dihydroxylysinonorleucine/its ketoamine, deH-hydroxylysinonorleucine/its ketoamine, and deH-histidinohydroxymerodesmosine were analyzed as their reduced forms, i.e., DHLNL, HLNL, and HHMD, respectively, and the mature trivalent cross-link, pyridinoline (Pyr), was simultaneously quantified by their fluorescence. All cross-links were quantified as mol mol−1 of collagen based on the value of 300 residues of hydroxyproline (Hyp) per collagen molecule. The Hyl content in collagen was calculated as Hyl Hyp−1 x 300. Results represent the mean values from triplicate biological samples in a single experiment.</p><!><p>Total RNA was extracted using TRIzol Reagent (Thermo Fisher Scientific; 15596-026). Quantitative RT-PCR was carried out using the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad; 1725274). The conditions for qRT-PCR were 95°C for 2 min, 95°C for 5 sec, and 60°C for 30 sec, for 40 cycles. Primers for Fkbp10 were purchased (Bio-Rad, qMmuCID0009528). Primer sequences for Ift20 were 5'-TGTGGAGCTCAAGGAGGAGT-3' and 5'-TGGCCTTCATCTTCTCGTTC-3'. Primer sequences for Plod2 were 5'-CATCCGAGAGTTCATTGCTCCAG-3' and 5'-GCGCTGTCTTTCAGGTGAGTAC-3'. Primer sequences for Gapdh were 5'-CGTCCCGTAGACAAAATGGT-3' and 5'-TCAATGAAGGGGTCGTTGAT-3'. Data were normalized to Gapdh levels and quantified using the 2−ΔΔCT method.</p><!><p>Cell lysates from skull tissues were subjected to SDS-PAGE (Bio-Rad; 4561036). Anti-IFT20 (Proteintech; 13615-AP, 1:1,000), FKBP65 (Proteintech; 12172-1-AP, 1:1,000) anti-GAPDH (Cell Signaling technology; 14C10, 1:5,000), and Goat anti-rabbit IgG HRP-conjugate (Millipore sigma; 12-348, 1:5,000) antibodies were used for western blotting. The Clarity Max ECL Substrate (Bio-Rad; 1705061) was used for chemiluminescent detection, and the signals were quantified with the image-J software.</p><!><p>A two-tailed Student's t test was used for the two groups. A p value of less than 0.05 was considered statistically significant.</p><!><p>To characterize the function of IFT-B complex in intramembranous bone formation in the face, we previously disrupted Ift20 in a neural crest-specific manner in mice (hereafter Ift20:Wnt1-Cre mice) and found that Ift20:Wnt1-Cre mice displayed craniofacial bone defects(11). Since Ift20:Wnt1-Cre mice died soon after birth due to the severe craniofacial abnormalities including cleft palate(11), this does not allow us to investigate the function of IFT20 in bones during postnatal and adult stages. In addition, while there is strong evidence that the primary cilia control embryonic bone development(47, 48), the focus on mice with IFT mutations has been on osteogenic proliferation and differentiation(6, 7). However, the role of IFT in biosynthesis of collagen, a key organizer of bone mineralization, is unknown. To address these questions, we utilized the osteocalcin-Cre driver to disrupt Ift20 in osteoblasts postnatally in mice (hereafter Ift20:Ocn-Cre mice). Consistent with craniofacial bone abnormalities observed in Ift20:Wnt1-Cre mice during embryogenesis(11), Ift20:Ocn-Cre mice displayed osteopenia-like phenotypes in skulls (Fig. 1A). Micro-CT analysis revealed that mineralization of trunk bones (e.g., femurs) was also attenuated in Ift20:Ocn-Cre mice (Supplemental Fig. 1). Picrosirius red staining in skull tissues further confirmed that the area of collagen matrices in Ift20:Ocn-Cre mice was smaller than that of controls (Fig. 1B), suggesting poor bone formation. These data suggest that, in addition to embryonic stages, IFT20 also plays a critical role in controlling bone formation in the adults.</p><!><p>To explore the role of IFT20 in bone formation, we biochemically characterized the major organic matrix, collagen, focusing on its post-translational modifications (PTMs) using skulls of Ift20:Ocn-Cre mice. The results shown in Fig 2 demonstrated that: (i) Collagen content (collagen/total proteins) (A) and Hyl content (Hyl/collagen) (B) were identical between WT and Ift20:Ocn-Cre mice; (ii) 26% and 64% more Hylald- derived cross-links, i.e. dihydroxylysinonorleucine (DHLNL) (C) and pyridinoline (Pyr) (D), respectively; (iii) In contrast, the Lysald-dederived cross-link, histidinohydroxymerodesmosine (HHMD) (E), was significantly lower in Ift20:Ocn-Cre mice by ~27%; and (iv) These changes in cross-links resulted in a significant increase by ~76% in the ratio of the Hylald- to Lysald-derived collagen cross-links in Ift20:Ocn-Cre mice (F). The HLNL cross-link (G) that can be derived from either Hylald or Lysald showed no difference between the WT and mutant groups (27). Interestingly, the total number of aldehydes involved in cross-linking showed no difference between the mutant and wild-type mice (H), indicating the major difference in cross-linking is the "type" not "quantity". The cross-linking pattern of Ift20:Ocn-Cre bone collagen also indicated that telopeptidyl Lys in this mutant was overhydroxylated leading to increases in the stable Hylald-derived cross-links and a decrease in the Lysald-derived cross-link.</p><p>To investigate whether these abnormal molecular phenotypes are also seen during embryonic stages, we analyzed skull tissues obtained from Ift20:Wnt1-Cre mice. The results were identical to those seen in the Ift20:Ocn-Cre mice, i.e., a significant increase in DHLNL and a decrease in HHMD when compared to controls (Supplemental Fig. 2). At this embryonic stage, a mature cross-link, Pyr, was below detection level. Together, these results suggest that IFT20 plays a critical role in collagen PTMs in craniofacial bone formation.</p><!><p>Since the cross-link analysis indicates that Ift20 disruption causes over-hydroxylation of telopeptidyl Lys, which may have caused the osteopenia-like phenotype (Fig. 1, Supplemental Fig. 1), we hypothesized that IFT20 may regulate the expression of the enzyme and/or its chaperone complex critical for telopeptidyl Lys hydroxylation. We first confirmed that ciliogenesis was completely disrupted in Ift20:Wnt1-Cre osteoblasts (Supplemental Fig. 3). Next, we analyzed the telopeptidyl LH, i.e. LH2, and associated chaperone complex(41, 45). The results showed, while the expression of LH2 encoded by Plod2 were comparable between WT and Ift20:Wnt1-Cre skulls, the expression of FKBP65 encoded by Fkbp10 was significantly increased in the latter (Fig. 3A). Protein levels of LH2 and FKBP65 were consistent with the respective gene expression (Fig. 3B). Other ER chaperone complex members such as HSP47 and Bip were comparable between WT and Ift20:Wnt1-Cre mice (Supplemental Fig. 4). To examine further whether or not the disruption of IFT20 is associated with increased FKBP65 expression in vivo, we superimposed ROSA26 EGFP reporter alleles in Ift20:Ocn-Cre mice. Consistent with our observation in Ift20:Wnt1-Cre mice (Fig. 3A, B), the levels of FKBP65 was significantly increased in a cell autonomous manner in Ift20:Ocn-Cre skulls (Fig. 3C). These results suggest that IFT20 plays a critical role in the regulation of FKBP65 expression presumably via a ciliary dependent function of IFT20 essential for regulating quality of craniofacial bone (Fig. 3D).</p><p>In summary, utilizing both Wnt1-Cre and Ocn-Cre drivers, we found that IFT20 regulates collagen biosynthesis in craniofacial and trunk bone formation. It would be of great interest to examine the cellular mechanisms by which ciliary-dependent signaling controls the expression of collagen PTM regulators in the future. Our study strongly indicates the importance of IFT to establish the bone matrix network, and may aid in uncovering the etiology of skeletal ciliopathies and, possibly, other craniofacial and bone diseases.</p>

PubMed Author Manuscript

Effect of surface vacancies on the adsorption of Pd and Pb on MgO(100)

AbstractTheoretical quantum mechanical calculations have been carried out to establish the effect of surface vacancies on the adsorption of Pd and Pb atoms on the defective MgO(100) surface. The investigated defects included neutral, singly and doubly charged O and Mg vacancies on the (100) surface of MgO. These vacancies played the role of Fsn+ and Vsn− (n = 0, 1, 2) adsorption centers for a single Pd or Pb atom. From the results of calculations, it is clear that the Pd- and Pb-atom adsorption at the Fsn+ and Vsn− centers shows different characteristics than at the regular O2− and Mg2+ centers. Drastic changes in geometric, energetic, and electronic parameters are evident in Pd/Vsn− and Pb/Vsn−. The effect of Fs0 and Fs+, which in practice are the most important vacancies, is smaller, yet the adsorption of Pd and Pb at these centers is more energetically favorable than at the regular O2− center. Of the two metals studied, the atom of Pd is bound by the Fs0 and Fs+ centers with higher adsorption energies.Graphical abstract Electronic supplementary materialThe online version of this article (10.1007/s00706-018-2159-1) contains supplementary material, which is available to authorized users.

effect_of_surface_vacancies_on_the_adsorption_of_pd_and_pb_on_mgo(100)

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

<p>Palladium supported on oxides has found numerous applications in heterogeneous catalysis [1–3]. The catalytic performance of Pd/oxide systems can be improved by coupling their Pd part with another metallic element [4, 5]. In the resulting bimetallic Pd-M/oxide catalysts, Pd is usually combined with a typical metal or a half metal (M=Al, Si, Zn, Ga, Ge, In, Sn, Sb, Te, Tl, Pb, or Bi) [5]. Recently, it has been reported that bimetallic Pd–Pb/MgO catalysts are more effective than monometallic Pd/MgO catalysts in performing aerobic oxidations of amines [6] and oxidative esterification of methacrolein with methanol [7, 8]. Understanding the enhanced catalytic performance of these bimetallic catalysts requires a detailed knowledge of several fundamental aspects of their metal-oxide interfaces. These aspects include, in particular, geometric and electronic features of interfaces and the strength of metal-oxide interaction. Ideally, the first step of such a characterization should concern small clusters of Pd and Pb, or even better single Pd and Pb atoms, at individual adsorption sites on a well-defined single-crystal MgO surface, such as the MgO(100) one. This surface is often regarded as a prototypical oxide surface in studies of metal adsorption, because it has a simple structure and well-defined stoichiometry [9]. Additionally, it is relatively easy to form defects on this surface [10].</p><p>Various experimental techniques can yield information on the structure and energetics of metal nanoparticles and films deposited on oxides [11] and many experimental efforts have indeed been undertaken to characterize both Pd/MgO(100) [12–17] and Pb/MgO(100) [18–22]. On the other hand, properties of single atoms adsorbed on oxide surfaces are available mostly from theoretical investigations based on computational quantum mechanical approaches [23]. Of Pd and Pb on MgO(100), only the former has become a subject for a large number of theoretical studies of single metal atom adsorption so far [15, 24–37]. To the best of our knowledge, no theoretical quantum mechanical investigations of Pb/MgO(100) have been reported until now.</p><p>This work is aimed at providing a theoretical quantum mechanical description for the adsorption of Pd and Pb on the MgO(100) surface with various point defects. Both oxygen (Fsn+) and magnesium (Vsn−) vacancies in three charge states (n = 0, 1, 2) have been taken into account. From experimental studies [38, 39], it is known that such defects may be formed on MgO(100), but with a significant differentiation in their concentrations. What is particularly important is that various defects occurring on the MgO(100) surface can act as anchoring sites for metal nanoparticles [38, 40], and additionally, they can modify the properties of deposited metal nanoparticles [38, 41]. Here, a set of essential geometric, energetic and electronic parameters for a single Pd or Pb atom adsorbed at the Fsn+ and Vsn− centers has been calculated to characterize the fundamental aspects of Pd- and Pb-atom adsorption on defective MgO(100). Due to the lack of any previous theoretical studies for Pb/MgO(100), it is vitally important to provide an insight into the effect of surface vacancies on Pb-atom adsorption at atomic level.</p><!><p>Essential parameters characterizing the adsorption of a single Pd atom at various centers on the defective MgO(100) surface</p><p>Results obtained from calculations in which the Pd atom was described by the LANL08(f) basis set are listed without parentheses, whereas the results from calculations utilizing the def2-TZVP basis set for Pd are in parentheses</p><p>Results for centers with the unbound Pd atom (Eads < 0) are not presented</p><!><p>It is instructive to compare the Pd-atom adsorption at the vacancies with that occurring at non-defective sites. Results describing the adsorption of a single Pd atom at the regular anionic O2− and cationic Mg2+ centers of defect-free MgO(100) surface are appended to Table 1. As evidenced by the Eads values of Pd/O2− and Pd/Mg2+, the Pd atom binds preferentially to the O2− center in the LS state. A small charge transfer to the metal atom appears for Pd/O2−, while the Pd atom remains essentially neutral at the Mg2+ center. The Pd-atom adsorption at O2− is less energetically favorable than at Fs0 and Fs+. The Pd/O2− structure also demonstrates a larger h value compared to those of Pd/Fs0 and Pd/Fs+. On the other hand, the Pd/O2− and Pd/Fs2+ structures are formed with very similar Eads energies, although the former exhibits a much larger h value.</p><p>An inspection of the results in Table 1 also reveals that the kind of the basis set assigned to the Pd atom most often has a rather minor effect on the calculated values of h, Eads and q. A discrepancy in the interpretation of the results obtained from LANL08(f) and def2-TZVP appears for Pd/Vs2− and Pd/O2−. The calculations employing the two basis sets designate different spin states as the energetically preferred state of Pd/Vs2−. In the case of Pd/O2− in the HS state, the calculations involving the LANL08(f) basis set predict an exothermic adsorption, in contrast to those carried out with def2-TZVP. However, the EadsHS value obtained from LANL08(f) is actually quite close to zero, and therefore, the significance of this discrepancy should not be overemphasized.</p><p>Our findings made for the Pd-atom adsorption are essentially in good agreement with conclusions reported in previous experimental [14–16] and theoretical [15, 24, 26–28, 33, 35, 36] studies of Pd/MgO(100). It is well-known that the defect-free MgO(100) surface is generally rather unreactive toward the adsorption of metal atoms [43]. The Mg2+ centers exhibit particularly low reactivity toward metal atoms [25]. In consequence, Pd atoms preferably occupy the O2− centers [28], with no significant charge transfer from or to the surface [27]. An experimental estimation of adsorption energy for Pd on MgO(100) is ca. 1.2 eV [14]. From an experimental measurement, a value of 2.22 Å was also deduced to be the height of an adsorbed Pd atom from the O2− center [26]. Our EadsLS and hLS values for Pd/O2− are very close to these experimental estimations. Similarly to metal adsorption on the defect-free MgO(100) surface, metal atoms on MgO(100) with defects also adsorb preferentially at centers where negative charge accumulates [33, 44]. More specifically, the Fs0 centers play the key role in the adsorption of Pd atoms [15, 16]. This is because these centers are the main part of vacancies formed on MgO(100), which was confirmed both experimentally [10] and theoretically [45, 46]. Besides the Fs0 centers, the Fs+ centers can also occur, but they are less likely due to their large formation energy [42]. Even larger formation energy was determined for the Fs2+ center [42]. Previous computational studies have shown that the Pd/Fs+ interaction is weaker than the Pd/Fs0 interaction but stronger than that of Pd/O2− [15, 33, 36]. Apart from rendering this trend correctly, our h and Eads values also reproduce quantitatively other theoretical results [15, 35, 36]. It has also been reported that the interaction between Pd and Vsn− centers is extremely strong [24]. According to an experimental study [10], the concentration of surface Mg vacancies seems, however, to be much lower than that of Fs0 and Fs+. Again, this is in line with large formation energies of Vsn− vacancies [42, 47].</p><p>This review of existing results for Pd/MgO clearly indicates that the computational methodology applied in this work leads to the correct description of Pd-atom adsorption on MgO(100) with surface vacancies. Thus, one can expect that the parameters characterizing the adsorption of Pb atom at the Fsn+ and Vsn− centers should also be predicted reliably.</p><!><p>Essential parameters characterizing the adsorption of a single Pb atom at various centers on the defective MgO(100) surface</p><p>Results obtained from calculations in which the Pb atom was described by the LANL08d basis set are listed without parentheses, whereas the results from calculations utilizing the def2-TZVP basis set for Pb are in parentheses</p><p>Results for centers with the unbound Pb atom (Eads < 0) are not presented</p><!><p>The kind of basis set assigned to metal atom affects the parameters of Pb-atom adsorption to a greater extent than the results for the Pd-atom adsorption. The greater discrepancies in the parameters obtained using LANL08d and def2-TZVP result from an inherent difference in the treatment of Pb atom with the two basis sets. These basis sets differ not only in the number of basis functions in their valence parts, but also in the size of their core parts treated with pseudopotentials. LANL08d is expected to yield less accurate results because (1) its quality is formally inferior to that of def2-TZVP and (2) a previous benchmark study confirmed its poorer performance [50]. Notwithstanding this difference, the application of either basis sets provides a qualitatively consistent picture of Pb-atom adsorption at the Fsn+ and Vsn− centers.</p><p>To establish the effect of surface vacancies on the Pb-atom adsorption, Table 2 also shows the h, Eads, and q parameters calculated for Pb/O2− and Pb/Mg2+. It is clear that the Pb-atom adsorption is possible only at the O2− center on the defect-free MgO(100) surface. Similarly to Pb/Fsn+, the Pb/O2− structure tends to conserve the triplet multiplicity of Pb and its EadsHS energy becomes more exothermic than EadsLS. On the other hand, the EadsHS value for Pb/O2− is smaller than those of Pb/Fs0 and Pb/Fs+. It proves that Pb atoms adsorb preferentially at the Fs0 centers on the defective MgO(100) surface. It worth reminding here that, of the considered Fsn+ and Vsn− vacancies, the Fs0 centers are most abundant on the defective MgO(100) surface.</p><p>For the Pb/Fsn+ structures, their propensity to change the spin state from HS to LS can be evaluated by calculating the difference between their EadsHS and EadsLS energies. The resulting HS → LS transition energies adopt smaller values than the excitation energy of a free Pb atom from its ground state to the lowest singlet state (0.95 and 0.89 eV at the B3LYP/LANL08d and B3LYP/def2-TZVP levels, respectively). Moreover, these transition energies are smaller than the HS → LS transition energy of Pb/O2−. It implies that the Fsn+ centers facilitate the HS → LS transition in the adsorbed Pb atom.</p><p>An experimental study concerning the growth of Pb film on well-defined oxide surfaces [18] reported a calorimetrically measured initial heat of adsorption of 1.07 eV for Pb/MgO(100) at 300 K. This value was an average of the bonding of Pb atoms to MgO(100) and Pb–Pb bonding within small Pb nanoparticles formed on MgO(100). For such nanoparticles, their Pb–MgO(100) bond strength was roughly estimated to be either 0.33 or 0.16 eV, depending on the kind of Pb nanoparticles adsorbed (whether two- or three-dimensional Pb nanoparticles). In a more recent study based on atomic beam/surface scattering measurements [22], a range from 0.72 to 0.81 eV was proposed to be the heat of Pb adsorption at terrace sites on MgO(100). Our EadsHS energy of Pb/O2− exceeds by ca. 0.3 eV the upper limit of this range.</p><!><p>Plots of HOMO contours for Pd/O2− and Pd/Fs0 in their LS state and for Pb/O2− and Pb/Fs0 in their HS state. These contours are plotted with an isovalue of 0.01 a.u. Magnesium, oxygen, palladium and lead are colored yellow, red, blue, and gray, respectively (color figure online)</p><!><p>The results reported in this work point out that the presence of vacancies on the MgO(100) surface, such as Fsn+ and Vsn−, has an important influence on the geometric, energetic, and electronic parameters characterizing the adsorption of Pd and Pb atoms. The Fs0 and Fs+ vacancies, which are most likely among the Fsn+ and Vsn− defects on MgO(100), constitute the centers at which the adsorption of single Pd or Pb atoms is more exothermic than at the regular O2− centers. The Eads values of Pd/Fs0 and Pd/Fs+ in their preferred spin states are at least 1 eV larger than the corresponding energies of Pb/Fs0 and Pb/Fs+. In that regard, the presence of Fs0 and Fs+ on MgO(100) does not change the energetic preference of Pd-atom adsorption over Pb-atom adsorption. Such preference was previously detected experimentally and is confirmed here computationally. The Pd/Fs0 and Pd/Fs+ structures favor the spin state with the maximum spin pairing, whereas Pb/Fs0 and Pb/Fs+ are most stable in their HS states. Due to its large atomic radius, the Pb atom at the Fs0 and Fs+ centers is adsorbed at only slightly smaller height than at the O2− center. This contrasts with the large reduction of h in the Pd/Fs0 and Pd/Fs+ structures, if compared to the h value of Pd/O2−. This reduction leads to larger increases in Eads and in the amount of electron charge transferred to the metal atom, as well as to a change in the shape of HOMO for Pd/Fs0 and Pd/Fs+. The Pd- and Pb-atom adsorption at the Vsn− vacancies, which are less abundant on MgO(100), is highly exothermic, far exceeding the Eads energies obtained for Pd/Fsn+ and Pb/Fsn+. In particular, the formation of Pb/Vs0 and Pb/Vs− structures is associated with extremely high Eads energies, which turn out to be sufficient to stabilize the LS state of these structures.</p><p>The presented quantum mechanical study of the surface vacancy effect is a tentative step in elucidating the properties of Pd–Pb/MgO catalysts. The findings made for Pb/Fsn+ and Pb/Vsn− may be of particular importance, because the Pb-atom adsorption on the defective MgO(100) surface has not been investigated theoretically so far.</p><!><p>The structures of Fsn+ and Vsn− centers with an adsorbed Pd or Pb atom were determined using a theoretical quantum mechanical approach based on the B3LYP computational method [51–53] and the embedded cluster model of surface [54]. These structures are denoted in this work by the abbreviation 'metal atom/adsorption center'. The aforementioned computational methodology was successfully used in many previous studies of adsorption on MgO(100), e.g., [41, 55, 56]. The Fsn+ centers were represented by two-layer [Mg13O12]n+ clusters surrounded by total ion model potentials of the nearest Mg2+ cations and embedded in a large array of ± 2 point charges. The Vsn− centers were modeled using two-layer [Mg12O13]n− clusters and an embedding environment comprised of total ion model potentials of Mg2+ and an array of ± 2 point charges. The Mg and O atoms of the [Mg13O12]n+ and [Mg12O13]n− clusters were described by the 6-31G basis set [57, 58]. Additional polarization and diffuse basis functions [58, 59] were ascribed to several Mg and O atoms directly involved in the interaction with a Pd or Pb atom. Further details of the aforementioned cluster models are given in Section S1 in Electronic Supplementary Material.</p><p>The adsorption of a single Pd or Pb atom at each investigated center was simulated by optimizing the height (h) of the metal atom from the surface layer of the adsorption center. The effect of surface relaxation induced by metal adsorption was also included in these calculations. Two sets of calculations differing in the kind of basis set ascribed to the metal atoms were performed to estimate basis set effects in the results of calculations. The first kind was the LANL08 basis set [60] in its LANL08(f) version for Pd and LANL08d for Pb. The def2-TZVP basis set [61] was the second kind of basis set assigned to the metal atoms. Two low-lying electronic states with different spin multiplicities were studied for the Pd- and Pb-atom adsorption. The low-spin state (LS) was characterized by the singlet multiplicity of the center with a Pd or Pb atom adsorbed, whereas the high-spin state (HS) assumed a triplet for each adsorbed metal atom.</p><p>To calculate the adsorption energy (Eads), the total energy of an adsorption center occupied with a metal atom was subtracted from a sum of the total energies of free metal atom in its ground state and the isolated surface center in its relaxed geometry. According to this definition, adsorption with a positive Eads value is an energetically favorable (exothermic) process. The electron charge (q) acquired by an adsorbed Pd or Pb atom was estimated by the partial charge of the atom. This partial charge was determined according to the Bader charge analysis [62].</p><p>All calculations except the Bader charge analysis were carried out using the GAUSSIAN 09 D.01 program [63]. The Bader charge analysis was done with the Multiwfn 3.4 program [64].</p><!><p>Supplementary material 1 (DOC 3089 kb)</p><p>Electronic supplementary material</p><p>The online version of this article (10.1007/s00706-018-2159-1) contains supplementary material, which is available to authorized users.</p>

PubMed Open Access

Photochemistry of Furyl- and Thienyldiazomethanes: Spectroscopic Characterization of Triplet 3-Thienylcarbene

Photolysis (\xce\xbb > 543 nm) of 3-thienyldiazomethane (1), matrix isolated in Ar or N2 at 10 K, yields triplet 3-thienylcarbene (13) and \xce\xb1-thial-methylenecyclopropene (9). Carbene 13 was characterized by IR, UV/vis, and EPR spectroscopy. The conformational isomers of 3-thienylcarbene (s-E and s-Z) exhibit an unusually large difference in zero-field splitting parameters in the triplet EPR spectrum (|D/hc| = 0.508 cm\xe2\x88\x921, |E/hc| = 0.0554 cm\xe2\x88\x921; |D/hc| = 0.579 cm\xe2\x88\x921, |E/hc| = 0.0315 cm\xe2\x88\x921). Natural Bond Orbital (NBO) calculations reveal substantially differing spin densities in the 3-thienyl ring at the positions adjacent to the carbene center, which is one factor contributing to the large difference in D values. NBO calculations also reveal a stabilizing interaction between the sp orbital of the carbene carbon in the s-Z rotamer of 13 and the antibonding \xcf\x83 orbital between sulfur and the neighboring carbon\xe2\x80\x94an interaction that is not observed in the s-E rotamer of 13. In contrast to the EPR spectra, the electronic absorption spectra of the rotamers of triplet 3-thienylcarbene (13) are indistinguishable under our experimental conditions. The carbene exhibits a weak electronic absorption in the visible spectrum (\xce\xbbmax = 467 nm) that is characteristic of triplet arylcarbenes. Although studies of 2-thienyldiazomethane (2), 3-furyldiazomethane (3), or 2-furyldiazomethane (4) provided further insight into the photochemical interconversions among C5H4S or C5H4O isomers, these studies did not lead to the spectroscopic detection of the corresponding triplet carbenes (2-thienylcarbene (11), 3-furylcarbene (23), or 2-furylcarbene (22), respectively).

photochemistry_of_furyl-_and_thienyldiazomethanes:_spectroscopic_characterization_of_triplet_3-thien

INTRODUCTION<!>Background<!>Computed Energies of C5H4S and C5H4O Isomers<!>Primary Photochemistry of 3-Thienyldiazomethane (1)<!>Electronic Absorption Spectrum of Triplet 3-Thienyl-carbene (13)<!>Photochemistry of 3-Thienylcarbene (13) and Other C5H4S Isomers<!>Possible Thermal Chemistry of Triplet 3-Thienylcarbene (13)<!>Photochemistry of 2-Thienyldiazomethane (2)<!>Spectroscopic Data for C5H4S Isomers<!>Photochemistry of 3-Furyldiazomethane (3)<!>Photochemistry of 2-Furyldiazomethane (4)<!>Spectroscopic Data for C5H4O Isomers<!>EPR Spectrum and Electronic Structure of Triplet 3-Thienylcarbene (13)<!>Simulation<!>Spectroscopic Assignments<!>Conformational Isomerism<!>Absence of the Other Carbenes<!>CONCLUSIONS<!>General<!>Computational Methods<!>Preparation of Tosylhydrazones<!>Thiophene-3-carboxaldehyde Tosylhydrazone<!>Thiophene-2-carboxaldehyde Tosylhydrazone<!>Furan-3-carboxaldehyde Tosylhydrazone<!>Furan-2-carboxaldehyde Tosylhydrazone<!>Preparation of Tosylhydrazide Sodium Salts<!>Preparation of Diazo Compounds 1\xe2\x80\x934<!>Procedure for Determining Extinction Coefficients for Diazo Compounds 1\xe2\x80\x934<!>(3-Thienyl)diazomethane (1)<!>(2-Thienyl)diazomethane (2)<!>(3-Furyl)diazomethane (3)<!>(2-Furyl)diazomethane (4)

<p>The chemistry of aryl and heteroaryl carbenes constitutes a subject of longstanding interest in the field of organic reactive intermediates.1–3 Basic relationships concerning the structure, reactivity, and spectroscopy of these intermediates have been deduced through product analyses, matrix-isolation spectroscopy, time-resolved spectroscopy, and computational studies. For any given system, these relationships are strongly influenced by the electronic ground state of the carbene (singlet or triplet) and the magnitude of the singlet-triplet energy gap. In the singlet series, the chemistry and spectroscopy of arylchlorocarbenes (phenyl,4,5 pyridyl,6 furanyl,7,8 thienyl,9,10 and their benzo analogues11–14) have been extensively investigated. Our own interest in arylcarbenes focuses on the triplet series because these species—lacking the halogen substituent—are more directly relevant to the harsh reaction environments encountered in combustion or in astrochemistry.15 That the chemistry of atomic carbon is important in astronomical environments invites attention to aryl- and heteroaryl carbenes, as these intermediates may be formed upon addition of a carbon atom to a stable, closed-shell aryl substrate. In the current study, we focus on the isomeric furyl- and thienylcarbenes, which have eluded detection and characterization to the present time.16,17 These fundamental studies of reactive thienyl intermediates are also germane to an understanding of the electronic structure of doped states of thiophene derivatives—materials that have achieved wide use in conducting polymers and other electronic applications.18–20</p><!><p>Shechter and co-workers reported detailed product analyses associated with thermolysis of the isomeric thienyldiazomethanes (1 and 2) and furyldiazomethanes (3 and 4) (Scheme 1).21,22 The intermediacy of the corresponding thienyl- and furylcarbenes was inferred from the isolation of the products of insertion into the C–H bond of cyclooctane and the formal products of dimerization. Fragmentation of the ring, to afford ring-opened products, was also observed. Shevlin and workers later postulated the intermediacy of 2- and 3-thienylcarbene in the reaction of atomic 13C with thiophene.23,24 Saito and co-workers devised intriguing systems in which the thermal furylcarbene fragmentation reaction is driven in the reverse direction under photochemical conditions. Thus, an acyclic enynal or enynone will undergo photocyclization to a 2-furylcarbene derivative, which can be trapped in either an inter- or intramolecular fashion.25 Thermal cyclization reactions of azo-ene-ynes are analogous, proceeding via a heteroaryl carbene intermediate.26–28 Albers and Sander elucidated key aspects of the photochemistry and spectroscopy of C5H4S and C5H4O isomers through their study of the photochemistry of 3-thienyldiazomethane (1), 3-furyldiazomethane (3), and 2-furyldiazomethane (4) under matrix isolation conditions.16,17 None of the carbene intermediates, however, were detected. In terms of computational studies, McKee, Shevlin, and Zottola described an insightful, comprehensive study of the C5H4S potential energy surface.24 The mechanism of ring-opening of 2-furylcarbene, 2-thienylcarbene, and related compounds has been the subject of several computational and theoretical investigations.7,24,29–31 Herges29 described the reaction as a coarctate transformation, while Birney31 interpreted it as a pseudopericyclic process.</p><!><p>To provide context for interpreting our experimental observations, we performed computational studies of the C5H4S and C5H4O potential energy surfaces at the B3LYP/6-31G* level of theory. Structures and relative energies are depicted in Schemes 2 and 3, and additional details are provided as Supporting Information. This methodology is adequate for providing qualitatively reliable predictions of infrared spectra and relative energies across a range of molecular structures. In terms of relative energies, Density Functional Theory (DFT) methods often overemphasize delocalization in conjugated π-electron systems,32–34 and the B3LYP functional biases the calculation of singlet–triplet energy gaps by ca. 1–3 kcal/mol by underestimating the stability of the singlet, relative to the triplet.35,36 Because the singlet–triplet energy gaps of the thienylcarbenes are of particular interest to us, we sought to corroborate the DFT predictions using ab initio methods. The results of coupled-cluster calculations, performed at a moderate level of theory (CCSD/cc-pVTZ), show good agreement with those obtained by using density functional theory for 2- and 3-thienylcarbene (Scheme 4). Both computational methods predict a triplet electronic ground state for both carbenes.</p><p>Of the isomers that we investigated, the structurally analogous thioaldehyde 5 (C5H4S) and aldehyde 16 (C5H4O) are the lowest energy structures on their respective potential energy surfaces. Portions of these surfaces have been investigated, previously, although the issue of conformational isomerism (s-E, s-Z) in thioaldehydes (5, 6) or aldehydes (16, 17) has not been explored in detail.16,17,24 The (s-Z) rotamer is commonly drawn in mechanistic schemes, but there is only one case throughout the photochemistry of diazo compounds 1, 2, 3, and 4 where an (s-Z) rotamer of any pentenyne (s-Z-6) is actually observed in the matrix (see below). The computed IR spectra of the (s-Z) and (s-E) rotamers are quite different and allow clear analysis and differentiation of the peaks present in the experimental spectrum.</p><!><p>Irradiation of 3-thienyldiazomethane (1) (λ > 534 nm; N2, 10 K) gives a mixture of (s-Z)-α-thial-methylenecyclopropene (9), (s-E)-3-thienylcarbene (s-E-13), (s-Z)-3-thienylcarbene (s-Z-13), and a minor amount of a species tentatively assigned as 1H-2-thiabicyclo[3.1.0]hexa-3,5-diene (12) (Scheme 5; Figure S1).37 IR assignments are based upon comparison of experimental spectra with computed IR spectra (B3LYP/6-31G*), as well as comparison with previously reported spectral data of 9 and 12.16,38 EPR and UV/vis experiments, performed under analogous conditions, provide strong support for the assignment of triplet 3-thienylcarbene (s-E- and s-Z-13). The EPR spectrum of 13 affords direct evidence for the triplet species, and the spectroscopic features are generally consistent with those expected for an arylcarbene (Figure 1).39–41 A detailed analysis of the EPR spectrum will be provided as part of the Discussion section. The electronic absorption spectrum exhibits the weak visible absorption features that are characteristic of triplet aryl carbenes (and the benzyl radical) (Figure 2).39 The spectroscopic assignment of triplet 3-thienylcarbene (13) is further supported by the wavelength-dependent photochemistry, in which the IR, UV/vis, and EPR signals attributed to 13 disappear upon photoexcitation into the visible absorption feature at λmax ca. 467 nm (Figures 2 and 3).</p><p>Comparison of the electronic absorption spectra of 3-thienyldiazomethane (1) and triplet 3-thienylcarbene (13) shows that diazo compound 1 absorbs at slightly longer wavelength (broad absorption with λmax = 494 nm) than carbene 13 (λmax = 467 nm). (The 494-nm absorption of diazo compound 1, which was measured in acetonitrile solution, is not observable in the matrix spectrum depicted in Figure 2 because of the low concentration.) It is possible, therefore, to irradiate diazo compound 1 under long-wavelength, broadband conditions without inducing secondary photochemistry in the incipient 3-thienylcarbene (13). Long-wavelength irradiation of 3-thienyldiazomethane (1) exhibits a subtle wavelength dependence, which we tentatively ascribe to differential photoreactivity of s-E and s-Z conformers of the diazo compound. Irradiation of diazo compound 1 at λ > 571 nm results in a mixture of s-E and s-Z conformers of triplet 3-thienylcarbene (13), with the s-Z conformer growing more quickly, as measured by the greater intensity of the X2 and Y2 signals in the EPR spectrum (Figure 1 and Figure S8). Both conformers continue to grow upon irradiation at slightly shorter wavelength (λ > 534, > 497, and > 472 nm), but the X2 and Y2 signals of the s-E conformer grow more quickly, such that the s-E- conformer of triplet 3-thienylcarbene (13) becomes the major conformer in the EPR spectrum (Figure 1). Since both conformers appear to be stable to the irradiation conditions, and each disappears rapidly upon irradiation at λ > 444 nm or λ = 467 ± 10 nm, we ascribe the differential rates of formation to the differential absorption and/or quantum yield for carbene formation from the conformational isomers of 3-thienyldiazomethane (1). (TD-DFT calculations predict the electronic absorption spectra for the conformational isomers of 3-thienyldiazomethane (1) to be similar (see the Supporting Information).)</p><p>With the EPR and visible spectra of triplet 3-thienylcarbene (13) in hand, we expended considerable effort in an attempt to optimize the experimental conditions to enhance the production of carbene 13 and/or minimize the formation of methylenecyclopropene derivative 9. (These efforts included extensive studies of the wavelength dependence of the photolysis of 3-thienyldiazomethane (1), as well as broadband "flash" irradiation.42) Although our attempts were largely unsuccessful, they help us understand the photochemical reactivity of this system. An important observation is that the methylenecyclopropene derivative (9) is always formed, even under conditions where triplet 3-thienylcarbene (13) is stable to the irradiation conditions. Plausible mechanisms for the formation of 9, under these conditions, involve a hot ground-state reaction of carbene 13 or a reaction in the excited state of diazo compound 1. Both explanations have ample precedent in the field of carbene chemistry. The first mechanistic scenario posits that N2 loss from the excited state of 3-thienyldiazomethane (1) yields carbene 13 with excess vibrational energy, and that 9 arises from a hot ground-state reaction of carbene 13. To probe for the involvement of a hot ground-state carbene, we performed the photolysis of diazo compound 1 in Ar and N2 matrices, as well as a 2-methyltetrahydrofuran (2-MeTHF) glass. These media are increasingly effective in vibrational cooling of guest molecules, owing to an increasing density of vibrational states.43,44 We found, however, that the matrix medium does not influence the photochemistry, as judged by the fact that the intensity of the EPR signal of triplet 3-thienylcarbene (13) was not enhanced (relative to Ar) in either an N2 matrix or a 2-MeTHF glass. Thus, we do not favor the mechanism involving hot ground-state 3-thienylcarbene (13). Such an explanation would also seem to be at odds with the relatively high thermal barrier (>30 kcal/mol) separating singlet 3-thienylcarbene (13) and α-thial-methylenecyclopropene (9).24 The more likely explanation, therefore, involves the excited state of the diazo compound. As observed in several other systems,45,46 the excited state of diazo compound 1 may partition between two pathways: N2 loss, to give 3-thienylcarbene (13), and N2 loss with concomitant rearrangement, to give α-thial-methylenecyclopropene (9) without the intervention of the carbene.</p><!><p>3-Thienylcarbene (13) exhibits a weak electronic absorption in the visible region of the spectrum (λmax = 467 nm) that is characteristic of triplet arylcarbenes (and the benzyl radical) (Figure 2).39 The vibronic structure associated with the corresponding electronic transition in triplet phenylcarbene (λmax = 430 nm) has been analyzed in detail.47 Time-dependent density functional theory calculations (TD-DFT) do a reasonably good job of reproducing the general features of the absorption spectrum of triplet phenylcarbene (Table 1), although the energy of the visible excitation is overestimated. Analogous calculations for triplet 3-thienylcarbene (13) predict the visible absorption to be red-shifted relative to phenylcarbene (Table 1), in accord with experimental observation. The absorption spectra of the rotamers of triplet 3-thienylcarbene (13) are indistinguishable under our experimental conditions—a result that is also borne out by the TD-DFT calculations. In this respect, triplet 3-thienylcarbene (13) is unlike the related singlet arylchlorocarbenes (furanyl,7,8 thienyl,9,10 and their benzo analogues11–13), in which the absorption spectra of the conformational isomers exhibit significant differences. In the lowest singlet state, however, the absorption spectra of the conformers of 3-thienylcarbene are predicted to be readily distinguishable (λmax 995 nm vs 1045 nm; Table 1).</p><!><p>Photoexcitation into the visible absorption feature (λmax ca. 467 nm) of triplet 3-thienylcarbene (13), using either broadband (λ > 444 nm) or narrow-band (λ = 467 ± 10 nm) irradiation conditions, leads to the rapid disappearance of the IR, UV/vis, and EPR signals of carbene 13 and the growth of (s-Z)-α-thial-methylenecyclopropene (9), as well as the growth of a small amount of 1H-2-thiabicyclo[3.1.0]hexa-3,5-diene (12) (Figures 3 and S3). This behavior explains the inability of Albers and Sander to observe carbene 13 in their study of the photochemistry of 3-thienyldiazomethane (1).16 Our findings establish that carbene 13 is not stable to the photolysis conditions used in the earlier experiment (λ > 435 nm).</p><p>The UV/vis spectrum of 9 exhibits an absorption at ca. 300–400 nm (Figure S11). Irradiation into this absorption feature (λ > 363 nm) results in photoisomerization of the s-Z conformer to the s-E conformer (Figure S4). At slightly shorter wavelength (λ > 330 nm), the IR bands of the s-E conformer continue to grow, and the IR bands of the acyclic isomers, pent-2-en-4-ynethial (5 and 6), slowly appear (Figure S5). At λ > 280 nm, another acyclic isomer, propargyl thioketene (8), appears in the IR spectrum, accompanied by the decrease in the IR absorptions of 5, 6, and 9 (Figure S6). Our findings corroborate those described earlier by Sander and co-workers,16 while adding some additional detail in terms of the conformational isomerism of pent-2-en-4-ynethial (5 and 6) and methylenecyclopropene derivatives (9). Our discovery of the shorter-wavelength chemistry that affords thioketene 8 is also new.</p><!><p>Allowing a matrix containing triplet 3-thienylcarbene (13) to stand in the dark at 15 K for an extended period of time (30–80 h) affords a small, but reproducible, decrease in the EPR signal of (s-E)-13 (Figures 4 and S10) and the visible absorption of 13 (Figure S12). These data suggest that the (s-E) conformer of triplet 3-thienylcarbene (13) undergoes a very slow thermal reaction at 15 K. IR experiments were performed under identical conditions to try to determine the product of this reaction, but the low concentration of triplet 3-thienylcarbene (13) in the matrix, and the presence of several other photoproducts, did not permit us to quantify carbene disappearance or product growth. In general, measurements of reaction kinetics—whether by EPR, UV/vis, or IR—were problematic because of a combination of low signal intensity and very slow reaction rates. Thermal reactions of reactive intermediates trapped in matrices at cryogenic temperatures are not uncommon, and these processes typically occur via a quantum mechanical tunneling mechanism48–50—even in cases where the reaction is formally forbidden by virtue of a change in spin multiplicity.51,52 We consider the cyclization of (s-E) 3-thienylcarbene (13) to 2-thiabicyclo[3.1.0]hexa-3,5-diene (12) to be a likely candidate for such a process, although we cannot exclude other possibilities (Scheme 6). The rearrangement of triplet 13 to singlet 12, which does not involve extensive motion of the heavy atoms, is computed to be slightly exothermic (ca. 2 kcal/mol).</p><!><p>Irradiation of 2-thienyldiazomethane (2), under the same conditions that led to the successful generation of triplet 3-thienylcarbene (13) (λ > 534 nm; Ar, 10 K), failed to afford IR or EPR features attributable to triplet 2-thienylcarbene (11) (Scheme 7). In accord with the results of earlier studies,16 the ring-opened product, Z-pent-2-en-4-ynethial (6), is the only species observed by IR spectroscopy (Figure S13). Careful examination of the spectrum establishes that both s-E and s-Z rotamers of 6 are present. Further irradiation (λ > 363 nm) results in cis–trans photoisomerization of Z-pent-2-en-4-ynethial (6) and E-pent-2-en-4-ynethial (5) (present only as the s-E conformer; Figure S14). Irradiation at λ > 237 nm affords propargyl thioketene (8) (Figure S15).</p><p>The nature of the electronic ground state of 2-thienylcarbene (11)—singlet or triplet—remains unclear at this time. Our DFT calculations predict a triplet ground state with a small singlet–triplet gap (1–2 kcal/mol) for both conformers of 2-thienylcarbene (11) (Scheme 2). We are not overly confident in the prediction of the ground state multiplicity, since the two states lie so close in energy and this computational methodology typically underestimates the energy of the singlet, relative to the triplet, by 1–3 kcal/mol.35,36 McKee's calculations predict a singlet ground state for carbene 11 with a singlet–triplet gap of 4.1 kcal/mol,24 but we are not overly confident in that prediction, either. Energies reported in the earlier study were estimated by using an additivity scheme that utilized both QCISD(T) and MP2 energies. The application of MP2 methodology to carbenes, however, is problematic because of spin contamination. It is evident from the data in Table 1 of ref 24 that the QCISD(T) single-point calculation predicts a triplet ground state for 11, while two different MP2 single-point calculations predict a singlet ground state.53 We are inclined to discount the additivity scheme because of its reliance on MP2 data, and focus on the results afforded by CCSD (Scheme 4), QCISD(T),24 or B3LYP (Scheme 4), which each predict a triplet ground state with small singlet–triplet gap.</p><!><p>3-Thienylcarbene (s-E-13): IR (N2, 10 K) 744 s cm−1; UV/vis (Ar, 10 K) λmax 441, 449, 457, 467 nm; EPR (Ar, 15 K), |D/hc| = 0.508 cm−1, |E/hc| = 0.0554 cm−1, Z1 = 1954.7 G, X2 = 4250.0 G, Y2 = 6550.4 G, Z2 = 8823.2 G, microwave frequency = 9.491 GHz. 3-Thienylcarbene (s-Z-13): IR (N2, 10 K) 744 s cm−1; UV/vis (Ar, 10 K) λmax 441, 449, 457, 467 nm; EPR (Ar, 15 K), |D/hc| = 0.579 cm−1, |E/hc| = 0.0315 cm−1, Z1 = 2770.0 G, X2 = 5000.0 G, Y2 = 6337.0 G, Z2 = 9600.0 G, microwave frequency = 9.491 GHz. (s-E)-E-Pent-2-en-4-ynethial (5): IR (Ar, 10 K) 3319 s, 1575 s, 1362 w, 1255 m, 1163 m, 1159 m, 976 w, 971 w, 962 m, 776 w, 694 w, 643 w, 640 w, 631 w cm−1. (s-E)-Z-Pent-2-en-4-ynethial (6): IR (Ar, 10 K) 3317 s, 2109 w, 1560 s, 1404 s, 1258 w, 1218 m, 1130 s, 1031 s, 862 w, 854 w, 777 s, 700 m, 643 s, 629 m, 465 w cm−1. (s-Z)-Z-Pent-2-en-4-ynethial (6): IR (Ar, 10 K) 3327 s, 2109 w, 1554 m, 1410 m, 1364 w, 1257 m, 1128 m, 1134 m, 944 w, 858 w, 638 s, 629 m, 465 w cm−1. Propargyl thioketene (8): IR (N2, 10 K) 3316 m, 1779 s, 1318 w, 1253 w, 645 w, 637 w, 613 w cm−1; IR (Ar, 10 K) 3324 w, 1781 s, 1315 w, 1245 w cm−1. (s-Z)-α-Thial-methylenecyclopropene (9): IR (N2, 10 K) 3141 w, 1718 s, 1498 w, 1474 m, 1367 m, 1358 m, 1189 w, 1169 m, 1009 w, 775 m, 696 w, 646 w, 629 w cm−1. (s-E)-α-Thial-methylenecyclopropene (9): IR (N2, 10 K) 3143 w, 1719 s, 1714 m, 1496 s, 1480 m, 1412 w, 1362 w, 1353 w, 1190 m, 1164 m, 1104 w, 1007 w, 912 w, 908 w, 843 w, 834 w, 718 w cm−1.</p><!><p>Irradiation of 3-furyldiazomethane (3), under the same conditions that led to the successful generation of triplet 3-thienylcarbene (13) from 3-thienyldiazomethane (1) (λ > 571 nm or > 534 nm; Ar, 10 K), failed to afford IR or EPR features attributable to triplet 3-furylcarbene (22) (Scheme 8). Long-wavelength irradiation (λ > 571 nm) of 3 yields a mixture of (α-formyl)methylenecyclopropene (s-Z- and s-E-19) and an unidentified species (U) exhibiting an IR band in the acetylenic C–H stretching region (3429 m, 1636 m, 1476 m cm−1, Figure S16). Irradiation at λ > 399 nm drives (s-Z)-19 away, while (sE)-19 and U continue to grow (Figure S17). Subsequent irradiation (λ > 330 nm) causes 19 to decrease in intensity and a mixture of E-pent-2-en-4-ynal (16) and Z-pent-2-en-4-ynal (17) to appear. As in the case of the sulfur analogues, the pent-2-en-4-ynals (16 and 17) are present in the s-E conformation (Figure S18). The IR assignments for 16, 17, and 19 are in good agreement with those reported previously.17</p><p>The photochemical appearance and disappearance of compound U is reproducible across multiple experiments, yet the identity of this species eludes us. We initially suspected propargyl ketene (18)—the oxygen analogue of propargyl thioketene (8) that was observed in the photochemistry of the related sulfur-containing system. The observed IR absorptions of U, however, are inconsistent with the IR spectrum computed for ketene 18. (The apparent absence of ketene or carbonyl stretching vibrations in the spectrum of U is telling.) We considered several other C5H4O isomers that contain a terminal alkyne moiety (Scheme S2), but none of the computed IR spectra shows a good correlation with the experimentally observed bands of U. We also considered the possibility of a photofragmentation reaction, but the observed IR absorption at 3429 cm−1 does not correspond to that of either acetylene54 or diacetylene.</p><!><p>Under gentler conditions than those that led to the successful generation of triplet 3-thienylcarbene (13) (λ > 571 nm; Ar, 10 K), irradiation of (2-furyl)diazomethane (4) (λ > 613 nm; Ar, 10 K) failed to afford IR or EPR features attributable to triplet 2-furylcarbene (22) (Scheme 9). In accord with the results of earlier studies,17 irradiation of diazo compound 4 (λ > 613 nm; Ar, 10 K) causes fragmentation of the furan ring, yielding Z-pent-2-en-4-ynal (17) (Figure S19). Subsequent irradiation at λ > 444 nm gives rise to E-pent-2-en-4-ynal (16) (Scheme 9, Figure S20).</p><!><p>(s-E)-E-Pent-2-en-4-ynal (16): IR (Ar, 10 K) 3313 m, 1710 s, 1594 m, 1264 w, 1124 s, 963 m, 716 w, 641 m, 583 w cm−1. (s-E)-Z-Pent-2-en-4-ynal (17): IR (Ar, 10 K) 3324 m, 2845 w, 2826 w, 2743 w, 1699 s, 1582 m, 1266 w, 1096 w, 909 m, 643 w cm−1. (s-Z)-(α-Formyl)methylenecyclopropene (19): IR (Ar, 10 K) 1749 s, 1647 s, 1494 m, 1344 w, 1114 w, 1080 w, 729 w, 705 m, 602 m cm−1. (s-E)-(α-Formyl)methylenecyclopropene (19): IR (Ar, 10 K) 1743 w, 1690 s, 1665 w, 1511 m, 1146 w, 1016 w, 799 w cm−1. Unknown (U): IR (Ar, 10 K) 3429 m, 1636 m, 1476 m cm−1.</p><!><p>The triplet EPR spectrum obtained upon irradiation of 3-thienyldiazomethane (1) (Figure 1) exhibits several unusual features, with respect to the spectra of triplet arylcarbenes.39,55–57 Conformational isomerism, a heavy atom effect of sulfur, and site effects in the matrix may all be manifest in the spectrum, and a proper interpretation is not readily apparent upon cursory inspection. The assignment of the individual EPR transitions to major and minor conformational isomers derives from the differential rate of appearance as a function of photolysis wavelength (λ > 571 vs λ > 472 nm; Figure 1 and Figure S8), as well as comparison with EPR spectra of other 2- and 3-thienylcarbene derivatives obtained in subsequent investigations.58,59 The zero-field splitting parameters of the major and minor isomers were calculated in the usual way, using the best fit of the EPR transitions with the spin Hamiltonian under the assumption that gx = gy = gz = ge.60 The striking result is the large difference in magnitude of the D values between the major (D = 0.508 cm−1) and minor (D = 0.579 cm−1) isomers observed in the triplet EPR spectrum.</p><!><p>Simulation of the triplet EPR spectrum, using the program XSophe (Bruker), was accomplished by manually varying values of D, E, and g to give an optimal fit. The experimental and simulated spectra exhibit excellent agreement (Figure 5). The best fit was obtained with g = 2.0 and the zero-field parameters D and E given in Table 2. In the experimental spectrum, the major isomer exhibits subtle features in the X2 and Y2 transitions, which we attribute to inequivalent sites in the argon matrix. These features are well-reproduced, in the simulated spectrum, by two species (A and A′) that differ only slightly in terms of E value (Table 2). Although the g = 2 signal in the experimental spectrum appears to be intense, its contribution in terms of percent of spins is negligible (0.3%), as established by the simulation. The quality of the simulated spectrum, and the excellent agreement of the zero-field parameters determined from the experimental spectrum and from the simulated spectra give confidence in the correctness of the analysis.</p><p>That the EPR spectrum of triplet 3-thienylcarbene (13) could be interpreted and simulated by using g = 2.0 was not obvious to us, at the outset, because of the possible influence of a heavy-atom effect by sulfur. Sulfur-containing radicals have been widely studied in biological systems.61 In cases where an unpaired electron is localized on sulfur, g values range from 1.96 to 2.073.62 In the case of carbene 13, the fact that little spin density resides at sulfur (see below), and that the nature of the EPR spectrum is much more sensitive to the values of D and E than to the value of g, suggest that any manifestation of spin–orbit coupling on g, through the heavy-atom effect of sulfur, is minimal.</p><!><p>The preceding analysis provides the zero-field splitting parameters for the major and minor species observed in the matrix, but it does not establish the spectroscopic assignments. In making the assignments, we consider several interpretations of the EPR spectrum (Figure 4). In the first, which is the assignment that we ultimately favor, we hypothesize that the transitions for the major and minor isomers arise from s-E/s-Z conformational isomerism of triplet 3-thienylcarbene (13). (In other words, the EPR spectra of the conformational isomers are quite different.) In this scenario, the spectral doubling of the X2 and Y2 transitions is attributed to matrix site splitting.</p><p>In the second interpretation of the EPR spectrum, we hypothesize that the transitions for the major isomer—for which the X2 and Y2 transitions exhibit features of spectral doubling—arise from conformational isomers of triplet 3-thienylcarbene (13). (In other words, the EPR spectra of the conformational isomers are quite similar.) We hypothesize that the transitions for the minor isomer—which are quite different—arise from a different triplet species. In our mind, a plausible candidate is 4-thia-2,5-cyclohexadienylidene (10) (Scheme 10), which could be readily envisioned to form upon photochemical or hot molecule rearrangement of 3-thienylcarbene (13) and bicyclic compound 12. DFT calculations by McKee et al.24 and by us (Scheme 2), however, predict a singlet ground state for 4-thia-2,5-cyclohexadienylidene (10). We therefore reject the assignment of 10 as a carrier of a triplet EPR signal.</p><p>In the third interpretation of the EPR spectrum, we hypothesize that the transitions for the major and minor isomers arise from the presence of a mixture of triplet 2-thienylcarbene (11) and 3-thienylcarbene (13). (In this scenario, the doubling of spectral transitions might arise through either s-E/s-Z conformational isomerism or multiple matrix sites.) That triplet 2-thienylcarbene (11) cannot be generated by direct irradiation of (2-thienyl)diazomethane (2) does not exclude the possibility that triplet 11 might be formed and observable under different reaction conditions (e.g., photochemical or hot molecule rearrangement of 3-thienylcarbene (13) or 4-thiacyclohexa-1,2,5-triene (7); Scheme 10). Although this scenario seems improbable—given the very low barrier for ring-opening of singlet 2-thienylcarbene (11)—we cannot rigorously exclude it.</p><!><p>Here, we follow the analysis of Roth and co-workers assigning geometric isomers for triplet arylcarbenes.55–57 If the conformational isomers of a triplet carbene experience different dipolar coupling between the unpaired electrons, the conformers will exhibit different D values. Differences in dipolar coupling may arise as a consequence of differences in spin density at the β-positions of a triplet carbene in which one unpaired electron is delocalized across a π-electron system. Thus, we computed natural spin densities for both triplet 3-thienylcarbene (13) and 3-furylcarbene (23) (Table 3). The finding of a large difference in spin density at the β-positions of the carbene (C2 = +0.42, C4 = +0.13) is consistent with a large difference in the D values of the conformational isomers of 3-thienylcarbene (13) (0.508 cm−1 vs 0.579 cm−1). Empirical correlations establish that the larger D value arises when the sp orbital of a triplet carbene is oriented anti with respect to the β-position bearing the larger spin density.55–57 Since C2 is the β-position bearing higher spin density in triplet 3-thienylcarbene (13), the s-Z conformer of 13 is assigned as the isomer with the larger D value (minor isomer in Figure 5), and the s-E conformer of 13 is assigned as the isomer with the smaller D value (major isomer in Figure 5). It is interesting to note that the difference in D values between the two conformers of 3-thienylcarbene (ΔD = 0.071 cm−1) is substantially larger than the difference observed for conformers of most arylcarbenes (ΔD ≈ 0.02–0.03 cm−1), vinylcarbenes (ΔD ≈ 0.05 cm−1), and α-carbonylcarbenes (ΔD ≈ 0.05 cm−1).55–57 The value rivals that reported for the conformers of benzoyl phenyl carbene (ΔD = 0.08 cm−1).</p><p>In an effort to obtain independent evidence bearing on the assignment of the conformational isomers of triplet 3-thienylcarbene (13), we sought to utilize newly developing methods for the first-principles calculation of zero-field splitting parameters. Neese's ORCA program shows considerable promise for the analysis of a variety of high-spin organic reactive intermediates.63–66 Unfortunately, we find that the ORCA calculations for the zero-field splitting parameters of triplet 3-thienylcarbene (13) are not robust. The computed values of D and E are much too sensitive to the basis set and level of theory. These problems are not manifest in calculations of the corresponding 3-furylcarbene (23) and therefore appear to be related to the presence of the second-row atom (sulfur). For species composed only of first-row elements, the spin–spin interaction term makes the dominant (sole) contribution to D. These systems appear to be well-handled by ORCA. For species that include heavier elements, Neese has shown that the spin–orbit interaction term makes the dominant contribution to D.64</p><p>Comparison of 3-thienylcarbene (13) and 3-furylcarbene (23) is instructive. The computed zero-field splitting parameters of 3-furylcarbene (23) exhibit the expected effects of conformational isomerism (Table 3), and the prediction that the (s-Z) conformer displays the larger D value is consistent with the qualitative expectations described in the preceding section. The difference in D values between the two conformers of 3-furylcarbene (23) (ΔD = 0.025 cm−1), however, is significantly smaller than that of 3-thienylcarbene (13) (ΔD = 0.071 cm−c1). While the very good agreement between the D values for the (s-E) conformers of these carbenes is undoubtedly fortuitous (0.508 cm−1 observed for 13; 0.506 cm−1 computed for 23), it serves to focus attention on the fact the experimental and computed D values for the (s-Z) conformers differ substantially (0.579 cm−1 observed for 13; 0.531 cm−1 computed for 23). Although the computed spin densities in both systems are quite similar (Table 2), there appears to be an interaction in the (s-Z) conformer of triplet 3-thienylcarbene (13) that renders it uniquely different, in terms of its zero-field splitting parameter D.</p><p>We employed NBO calculations to investigate the possible electronic interactions between the sulfur heteroatom and the triplet carbene center in 3-thienylcarbene (13). Perhaps the most important conclusion from these studies is the absence of a readily identifiable, dominant interaction between these centers. The analysis does reveal a weak interaction between the filled sp orbital at the carbene carbon and the empty antibonding σ orbital of the C2–S1 bond in the s-Z isomer (Figure 6). This interaction was absent in the s-E isomer. The energy of this interaction is not large (0.37 kcal/mol), but it does reveal a subtle distinction in the electronic structure of the conformational isomers.</p><p>In summary, our analysis of conformational isomerism in triplet 3-thienylcarbene (13) is commensurate with a conventional interpretation, in which differences in spin density at the nonequivalent ortho positions give rise to differences in dipolar spin–spin coupling. NBO analyses provide a tenuous hint concerning differential electronic interactions between sulfur and the carbene moiety in the s-E and s-Z conformers of triplet 13. It should be noted, however, that nothing in the analysis rigorously requires the assignment of conformers to be correct. The structural assignment is based upon model systems that do not contain a heavy atom.57 Literature precedent, although sparse, suggests that the spin–orbit contribution in a system that contains a heavy atom will be dominant.64 It is conceivable that a significant spin–orbit contribution could reverse the predictions of which conformer has the larger or smaller D value, thereby reversing the assignments of s-E and s-Z conformers of triplet 3-thienylcarbene (13).</p><!><p>The inability to generate and characterize triplet 3-furylcarbene (23) was the biggest disappointment for us in the current investigation. The electronic absorption spectrum of 3-furyldiazomethane (3) is quite similar to the corresponding sulfur analogue (1), and we did not anticipate a significant difference in the photochemistry of these diazo compounds. For both oxygen- and sulfur-containing carbenes (23 and 13), DFT calculations predict triplet ground states for both s-E and s-Z conformers, with small singlet–triplet energy gaps (ca. 2–5 kcal/mol) (Schemes 2–4). The computed singlet–triplet gaps of the 3-furylcarbenes (23) are slightly smaller than those of the 3-thienylcarbenes (13)—the species that are spectroscopically observed. One conclusion to be gleaned from the computational data shown in Schemes 2 and 3 is that the methylenecyclopropene derivatives, which are formed as the major products during the initial photolysis, lie significantly lower in energy than the singlet carbenes for the oxygen series (ca. 24 kcal/mol lower), compared to the sulfur series (ca. 14 kcal/mol lower). This situation is consistent with a scenario in which a vibrationally hot 3-furylcarbene (23) would be more prone to suffer rearrangement to methylenecyclopropene (19) than the corresponding 3-thienylcarbene (13)—relative to undergoing vibrational cooling by the matrix.</p><p>The inability to generate and characterize 2-thienylcarbene (11) or 2-furylcarbene (22) is less surprising. DFT calculations, by us and by others,30,31 predict a singlet ground state for both conformers of 2-furylcarbene (22). The recent analysis by Herges and Haley reveals a substantial thermodynamic stabilization of heteroaryl carbenes.28 Ring-opening of 2-thienylcarbene (11) or 2-furylcarbene (22) to the corresponding pentenynal is exothermic, relative to the singlet carbenes, by 20 kcal/mol in the sulfur case and 30 kcal/mol in the oxygen case. The computed barriers for ring-opening of syn- and anti-2-furylcarbene (22) are very low (2–4 kcal/mol).30,31 Herges29 described the reaction as a coarctate transformation, while Birney31 interpreted it as a pseudopericyclic process. Independent of the classification, in terms of orbital symmetry, the reaction is one that may be envisioned to occur via a tunneling mechanism (at least at cryogenic temperature). Our experiments, of course, do not establish that either 2-thienylcarbene (11) or 2-furylcarbene (22) is directly involved in the photochemistry of the corresponding diazo compounds. The literature contains many examples in which reactions occur in an excited state of a diazo compound, circumventing a carbene intermediate altogether.45,46</p><!><p>Triplet 3-thienylcarbene (13) has been generated upon irradiation of 3-thienyldiazomethane (1) (λ > 534 nm) and characterized by IR, UV/vis, and EPR spectroscopy. The s-E and s-Z conformers of the triplet carbene exhibit substantially different zero-field splitting parameters (D = 0.508 and 0.579 cm−1, respectively), which arise as a consequence of a large difference in spin density at the two ortho positions in the thiophene ring. Despite finding experimental conditions for the successful generation of triplet 3-thienylcarbene, the conditions were not transferable for generating triplet 2-thienylcarbene (11) or the isomeric 2- or 3-furylcarbenes (22, 23).</p><!><p>1H NMR spectra (300 MHz) were obtained in Me2SO-d6; chemical shifts (δ) are reported as ppm downfield from internal SiMe4. Mass spectra and exact mass measurement (EMM) were obtained by using electrospray ionization (ESI). Thiophene-3-carboxaldehyde, thiophene-2-carboxaldehyde, furan-3-carboxaldehyde, and furan-2-carboxaldehyde were purchased from commercial sources and purified by vacuum distillation. The matrix isolation apparatus and technique have been described previously,67,68 and additional details are provided as Supporting Information.</p><!><p>Optimized geometries, energies, and infrared intensities were obtained at the B3LYP/6-31G* level of theory, using the Gaussian software package.69 The nature of stationary points was confirmed by calculation of the harmonic vibrational frequencies, which also provided zero-point vibrational energy (ZPVE) corrections. Vibrational frequencies were not scaled. Geometries, harmonic vibrational frequencies, and singlet–triplet energy gaps for 2- and 3-thienylcarbene (11 and 13) were also computed by using coupled-cluster methodology, CCSD,70 which includes single and double excitations. The correlation-consistent cc-pVTZ basis set was used.71–73 All CCSD calculations were done in the frozen-core approximation with the CFOUR program system.74 Natural bond orbital (NBO) calculations were performed at the B3LYP/6-31G* level of theory using the NBO program.75 Electronic absorption spectra were computed at the B3LYP/6-31G* geometries, using time-dependent density functional theory methods (M06 and CAM-B3LYP) and the aug-cc-pVTZ basis set, as implemented in Gaussian09.69 First-principles calculations of zero-field splitting parameters D and E were performed at the B3LYP/6-31G* geometries, using the B3LYP functional and the EPRIII basis set, as implemented in the ORCA program.76 Simulations of EPR spectra of randomly oriented triplets were performed with use of the XSophe program,77 as supplied by Bruker Instruments. The matrix diagonalization method was utilized (with 1600 partitions and 8 segments).</p><!><p>The tosylhydrazone precursors to compounds 1, 2, 3, and 4 were synthesized by using a modified method given by Katritzky.78 p-Toluenesulfonhydrazide (4.65 g, 25 mmol) was added to a solution of the corresponding aldehyde (25 mmol) in 30 mL of methanol and refluxed for 12 h. The solution was diluted with water (50 mL) and the precipitate was collected by suction filtration. The crude product was recrystallized from methanol.</p><!><p>White crystals, yield 70%; mp 155–157 °C (lit.22 mp 157.5–159 °C); 1H NMR (Me2SO-d6) δ 2.35 (s, 3H), 7.27 (d, J = 4.6 Hz, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.56 (dd, J = 4.9, 2.8 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.80 (d, J = 1.9 Hz, 1H), 7.93 (s, 1H), 11.28 (s, 1H); MS (ESI) (MH+) 281.1; MS (EMM) (MH+) calcd 281.0413, measured 281.0413.</p><!><p>White crystals, yield 83%; mp 145–146 °C (lit.22 mp 142–143.5 °C); 1H NMR (Me2SO-d6) δ 2.29 (s, 3H), 7.06 (dd, J = 4.9, 3.6 Hz, 1H), 7.35 (dd, J = 3.5, 1.1 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 4.1 Hz, 1H), 7.71 (d, J = 8.2 Hz, 2H), 8.08 (s, 1H); MS (ESI) (MH+) 281.1; MS (EMM) (MH+) calcd 281.0413, measured 281.0410.</p><!><p>Tan crystals, yield 36%; mp 118–120 °C (lit.22 mp 116–119 °C); 1H NMR (Me2SO-d6) δ 2.36 (s, 3H), 6.62 (s, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.68 (s, 1H), 7.73 (d, J = 8.1 Hz, 2H), 7.85 (s, 1H), 8.02 (s, 1H), 11.26 (s, 1H); MS (ESI) (MH+) 265.1 MS (EMM) (MH+) calcd 265.0642, measured 265.0638.</p><!><p>Light tan crystals, yield 44%; mp 113–115 °C (lit.22 mp 125–126 °C); 1H NMR (Me2SO-d6) δ 2.31 (s, 3H), 6.55 (dd, J = 3.3, 1.9 Hz, 1H), 6.79 (d, J = 3.2 Hz, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 1.6 Hz, 1H), 7.77 (s, 1H), 11.43 (s, 1H); MS (ESI) (MH+) 265.1; MS (EMM) (MH+) calcd 265.0642, measured 265.0654.</p><!><p>Sodium hydride (1.1 equiv, 60% suspension in mineral oil) was added to the corresponding tosylhydrazone (1 equiv) in THF and the mixture was allowed to stir at room temperature for 1 h. The resulting precipitate was filtered and dried on a vacuum line. This reaction was run on a 500 mg scale with respect to the tosylhydrazone.</p><!><p>The diazo compounds were generated by heating the corresponding tosylhydrazone sodium salts to 110 °C under vacuum. The highly colored diazo compound was collected on a coldfinger at −78 °C and rinsed into a deposition tube with CH2Cl2. After solvent removal under vacuum at −41 °C, the diazo compound was deposited onto a cold window (IR, UV/vis) or copper rod (EPR) with a constant flow of argon or nitrogen. This procedure yielded the matrix isolated samples of diazo compounds 1, 2, 3, or 4.</p><!><p>Diazo compounds 1–4 were formed via the procedure given above, but were rinsed from the coldfinger with CD3CN instead of CH2Cl2 to permit the use of 1H NMR spectroscopy. An NMR sample was made with 1 mL of the original sample and the remaining sample was used to make stock solutions at varying concentrations. The NMR solution was spiked with a known amount of benzene to serve as an internal standard. This allowed the molar quantity of diazo compound to be determined, based upon the integration of the peaks in the spectrum. Finally, dividing the moles of diazo compound in the original sample by the 1 mL sample size used for NMR spectroscopy, the molarity of the original solution could be determined. The different stock solutions were used to determine λmax in the visible and ultraviolet regions of the UV/vis spectrum and the Beer–Lambert law was used to determine extinction coefficients. Samples and solutions were kept in dry ice when they were not being used, in order to minimize decomposition. Solutions slowly warmed to room temperature during the course of NMR or UV/vis spectroscopic measurements.</p><!><p>Red liquid. UV/vis (CH3CN) λmax (nm) (ε) (L mol−1 cm−1) 494 (5.98), 260 (2820), 229 (2950), 192 (2010); IR (N2, 10 K) 2065 s, 1597 w, 1544 w, 1534 w, 1435 w, 1404w, 859 w, 841 w, 763 m, 709 w, 622 w, 485 w, 445 w cm−1; 1H NMR (CD3CN) δ 5.31 (s, 1H), 6.78 (dd, J = 1.3, 5.0 Hz, 1H), 6.83 (dd, J = 1.3, 3.0 Hz, 1H), 7.44 (dd, J = 3.0, 5.0 Hz, 1H).</p><!><p>Red liquid. UV/vis (CH3CN) λmax (nm) (ε) (L mol−1 cm−1) 502 (8.13), 297 (8620), 204 (6120); IR (Ar, 10 K) 2071 s, 1597 w, 1523 w, 1449 w, 1381 w, 1306 w, 1077 w, 803 w, 680 w cm−1; 1H NMR (CD3CN) δ 5.54 (s, 1H), 6.86 (dd, J = 1.0, 3.5 Hz, 1H), 6.99 (dd, J = 3.5, 5.0 Hz, 1H), 7.15 (dd, J = 1.0, 5.0 Hz, 1H).</p><!><p>Orange-red liquid. UV/vis (CH3CN) λmax (nm) (ε) (L mol−1 cm−1) 491 (8.51), 251 (8050), 211 (5260); IR (Ar, 10 K) 2066 s, 1594 w, 1509 w, 1416 w, 1368 w, 1170 w, 1065 w, 1028 w, 874 w, 763 w, 589 w cm−1; 1H NMR (CD3CN) δ 5.03 (s, 1H), 6.29 (s, 1H), 7.34 (m, 1H), 7.49 (m, 1H—very small coupling). (The NMR sample was spiked with a known quantity of benzene to enable the determination of the solution concentration of diazo compound. The 1H NMR resonance of benzene interferes with the 7.34-ppm resonance of diazo compound 3, precluding an accurate determination of chemical shift/integration/coupling constant.)</p><!><p>Orange-red liquid. UV/vis (CH3CN) λmax (nm) (ε) (L mol−1 cm−1) 496 (16.3), 278 (10800); IR (Ar, 10 K) 2076 s, 1593 w, 1516 w, 1428 w, 1007 w, 927 w, 884 w, 767 w, 713 w, 657 w, 593 w cm−1; 1H NMR (CD3CN) δ 5.36 (s, 1H), 6.03 (d, J = 3.3 Hz, 1H), 6.45 (dd, J = 2.0, 3.3 Hz, 1H), 7.42 (dd, J = 0.7, 2.0 Hz, 1H).</p>

PubMed Author Manuscript

Multilevel X-Pol: A Fragment-based Method with Mixed Quantum Mechanical Representations of Different Fragments

The explicit polarization (X-Pol) method is a fragment-based quantum mechanical model, in which a macromolecular system in solution is partitioned into monomer fragments. The present study extends the original X-Pol method, where all fragments are treated using the same electronic structure theory, to a multilevel representations, called multilevel X-Pol, in which different electronic structure methods are used to describe different fragments. The multilevel X-Pol method has been implemented into Gaussian 09. A key ingredient that is used to couple interfragment electrostatic interactions at different levels of theory is the use of the response density for post-self-consistent-field energy (The response density is also called the generalized density). The method is useful for treating fragments in a small region of the system such as the solute molecules or the substrate and amino acids in the active site of an enzyme with a high-level theory, and the fragments in the rest of the system by a lower-level and computationally more efficient method. The method is illustrated here by applications to hydrogen bonding complexes in which one fragment is treated with the hybrid M06 density functional, M\xc3\xb8ller-Plesset perturbation theory, or coupled cluster theory, and the other fragments are treated by Hartree-Fock theory or the B3LYP or M06 hybrid density functionals.

multilevel_x-pol:_a_fragment-based_method_with_mixed_quantum_mechanical_representations_of_different

1. Introduction<!>2. Theoretical Background<!>2.1. The X-Pol Method<!>2.2. Multilevel X-Pol<!>2.3. Iterative Updating (IU) Method<!>3. Computational details<!>4. Results and discussion<!>5. Concluding remarks

<p>The early development of atomistic potential energy functions1 for polypeptides and the current coarse-grained models2 by Scheraga and coworkers have profoundly influenced the field of computational biology. In recent years, a number of fragment-based quantum mechanical methods have been explored.3–22 In these methods, a large system is partitioned into monomer blocks also called fragments, which may be separate individual molecules or covalently connected species such as amino acid residues in a protein. Fragment-based methods are computationally efficient, which enables electronic structure calculations to be applied to condensed-phase and biomolecular systems to gain a deeper understanding of intermolecular interactions such as polarization and charge transfer.23–32 Although linear scaling quantum mechanical calculations of proteins have been carried out,5,33–36 further approximations are needed to treat intermolecular electrostatic interactions in order to overcome the sampling computational bottleneck for statistical mechanical properties. To this end, the explicit polarization (X-Pol) method, making use of block localization of molecular orbitals within individual fragments,8,10,37,38 was developed for statistical mechanical Monte Carlo and molecular dynamics simulations of condensed phase9,39 and biomolecular systems.40 The block-localization scheme in the X-Pol method can also be applied to density functional theory.29–30,41–42</p><p>In many applications, one is particularly interested in the properties of a small region of the system, which could be the solute molecule in solution or the active site of an enzyme, and, a high level of theory is needed to yield accurate results for this region of the system. Yet, it is also important to incorporate explicitly the instantaneous polarization of the rest of the system. One approach is to use the method of combined quantum mechanics and molecular mechanics (QM/MM) with the former treating the small region of interest and the latter representing the solvent and protein environment;43–44 however, most molecular mechanics treatments do not include the mutual polarization effects.45 Beyond traditional QM/MM approaches, a mixed multilevel fragment-based quantum mechanical method will allow the environmental region also to be modeled by an electronic structure method.46 The present paper describes such a multilevel method to represent different fragments with different quantum mechanical methods within the X-Pol formalism. The present multilevel X-Pol procedure has been implemented into the Gaussian 09 program47 and it is sufficiently general that any theoretical method available in that program can be combined to represent any of the different fragments. Thus, the present approach differs from the strategy in other fragment-based molecular orbital methods to treat different level of theory separately, such as FMO-MP2,48 FMO-DFT,49 FMO-coupled cluster,50 FMO-multiconfiguration,51 or multilayer with different basis sets;52 they are all treated in the same footing here. Our method also represents a general strategy for a multilayer QM/QM coupling to study chemical reactions and intermolecular interactions.18,52–54</p><p>In the following, we first briefly summarize the X-Pol method, which is followed by a discussion of the multilevel X-Pol strategy. The computational details are given in Section 3, and Section 4 presents applications of the multilevel X-Pol method to two hydrogen bonding systems, one involving acetic acid and water and the other being a Zundel ion-water cluster. Section 5 summarizes the main findings of the present study.</p><!><p>X-Pol theory has a hierarchy of three elements: (1) the construction of the total molecular wave function, (2) the formulation of an effective Hamiltonian, and (3) the reduction of computational costs in electronic integral evaluation.8–10,38 We first briefly summarize the X-Pol method for the case in which all fragments are treated at the same self-consistent field (SCF) level, and discuss some methods that can be used to include exchange, dispersion and charge transfer contributions. Then, we describe the procedure for a multilevel X-Pol approach with mixed theoretical levels, focusing our attention on post-SCF methods. Throughout this paper, we consider systems that do not have covalent bond connections between different fragments, but the generalization for treating covalently connected fragments can be achieved using methods described previously,10 in particular by making use of the generalized hybrid orbital (GHO) scheme developed for combined QM/MM simulations at various levels of theory.55–59</p><!><p>In X-Pol, a macromolecular system is partitioned into N monomer blocks, also called fragments, and the total wave function Ψ of the system is written as a Hartree product of the antisymmetric wave functions of individual fragments:8 (1)Ψ=∏A=1NΨA where ΨA is the wave function of fragment A, which may be approximated by a single determinant or by a multi-configurational wave function. The wave functions for different monomers do not have to be approximated using the same method or represented at the same level of theory.</p><p>The effective X-Pol Hamiltonian for the system is (2)Ĥ=∑ANĤAo+12∑AN∑B≠AN(ĤABint+EABXD) where ĤAo is the electronic Hamiltonian for an isolated fragment A, and ĤABint and EABXD represent electrostatic and exchange-dispersion (XD) interactions between fragments A and B.8,10,38 The interaction Hamiltonian ĤABint depends on both electronic and nuclear degrees of freedom, and it can be viewed as an electrostatic embedding of the QM fragment A in the external field of fragment B: (3)ĤABint=−∑i=1MAeΦEB(riA)+∑α=1NAZαAΦEB(RαA) where MA and NA are, respectively, the number of electrons and nuclei of fragment A, riA and RαA are the corresponding positions for electron i and nucleus α, ZαA is a nuclear charge, and ΦEB(rxA) is the electrostatic potential at rxA due to the external charge density of fragment B: (4)ΦEB(rxA)=∫ρB(r')|rxA−r'|dr' where ρB(r')=−ρeleB(r')+∑bZbBδ(r'−RbB) is the total charge density of fragment B, including both the smooth electron density ρeleB(r') and the nuclear charges {ZbB} at {RbB}. In the present multilevel X-Pol, the embedding potential ΦEB(rxA) is modeled by partial atomic charges {qbB[ρeleB]} derived from the corresponding charge density, ρeleB, for example by population analysis (Mulliken or Löwdin charges) or by electrostatic potential fitting (CHELPG or Merz-Kollman schemes), and this simplifies it to (5)ΦEB(rxA)=∑bqbB|rxA−RbB| The theory can be extended to use higher multipole moments60 or even the full charge distribution of the fragments (eq 4) to compute the electrostatic potential,61 but that will not be considered here.</p><p>The total X-Pol energy is given as follows: (6)E[{ρ}]=<Ψ|H̑|Ψ>=∑ANEA+12∑AN∑B≠ANEABint[ρA,{qbB}]+EABXD where EA is the energy of fragment A (note that EA is different from the gas-phase energy EAo because the wave function ΨA has been polarized by the rest of the system in X-Pol), and EABint[ρA,{qbB}] is the electrostatic interaction energy between fragments A and B, akin to that used in a QM/MM method.43–44</p><p>The energy term EABXD in eq 4 accounts for the effects of the approximation used in eq 1, which by construction neglects short-range exchange repulsion and long-range dispersion interactions as well as charge transfer contributions. In the original X-Pol method,8,10 exchange repulsion is represented by a pairwise RIJ−12 dependence and the attractive non-covalent interaction by a pairwise RIJ−6 term as in the Lennard-Jones potential, where RIJ is an interatomic distance. In the present study, an exponential function for the repulsion, as in the Buckingham potential is used: (7)EABXD=∑INA∑JNBAIJe−BIJ·RIJ−CIJRIJ where the parameters AIJ, AIJ, and CIJ are determined using standard combining rules from atomic parameters such that AIJ = (AIAJ)1/2, BIJ = (BI + BJ)/2, and CIJ = (CICJ)1/2.</p><p>The effect of charge transfer is modeled indirectly. The strict block localization of molecular orbitals within individual monomers in X-Pol does not allow charge delocalization between different fragments (unless one uses a grand canonical formulation, which is not employed here).31 At distances longer than hydrogen bonding range, it is often a good approximation to neglect charge transfer, and interfragment electrostatic interactions can then be adequately described by the electrostatic embedding scheme43–44 using the Coulomb potential (eq 5). However, at short interfragment distances where there is significant orbital overlap, one needs to take into account the energy component due to charge delocalization (sometimes also called charge transfer).30–31,61 In the present work, we account for charge transfer only empirically, in particular (in the spirit often used in molecular mechanics)62 by modeling the charge delocalization energy with enhanced electrostatic polarization. Consequently, the electrostatic potential ΦEB(rxA) in eq 5 is recognized as an effective potential that mimics both long-range Coulomb (electrostatic) interactions and short-range charge delocalization contributions, and this can be achieved to some extent by optimizing the parameters in the EABXD term (eq 7) and possibly the charge model63 {qbB[ρeleB]} (eq 5) to best reproduce hydrogen bonding interactions for a set of bimolecular complexes38 (however such optimization is beyond the scope of the present article).</p><p>Beyond the empirical approximations, a variational many-body expansion approach in X-Pol has been described, which includes exchange repulsion, charge delocalization and dispersion terms explicitly.64 Individually, one way to improve on the repulsive potential is to antisymmetrize the X-Pol Hartree-product wave function;61,65–68 this yields X-Pol with full eXchange, called X-Pol-X.69 When the monomers are treated by Hartree-Fock theory, this calculation can be accomplished by using the formalisms of block-localized wave function (BLW)61,67–68 or the SCF-MI method,65 and this procedure has been extended to density functional theory.29–30,42 To treat dispersion interactions, multiconfigurational methods and perturbation theories can be used; for example, one can adopt symmetry adapted perturbation theory (SAPT) as a post-SCF correction to the X-Pol energy,70 as has been done recently by Jacobson and Herbert.22 Both exchange-repulsion and dispersion interactions are of short range on the length scale of solutions and biopolymer systems, and only the close neighbors need to be explicitly considered.69</p><p>Charge delocalization effects can also be estimated using a grand canonical ensemble,31 or by using the method of interaction energy expansion introduced by Stoll and Preuss,71 which has been adopted by Kitaura and coworkers in a fragment molecular orbital implementation.11 Of course, a straightforward way of including charge delocalization effects is to use larger fragments that include charge transfer partners.28 Another approach, which has been recast in several ways, is the molecular fractionation with conjugated caps (MFCC) approach by Zhang and coworkers.13,72 In MFCC, the individual fragments are capped with a structure representative of the local functional group of the original system, and the total energy is obtained by subtracting the energies that account for the common fragments used in the "caps".73–74 In both cases, the total energy can be conveniently determined using this addition-subtraction scheme; however, the total molecular wave function is no longer available, making energy gradient calculations more challenging. In this regard, we have developed a generalized explicit polarization (GX-Pol) method on the basis of a multiconfiguration self-consistent field (MCSCF) wave function that makes use of dimeric, charge-delocalized fragments.30,75 In the present study, we do not include the explicit treatment of charge delocalization energy, but this can be addressed in a separate study.</p><!><p>The method outlined in the previous section has been implemented with all fragments treated at the same theoretical level (semiempirical,8,10,37 Hartree-Fock (HF), or density functional theory (DFT)38). Here, we consider a system partitioned into N fragments, of which N' fragments are treated by a method denoted as high level (HL) and the remainder N − N' fragments are modeled with a different approach specified as low level (LL). The former fragments are called HL fragments, and the latter are called LL fragments. Generalization to any number of levels is straightforward, but for convenience, we restrict the following discussions to two levels. This division highlights the need for high accuracy in a small (HL) region of interest, such as the solute molecule in a solution or the active site of an enzyme, while retaining the need for a computationally efficient way to include polarization effects in the remainder of the system. In the present illustration of the method, the LL method is restricted to either HF or DFT.</p><p>A variety of methods can be used for fragments in the HL region, and they are divided into two categories: SCF and post-SCF. For methods such as DFT and multiconfiguration SCF (MCSCF), the treatment is the same as described previously38 for single-level X-Pol based on ab initio Hartree-Fock calculations or DFT. The second category includes post-SCF methods such as configuration interaction (CI), coupled cluster (CC) and Møller-Plesset (MP) perturbation theory; when such methods are employed for HL fragments, the total X-Pol energy is written as (8)E[{ρ}]=∑AN(EASCF+12∑B≠ANEABint[ρA,{qbB}]+EABXD)+∑AN'EAcorr=EtotSCF+Etotcorr where EASCF is the SCF energy of the reference wave function, EAcorr is the post-SCF correction for fragment A, and EtotSCF and Etotcorr are the total SCF energy and the total post-SCF correlation energy. Note that the SCF energy EASCF can be obtained either from a single determinant reference wave function in a CI, CC, or MP2 calculation or a multiconfiguration wave function in multireference CI, or CASPT2, etc. calculations. The main difference of this energy expression from that of eq 6 is that the total multilevel X-Pol energy is no longer written as the expectation value of an X-Pol wave function.</p><p>In using eq 8, the computation involves an initial optimization of the X-Pol SCF wave function, followed by determining the post-SCF energy corrections for fragments in the HL region. In the SCF procedure, the charge densities of the HL fragments that polarize other fragments are the response densities corresponding to the post-SCF calculation.76–78 The response density (which is also called the generalized density and relaxed density) is the sum of the SCF density and the relaxation density due to the post-SCF procedure, which is obtained using the Z-vector method,79 including a single coupled perturbed HF calculation for the occupied and virtual molecular orbital (MO) block, independent of the specific post-SCF method.77 The response density procedure allows the use of methods (such as MP perturbation theory) for which the energy does not correspond to a wave function expectation value; it is also more accurate for computing one-particle properties using CC and other post-SCF methods. The response density is obtained by adding the relaxation density to the SCF density and transforming into the atomic basis for population analysis and computation of one-particle properties including the electrostatic potential: (9)PμνA,HL=PμνA,SCF+PμνA,rel where PμνA,SCF and PμνA,rel are the SCF and relaxation densities for fragment A in the HL region. If Mulliken population analysis (MPA) is used,80 the partial atomic charges in eq 8 for HL fragments can be written as (10)qaA,HL=ZaA−∑μ∈aνPμνA,HLSμνA=qaA,SCF+qaA,rel</p><p>The elements of the effective Hamiltonians (Fock or Kohn-Sham matrices), both for the HL and LL fragments, in a multilevel X-Pol method can be written similarly as22,81 (11)FμνA,Xpol=∂ESCF[{ρ}]∂PμνA,SCF=FμνA,o−12∑B≠A∑b∈BqbB(IbB)μνA+12∑a∈AXaA(ΛaA)μν where FμνA,o the Fock matrix element for an isolated fragment A, qbB is the partial charge on atom b in fragment B and it is understood that qbB≡qbB,HL for fragments in the HL region, IbB is the matrix of the pair potential in atomic basis as defined by (12)(IbB)μνA=<μ|1|r−RbB||ν> and XaA is a vector arising from the derivative of the interaction energy with respect to partial atomic charge of atom a: (13)XaA=∑B≠A(∑λσPλσB(IaA)λσB+∑b∈BZbB|RbB−RaA) Note that the notation PλσB is defined as PλσB≡PλσB,HL if B≤N', and as PλσB≡PλσB,SCF if B>N'. The elements of the response density matrix, ΛA, are given by (14)(ΛaA)μν=∂qaA∂PμνA,SCF=∂qaA,SCF∂PμνA,SCF where qaA is the atomic charge on atom a, and PμνA,SCF is an element of the density matrix of fragment A in the SCF optimization of the X-Pol fragment wave function. The charge derivatives have been given for a number of charge models.22,81 The interpretation of eqs 11–14 is that the wave functions for all fragments are optimized at the SCF level, but their polarization, by virtue of setting qbB to qbB,SCF+qbB,rel for B≤N', includes contributions from the relaxation density corresponding to the post-SCF energy in the HL region.</p><!><p>In the standard X-Pol method, the SCF wave function of eq 1 is optimized variationally by using eq 11.81 An alternative way of optimizing the SCF wave function, which is non-variational, is to consider each fragment as an isolated molecule embedded in the electrostatic field of the rest of the system. Then, the Fock matrix for each fragment can be written separately as follows:8–10 (15)FA,IU=FA,o−∑B≠A∑b∈BqbB(IbB)A where FA,o is the Fock matrix of fragment A, qbB is a column vector of atomic charges of fragment B stretched to the dimension of the orbital basis and (IbB)A is the matrix of pair potential (eq 12). The total electronic energy of the system can then be determined iteratively by a double self-consistent-field (DSCF) procedure.8–10,82 Starting with an initial guess of the one-electron density matrix for each fragment, one loops over all fragments in the system and performs SCF optimization of the wave function for each fragment in the presence of the instantaneous external charges of all other fragments (through (IbB)A). This is iterated (the SCF for the system) with an updated external potential until the total electronic energy and the charge density are converged. This iterative updating (IU) procedure is straightforward and was the approach proposed for fragment calculations in Ref. 8 and adopted in the subsequent FMO implementation.11,83 Such an iterative updating procedure can be found in many applications both in electronic structure theory82 and combined QM/MM approaches that include MM polarization.45 A main short coming of the above approach is that the Fock matrix in eq 15 is not variationally optimized,18,81 and it is not suitable for efficiently computing energy gradients. In this study, we use the superscript IU for the non-variational iterative updating procedure in eq 15, and simply Xpol for the variational method employing eq 11.</p><p>Note that both the sequential and variational optimization of the X-Pol wave function can be carried out by DSCF iterations, although they can also be done, if desired, as a single large SCF problem. In the illustrations of the multilevel X-Pol method presented below, we will we compare the energy difference between the two optimization procedures.</p><!><p>The goal of this study is to illustrate that the multilevel X-Pol fragment-based quantum mechanical model can be implemented with an arbitrary combination of different electronic structural methods for different fragments. The X-Pol method has been implemented into a locally modified version of the Gaussian-09 program.47 In this program, the response density78 can be computed for a range of post-SCF methods, including MPn, QCISD, CCD, CCSD, CID, CISD, BD, and SAC-CI,47 thus, any of these—as well as SCF methods—can be used to represent a given fragment in multilevel X-Pol.</p><p>We choose two hydrogen bonding complexes, (1) acetic acid (fragment A) and water (fragment B), and (2) H5O2+ (fragment A) and four water molecules (four water fragments as B for a total of five fragments). The complexes and monomer structures are optimized using the hybrid M06/MG3S DFT, which are then used in all subsequent single-point energy calculations with various multilevel X-Pol methods. In the present study, we have used the hybrid density functional theory M06, second order Møller-Plesset perturbation theory (MP2), and coupled-cluster with singles and doubles (CCSD) method for acetic acid and the H5O2+ ion, and we employed Hartree-Fock (HF), B3LYP84 and M0685 density functional method for water. In all X-Pol calculations, the 6-31G(d) basis set was used.</p><p>The binding energy ΔEb for the complex is defined as (16)ΔEb=EAB−EAo−EBo In X-Pol, the binding energy can be decomposed into an intramolecular distortion term ΔEdist, including both the energy change due to geometric variation and the energy cost needed to polarize the electron density, and an intermolecular interaction contribution.30,43,61 The latter can be further separated into an electrostatic component ΔEint and an exchange-dispersion energy term ΔEXD. In this energy decomposition scheme, we rewrite eq 16 as (17)ΔEb=ΔEdist+ΔEint+ΔEXD These terms are defined as follows: (18)ΔEdist=∑AN(EA−EAo) where EA and EAo are the intra-monomer components of the energies of monomer A in the complex or in isolation, (19)ΔEXD=∑A>BNEABXD and (20)ΔEint=∑A>BNΔEABint=∑A>BN12[ΔEAint(B)+ΔEBint(A)] where ΔEABint is the interaction energy between monomers A and B, and ΔEXint(Y) is the electrostatic interaction energy of the "QM" fragment X polarized by the external potential of monomer Y. Although ΔEXint(Y) and ΔEYint(X) describe the same interaction between monomers X and Y, they are not symmetric unless the same theoretical model is used for both monomers and the Coulomb integrals are explicitly computed over all basis functions. For convenience of discussion, we also define the total electrostatic component of binding energy as (21)ΔEele=ΔEdist+ΔEint</p><!><p>Tables 1 shows the computed electrostatic interaction energies between acetic acid and water for the optimized configuration shown in Figure 1 using the sequential optimization approach in multilevel X-Pol. Two charge models are used in this work, those from Mulliken population analysis (MPA) and those from Merz-Kollman electrostatic potential fitting (MK). The corresponding results obtained using variational optimization in multilevel X-Pol are given in Table 2. In this case, only the MPA charges are used.</p><p>The total interaction energy between an acetic acid and a water molecule at the configuration shown in Figure 1 is −6.9 kcal/mol from M06/MG3S optimization, which is reduced slightly to −6.6 kcal/mol using CCSD(T)/MG3S//M06/MG3S. The electrostatic interaction energy from X-Pol by iterative updating method using M06/6-31G(d) for both fragments is −7.7 kcal/mol when the MPA charges are used, and it is reduced to −7.0 kcal/mol when the MK charges are used. With a different combination in the multilevel X-Pol method in which acetic acid is treated by CCSD(T) and water by M06, the computed electrostatic interaction energies are −7.6 and −7.2 kcal/mol with the MPA and MK charges, respectively, similar to the single level results. Switching to the variational X-Pol method, we obtained an electrostatic interaction energy of −9.0 kcal/mol using the M06 representation of both monomers and the MPA charges. In this case, the variational optimization of the Kohn-Sham orbitals lowers the energy by 1.8 kcal/mol for this bimolecular complex. Similar trends are found in other multilevel X-Pol combinations in Table 2. The results in Table 1 do not include the exchange repulsion energy, charge transfer contributions, or correlation effects that result from wave function delocalization in a full KS-DFT calculation. The latter two effects are not fully separable in energy decomposition analyses, but both make stabilizing contributions to the bimolecular complex, which tend to partially compensate the strong exchange repulsion energies. If we optimize the empirical parameters in eq 7 for the M06 and B3LYP combination in multilevel X-Pol, we obtained a energy of of 2.1 kcal/mol for the ΔEXD term, which is applied to all multilevel X-Pol methods in Table 2 to yield the total binding energies ΔEb.</p><p>Table 1 shows that the MPA charges tend to provide stronger electrostatic polarization effect than do the MK charges. This results in overall interaction energies that are greater in magnitude. Both ΔEAint(B) and ΔEBint(A) describe the electrostatic interaction energy between fragments A and B, but they differ numerically because the former specifies embedding of fragment A in the classical field of fragment B whereas ΔEBint(A) gives the embedding energy of fragment B in the electrostatic field of fragment A. Across the series of five different combinations shown in Tables 1, the computed ΔEAint(B) values are greater than the ΔEBint(A) terms by using IU optimization of the wave function within an electrostatic embedding picture; however, the ordering is reversed in the variational X-Pol method. Furthermore, the electrostatic polarization is significantly stronger by variational optimization than by IU optimization of the wave function, both in single level and in multilevel X-Pol. On one hand, the difference between the ΔEAint(B) and ΔEBint(A) terms highlights the asymmetry in the representation of two fragments in a QM/MM type of treatment, and the average of the two terms is defined as the X-Pol dimer interaction energy (eq 20).8–9 On the other hand, the difference between the IU and variational optimization procedures for the X-Pol wave function shows the importance of correctly accounting for the mutual polarization effects among different fragments that minimize the adiabatic ground state energy. Note that few existing fragment-based methods optimize the fragment wave functions variationally.</p><p>Table 3 gives the interaction energies between the Zundel ion H5O2+ and four water molecules computed with various theoretical models using the optimized structure with M06/MG3S86 (Figure 2). The optimized structure for the complex is very similar to that optimized using B3LYP/6-311+G(dp) from ref 87.</p><p>It is interesting to first compare various methods of estimating the exchange repulsion–dispersion contributions to the energy of binding in this case. The exchange repulsion energy can be obtained as the difference between the energy from the antisymmetrized X-Pol wave function and that from the X-Pol at the Hartree-Fock level. We found that the results depend noticeably on the basis set and the charge model used for electrostatic coupling between different fragments (eq 11). The estimated exchange energies are 30.0 and 28.5 kcal/mol using iterative updating optimization in X-Pol with the MK and MPA charges, respectively. This increases to 35.8 kcal/mol using the variational X-Pol wave function and the MPA charges. In these cases, the 6-31G(d) basis is used.</p><p>To gain more insights into the magnitude of the contributions from inter-monomer exchange, dispersion and charge transfer on the hydrogen bonding interactions in the Zundel ion complex, we have carried out an interaction energy decomposition analysis using the block-localized wave function method (BLW-ED) using a larger basis set.30,61 At the HF/aug-cc-pVDZ level, the exchange repulsion and charge transfer contributions to the energy of binding are estimated to be 38.8 and −13.3 kcal/mol, respectively, for a net contribution of 25.5 kcal/mol, and the total binding energy is −62.4 kcal/mol. If one uses the difference between the CCSD(T) binding energy (−69.7 kcal/mol) and that at the HF level (−62.4 kcal/mol) as a rough estimate of the dispersion contribution, a value of −7.3 kcal/mol is obtained. Then, the overall EABXD term including the effect of charge transfer may be estimated as 18.2 kcal/mol (that is, 25.5 minus 7.3 kcal/mol).</p><p>We optimized the Lennard-Jones parameters separately for the oxonium ion system with the M06 density functional for H5O2+ and the B3LYP functional for (H2O)4, and we obtained AOO = 1.5221×105 kcal/mol, BOO = 3.754 Å, and COO = 756.3 Å6 kcal/mol for the Buckingham potential (eq 7). Then, eq 7 yields a value of 18.4 kcal/mol for ΔEXD, in good agreement with the above analysis. Although the ΔEXD term ought be reoptimized for each multilevel X-Pol model, we have used to same Buckingham energy for all combinations listed in Table 3, and the total binding energies in the last column of Table 3 are reasonable in comparison with the CCSD(T) value (at the M06/MG3S geometry) of −69.7 kcal/mol. For comparison, the corresponding multilevel X-Pol values without inclusion of the ΔEXD term are significant greater than the full QM result, ranging from −83 to −92 kcal/mol.</p><p>For multilevel X-Pol in which both HL and LL energies are obtained at the SCF level, the energy of binding from the variational approach will be more negative than that obtained using the non-variational (iterative updating) procedure, which is also used in the fragment molecular orbital model.11–12 The results using the M06 density functional for the HL fragment in Table 3 are indeed consistent with this expectation. However, if the HL energy is determined by a post-SCF theory as in MP2 and CCSD calculations in Table 3, there is no guarantee that the "variational" multilevel X-Pol energy is lower than that of the IU optimization result because only the reference wave function used in the post-SCF calculation is optimized. This is seen in the CCSD and M06 combination, which yields a binding energy smaller than that from the IU optimization method (both using the MPA charges). The reference wave functions for the individual fragments are more strongly distorted than in other cases.</p><!><p>The explicit polarization (X-Pol) method is a fragment-based quantum mechanical method, in which a macromolecular system is partitioned into monomer fragments and the total molecular wave function is written as a Hartree product of the antisymmetric wave functions for individual fragments. In the present study, a general formulation is presented to treat different fragments with different electronic structure methods. The current implementation of the multilevel X-Pol method in Gaussian-09 allows any method available in that program to be used to describe a given fragment. The key to the implementation is using the response density to compute the electrostatic coupling (and mutual polarization), in particular by using the response density in population analyses or in an electrostatic potential charge fitting procedure.</p><p>The computational method is illustrated by calculations on two hydrogen bonding complexes involving acetic acid and water, and the H5O2+ ion and four water molecules. Acetic acid and H5O2+ are treated using M06, MP2 and CCSD as the high-level theory, and these methods are paired with one of HF, M06 and B3LYP as the lower-level method. The present multilevel X-Pol method can be used to treat a small region of the system, such as the solute molecule in solution or the active site of an enzyme, with a high-level theory, and the remainder of the system with a more computationally efficient method.</p>

PubMed Author Manuscript

Fluctuations of water near extended hydrophobic and hydrophilic surfaces

We use molecular dynamics simulations of the SPC-E model of liquid water to derive probability distributions for water density fluctuations in probe volumes of different shapes and sizes, both in the bulk as well as near hydrophobic and hydrophilic surfaces. Our results are obtained with a biased sampling of coarse-grained densities that is easily combined with molecular dynamics integration algorithms. Our principal result is that the probability for density fluctuations of water near a hydrophobic surface, with or without surface-water attractions, is akin to density fluctuations at the water-vapor interface. Specifically, the probability of density depletion near the surface is significantly larger than that in bulk, and this enhanced probability is responsible for hydrophobic forces of assembly. In contrast, we find that the statistics of water density fluctuations near a model hydrophilic surface are similar to that in the bulk.

fluctuations_of_water_near_extended_hydrophobic_and_hydrophilic_surfaces

Introduction<!>Simulation Models<!>Umbrella sampling<!>Length-scale dependence of density fluctuations in bulk water<!>Water near hydrophobic surfaces and the effect of dispersive attractions<!>Fluctuations near hydrophobic and hydrophilic surfaces<!>Summary<!>

<p>According to theory, fluctuations of water density on sub-nanometer length scales obey Gaussian statistics,1–3 while those on larger length scales deviate significantly from this behavior.4 The deviations at large length-scales reflect the fact that water at standard conditions lies close to water-vapor coexistence.4,5 A large enough repellent surface can therefore induce the formation of a water-vapor-like interface, and as such, the probability of water depletion is enhanced near such a surface. In particular, the presence of this liquid-vapor-like interface facilitates large fluctuations in its vicinity compared to that in the bulk. This perspective is at the heart of current ideas about hydrophobic effects.6,7 In this paper, we use molecular simulations to further examine the validity of this perspective. We demonstrate marked similarities between water-vapor interfaces and water-oil interfaces for large enough oily surfaces with low radii of curvature.</p><p>The fact that a purely repulsive hydrophobic surface induces the formation of a vapor-liquid-like interface is well-established.4,8–10 Dispersive attractions between the hydrophobic surface and water can mask this effect, as the attractions move the interface to a mean position immediately adjacent to the hydrophobic surface. This removes any significant presence of vapor on average.10–13 As a result, it is difficult to detect the presence of a water-vapor-like interface by considering only the mean behavior of water density profiles. Rather, the presence of this interface is more directly reflected in fluctuations away from those profiles,6,14–19 which motivates our focus here on the statistics of these density fluctuations.</p><p>Several groups have studied the statistics of these fluctuations.3,20,21 This paper is distinguished from this earlier work by the fact that we report the statistics of large length-scale fluctuations, which are expected4 to be fundamentally different from the small length-scale fluctuations studied in the earlier work. Large length-scale water density fluctuations control pathways for assembling hydrophobically stabilized structures.4,22–29</p><p>The central quantity to be examined is the probability for finding N water molecules in a sub-volume v, Pv(N). Hummer and Pratt and their co-workers introduced the idea of studying this function as a route to understanding solvation.3,20,30,31 In pure solvent, large length-scale fluctuations are very rare, but these are the fluctuations of interest here. Therefore, to obtain reasonable statistical information, we must employ some form of biased non-Boltzmann sampling.32,33 The simulation setup and system details are described in the next section, after which we explain the specific sampling method that we employ. Results are then presented, beginning with those for the length-scale dependence of fluctuations in bulk water, followed by a discussion of the effect of dispersive attractions between the water molecules and a hydrophobic surface. Finally, we contrast fluctuations near a hydrophobic surface with those near a hydrophilic surface.</p><p>Our results are qualitatively consistent both with the Lum-Chandler-Weeks (LCW)4 theory for the length-scale dependence of hydrophobic effects and with recent computer simulation studies.18,34 This present work, however, is the first publication of quantitative computer simulation data on the nature of water density fluctuations that deviate far from the mean in an atomistic model of water.35 As such, it is the first test with an atomistic model of the underlying assumptions from which the LCW theory derives. Further, as a matter of technical interest, the numerical technique we use to determine reliable distributions for rare fluctuations is new and may prove useful in other contexts.</p><!><p>For the purposes of studying hydrophobic effects and juxtaposing hydrophobic and hydrophilic solvation, it is most important that the liquid be close to liquid-vapor equilibrium with a substantial surface tension, that it has a high dielectric constant and small compressibility, and that its molecules typically arrange with local tetrahedral order.6 There are many models that could be used to satisfy these criteria. We have used the SPC-E model.36 Given the many commonalities between behaviors we elucidate here and those found with models as simple as the lattice-gas,19,23 it seems unlikely that our findings would change significantly if we changed to another standard atomistic model of water.</p><p>Density fluctuations of SPC-E water in the canonical ensemble at 298K can be quantified using the LAMMPS molecular dynamics (MD) simulation package.37 Specifically, we evaluate the probability, Pv(N), of finding N water oxygens in a probe volume of interest, v. A straightforward canonical ensemble simulation, with an average water density, ρ = 1 g/cm3, would suppress large density fluctuations. To avoid this suppression, a particle-excluding field is introduced on one surface of the simulation box, with the box large enough that the net density is less than the bulk liquid density. Through this construction38,39 we ensure that the bulk liquid remains in the center of the box, that it is at coexistence with its vapor phase, and that a free liquid-vapor interface acts as a buffer in the event of a large density fluctuation. Solutes are placed near the center of the box, deep within the bulk liquid and far from the free water-vapor interface.</p><p>To model a hydrophilic solute, we consider water molecules within a particular sub-volume of bulk water, specifically a sub-volume of dimensions 3 × 24 × 24 Å3. Taking a configuration of equilibrated bulk liquid, we immobilize those water molecules that are within this sub-volume. The immobilized molecules are then the solute. Its surface is unlikely to disrupt the hydrogen bond network of the neighboring solvent because the surface is made up of fixed water molecules in a configuration that is typical of bulk water. Indeed, to the extent that fluctuations in water structure are unimportant, there is no cost in free energy to solvate this solute. In this sense, it is an ideal hydrophilic solute.</p><p>To model a hydrophobic solute, we replace the fixed waters in our model of a hydrophilic solute with methane-like oily particles. These oily particles are uncharged and interact with the surrounding solvent water molecules via a standard water-methane Lennard-Jones potential.40 The resulting potential energy field expels water from the volume occupied by the solute. It also attracts water with dispersive interactions. The solute constructed in this way and a typical configuration of solvent water are shown in Fig. 1.</p><p>In order to study the effect of dispersive attractions on water-density fluctuations, the Lennard-Jones (LJ) pair potential between the hydrophobic solute and the solvent is split into repulsive and attractive parts using the Weeks-Chandler-Andersen (WCA) prescription.41 The role of attractions can then be examined systematically with a scaling parameter λ, (1)uλ(r)=u0(r)+λΔu(r), where u0(r) and Δu(r) are the WCA repulsive and attractive branches of the LJ potential, respectively, σ = 3.905Å and ε = 0.118Kcal/mol were used as the LJ parameters for the oily particles,40 and Lorentz-Berthelot mixing rules were used to obtain water-solute interaction parameters.</p><!><p>Since we have chosen molecular dynamics to probe our system, it is convenient to use a biasing umbrella potential that produces continuous forces. The potential we use is a function of the entire set of water oxygen coordinates: {ri}, i = 1, 2, …M. The number of molecules, N, in a specific volume, v, is not a continuous function of {ri}, but the biasing potential we use to influence this number is a continuous function of these variables. In particular, we focus on the coarse-grained particle number: (2)N^({ri},v)=∫dr∑i=1MΦ(r−ri)hv(r), where hv(r) = 1 or 0, depending on whether or not r is in volume v, r has Cartesian coordinates x, y, z, and (3)Φ(r)=φ(x)φ(y)φ(z) with φ(x) being a normalized, truncated and shifted Gaussian-like distribution, (4)φ(x)∝[exp(−x2/2ξ2)−exp(−rc2/2ξ2)]θ(rc−|x|). The proportionality constant is the normalization constant, θ(x) is the Heaviside step function, and we have chosen the coarse-graining length ξ to be 0.1Å and the cut-off length rc to be 0.2Å.</p><p>In the limit ξ → 0, the dynamical variable Ñ({ri},v) is the actual number of water molecules in the volume v. But for finite ξ, Ñ ({ri};v) is a continuous and differentiable function of {ri}. We construct the biasing potential with this variable. In particular, we let (5)U({ri};κ,η)=κ2[N^({ri},v)−η]2, where κ is a positive constant. We have found it convenient to use κ = 0.25 kcal/mol. Simulating our system in the presence of this umbrella potential allows us to bias the system towards configurations with Ñ({ri},v) values near η. When η differs substantially from the mean value for the dynamical variable, these configurations will be very improbable in the unperturbed system. Nevertheless, these configurations can be accessed reversibly through a series of simulations that slowly change the control variable η.</p><p>By influencing the coarse-grained number of particles in the probe volume, the control variable η also influences the actual number of water molecules in that volume. We have picked a small value of the coarse graining length ξ to ensure that the latter influence is significant. As a result, with a series of simulations with different values of η, histograms for both the coarse-grained number and the actual number can be collected and then unbiased and stitched together within the framework of the weighted histogram analysis method (WHAM).42–44 This procedure yields the joint distribution function that the coarse grained particle number, Ñ({ri},v), has value Ñ and the actual particle number is N. The joint distribution, Pv(N, Ñ), can then be integrated to give the distribution of interest, Pv(N).45,46 With Pv(N) known, the free energy of solvation of a "hard" solute or cavity of volume v, Δµv, is also known because3,46 (6)βΔμv=−lnPv(0) where kBβ = 1/T is inverse temperature and kB is Boltzmann's constant. This formula holds irrespective of where the probe volume is placed, whether close to or far from a solute. The free energy of solvation depends, of course, on the location, shape and size of v, and we explore aspects of this dependence with the results reported below.</p><!><p>In Fig. 2, we show results for Pv(N) in different probe volumes in bulk water. Because Pv(N) is Gaussian for molecularly sized probe volumes,3 we present these results in comparison with Gaussians of the same mean, 〈N〉v, and the same variance, 〈(δN)2〉v. For small deviations from the mean, Pv(N) is essentially Gaussian, and for small volumes v, only small deviations from the mean N are possible. For large v, however, the wings of the distribution differ markedly from Gaussian for small N.</p><p>In pure water, the chance of observing these deviations is negligible, less than one part in many powers of ten. On the other hand, these deviations become accessible and even dominant near a sufficiently large and repellent solute particle. In particular, while the free energy to reduce N, namely −kBT ln [Pv(N)] is parabolic near the mean, it can vary linearly or sub-linearly with N in the wings of the distribution for a large enough volume v. See Fig. 2a for the case of a large cubic probe volume. The introduction of a perturbing potential, perhaps due to the presence of another solute, introduces a potential energy that scales linearly with N. If −kBT ln [Pv(N)] were parabolic for all N, the addition of such a potential energy would simply shift the parabola to a different mean. However, when −kBT ln [Pv(N)] varies linearly or sub-linearly with N in the wings of the distribution, a perturbing potential can favor low N to the point where low values become the most probable values. This type of shift in the distribution, which can occur only for large v, is responsible for many large length-scale hydrophobic effects.4</p><p>In the case of the large but thin rectangular volume considered in Fig. 2a, the wings of Pv(N) also exhibit deviations from Gaussian behavior.47 In this case, however, the distribution lies below the Gaussian. The differences between the distribution for the large cubic volume and that for the large thin volume reflects interfacial dominance of hydrophobic solvation in the large length-scale regime. Namely, the surface area of large thin volume is larger than that of the large cubic volume, and in the large length-scale regime, Δµv ≈ Av γ̃. Here, Av is the surface area52 of the volume v, and γ̃ is a free energy per unit area that depends weakly upon v. Figure 2b illustrates the accuracy of this approximation for the length scale regime considered. (The value of γ̃ is of the order of but smaller than the liquid-vapor surface tension, γ, as is expected for the size of volumes considered.49,51) Thus, the probabilities for emptying the large cubic and large thin probe volumes differ by about 25 orders of magnitude largely because of differing free energies of interface formation.</p><p>To elaborate, consider the solvation energy for the cavity v, relative to that of n independent smaller voids, say δv = 3 × 6 × 6 Å3: (7)ΔΔμv=Δμv−nΔμδv. Here, Δµδv denotes the solvation free energy for a single independent smaller void δv and the net volume v is composed of n such voids, i.e., n = v/δv. ΔΔµv is the free energy of hydrophobic assembly – the change in free energy as a result of assembling the cavity v from the n separated components. In the small length-scale regime, the net solvation energy would be nΔµδv, and this free energy would be a good approximation to the value of −kBT ln[Pnδv(0)], to the extent that Pnδv(N) is the Gaussian distribution centered at the mean value of N.53,54 As such, the extent to which Pv(N) deviates from the corresponding Gaussian, and the extent to which the resultant ΔΔµv is non-trivial, is the extent to which large length-scale effects are important.6 Figure 2a shows that these effects cause a favorable driving force to assemble the smaller voids into a cubic geometry, and they cause an unfavorable driving force to assemble the smaller voids into a thin geometry.</p><p>The fat tail of Pv(N) in the regime of small N, responsible for the favorable hydrophobic driving force of assembly, manifests the formation of a liquid-vapor interface. The existence of such tails are expected for large enough probe volumes in any liquid close to liquid-vapor phase coexistence,4,55 and they have been found in simulations of various models.56,57 Figure 2, however, provides the first demonstration of these tails in an atomistic model of bulk liquid water. Figure 2 also provides the first such demonstration that fat tails will disappear when a large volume is reshaped into a sufficiently constraining geometry.</p><!><p>Figure 3 shows normalized mean densities as a function of the distance, x, from the center of the idealized large flat hydrophobic solute (see Fig. 1). Several strengths of dispersive attractions between the solute and water are considered. See Eq. (1). For λ = 0, the mean density profile is sigmoidal, suggestive of a vapor-liquid interface. However, addition of a small amount of attraction results in a qualitatively different density profile. For λ = 0.4, there is a maximum in the density profile accompanied by layering. Further increasing the attractions leads to a more pronounced maximum and layering. This behavior is in accord with the qualitative predictions of LCW theory.10 Nevertheless, contrary to these predictions, it has been suggested that a layered density profile implies an absence of a liquid-vapor-like interface near an extended hydrophobic surface with dispersive attractions to water.11,58 Confusion on this point seems to reflect a singular focus on the mean density, but the mean by itself is not an obvious indicator of liquid-vapor-like interfaces. Interfaces are relatively soft so that a weak perturbation can affect the location of the interface and thus the mean density profile while not destroying the interface.57 In other words, in order to fully appreciate the effect that a hydrophobic solute has on the surrounding solvent, one should look at both the mean density and the density fluctuations.6</p><p>The statistics for these fluctuations can be obtained from the distribution of particle numbers in suitably chosen probe volumes. Figure 4a shows Pv(N) distributions for the thin rectangular probe volume v=(3 × 24 × 24) Å3 placed between x = 5Å and x = 8Å. With this position, there is no overlap between solute particles and water molecules in v, as inferred from their van der Waals' radii. The distributions for λ = 0 and λ = 0.4 are similar, with the probability of density depletion slightly lower for the latter case, but still significantly higher than that in the bulk.</p><p>The free energy to empty this probe volume adjacent to the large hydrophobic solute with λ = 0.4 is 57 kBT, whereas the free energy to empty this same v when it is in bulk and far from the hydrophobic surface is 147 kBT. See Fig. 4b. This large difference in free energies is due to interface formation. In Fig. 3, the presence of a liquid-vapor-like interface is evident in the mean density of solvent near the extended hydrophobic solute with λ = 0. Pv(0) for v adjacent to the solute is then essentially the probability to move the interface outwards by 3Å from x ≃ 5 Å to x ≃ 8 Å. The free energetic penalty, Δµv, for this process is 48kBT. See Fig. 4b. The corresponding Δµv for λ = 0.4 is 57kBT, only a 9kBT increase on turning the attractions on to λ = 0.4 and as much as 90kBT less than that required to form interfaces.</p><p>Hence, while the presence of a mean density maximum and layering at λ = 0.4 might lead one to question the presence of a liquid-vapor-like interface, the probabilities for fluctuations in density and the ease with which a volume near the hydrophobic surface can be vacated leaves no doubt as to its presence. Further, the presence of this interface is responsible for the hydrophobic force of assembly. In particular, because large solvent density fluctuations are more likely adjacent to a hydrophobic surface than in bulk, the free energy cost to reorganize solvent and thus solvate a cavity is significantly lower than that in bulk. Figure 4b shows that this effect is dominant until solute-solvent attractions are nearly 3 times the value of typical dispersive attractions between hydrophobic solutes and water.</p><!><p>Figure 5a shows Pv(N) for the probe volume v = (3 × 24 × 24) Å3 next to the hydrophobic solute (λ = 1), and compares it with that for the probe volume next to the hydrophilic solute. Near the hydrophilic solute, the probability is nearly identical to the Pv(N) for v in the bulk. On the other hand, near the hydrophobic solute, the tail in the probability and thus the probability of density depletion, is significantly higher than that in bulk.</p><p>Figure 5b shows the solvation free energy, Δµv, of the probe cavity as a function of the distance between the center of the solute and the center of the cavity volume. For the cavity v near the hydrophilic solute, this free energy is essentially equal to that for the cavity v in the bulk. On the other hand, Δµv for the cavity near the hydrophobic solute increases monotonically as the cavity is moved away from the solute and eventually plateaus at its bulk value. The variation of this free energy with respect distance from the solute shows that the considered hydrophobic surface affects density fluctuations in the water at a distance of up to ~ 10Å. This behavior is in agreement with recent simulation studies, reporting the free energy of solvating a molecularly sized WCA cavity near hydrophobic surfaces18 and also the potential of mean force for bringing two hydrophobic plates close together.34</p><!><p>With the results presented above, we have shown that: 1. For a typical large volume in pure water, Pv(N) exhibits fat tails at small N. These tails, never before demonstrated in an atomistic model of liquid water, manifest the formation of liquid-vapor interfaces. 2. For large volumes that do not exhibit these tails in bulk water, the solvation behavior is still governed by interfacial energetics, and Pv(N) does exhibit fat tails at small N when these volumes are placed adjacent to a hydrophobic surface. 3. These tails do not appear adjacent to hydrophilic surfaces. 4. These tails, reflecting relative softness of a liquid-vapor interface and enhanced probability of water depletion, imply that the free energy of a cavity adjacent to a hydrophobic surface is more favorable than that of a cavity in bulk.</p><p>These results bear directly on nano-scale assembly where two hydrophobic surfaces may approach each other, and at least one of these surfaces is large enough to induce the formation of the soft liquid-vapor-like interface. At a particular separation, the liquid between them will be sufficiently destabilized to make drying and hydrophobic assembly kinetically accessible. In contrast, density fluctuations near a hydrophilic surface are identical to those in the bulk and the vapor phase is not stabilized by the presence of a hydrophilic surface. Hydrophobic and hydrophilic surfaces thus differ fundamentally in the way they affect the fluctuations of water molecules in their proximity. It is not the mean density, but rather the statistics of fluctuations that is most important.</p><!><p>A typical probe volume, v = (3 × 24 × 24) Å3, is shown here adjacent to a hydrophobic surface (blue particles). The rendered water molecules (red and white) are in a typical equilibrium configuration taken from one of our simulations.</p><p>(a) Probability distribution of finding N water oxygens in a probe volume v in bulk water for a "small" cubic v = (6 × 6 × 6) Å3, a larger "cubic" v = (12 × 12 × 12) Å3 and a "thin" v = (3 × 24 × 24) Å3.47 The solid line refers to the Gaussian distribution with the same mean and variance; δN = N − 〈N〉v, where 〈N〉v = ρv is the mean number of oxygen centers in the probe volume v. (b) The solvation free energy, Δµv, in units of kBT, per unit surface area, Av, for probe cavities with different thicknesses and square cross-sections, but the same large volume [(12Å)3 = 1728Å3]. The dashed48 and the dotted49 lines indicate reported values of the the surface tension of SPC-E water.50,51 For both (a) and (b), statistical error estimates for our simulation results are smaller than the size of the symbols used.</p><p>Mean water density 〈ρ(x)〉, relative to its bulk liquid value, ρ, perpendicular to extended hydrophobic solutes with different strengths of solute-solvent attractions, λ.</p><p>(a) Pv(N) for probe volumes v = (3 × 24 × 24) Å3 adjacent to hydrophobic solutes with different attractive solute-solvent couplings, λ. (b) The corresponding solvation free energies for the empty volume v. The arrow shows the value of this free energy when the probe volume is placed in bulk rather than adjacent to the solute. The error bars on the last three points in (b) are standard deviation error estimates for the simulation results for the free energy. Error estimates for all other results shown in both (a) and (b) are smaller than the symbols used.</p><p>(a) Pv(N) for v = (3 × 24 × 24) Å3 in bulk, adjacent to the hydrophilic solute, and adjacent to the hydrophobic solute with solute-solvent attraction parameter λ = 1. (b) The change in solvation free energies of the probe volume v at different parallel positions from the solutes.</p>

PubMed Author Manuscript

Traceless Tandem Lesion Formation in DNA From a Nitrogen-Centered Purine Radical

Nitrogen-centered nucleoside radicals are commonly produced reactive intermediates in DNA exposed to \xce\xb3-radiolysis and oxidants, but their reactivity is not well understood. Examination of the reactivity of independently generated 2\xe2\x80\xb2-deoxyadenosin-N6-yl radical (dA\xe2\x80\xa2) reveals that it is an initiator of tandem lesions, an important form of DNA damage that is a hallmark of \xce\xb3-radiolysis. dA\xe2\x80\xa2 yields O2 dependent tandem lesions by abstracting a hydrogen atom from the C5-methyl group of a 5\xe2\x80\xb2-adjacent thymidine to form 5-(2\xe2\x80\xb2-deoxyuridinyl)methyl radical (T\xe2\x80\xa2). The subsequently formed thymidine peroxyl radical adds to the 5\xe2\x80\xb2-adjacent dG, ultimately producing a 5\xe2\x80\xb2-OxodGuo-fdU tandem lesion. Importantly, the initial hydrogen abstraction repairs dA\xe2\x80\xa2 to form dA. Thus, the involvement of dA\xe2\x80\xa2 in tandem lesion formation is traceless by product analysis. The tandem lesion structure, as well as the proposed mechanism, are supported by LC-MS/MS, isotopic labeling, chemical reactivity experiments, and independent generation of T\xe2\x80\xa2. Tandem lesion formation efficiency is dependent on the ease of ionization of the 5\xe2\x80\xb2-flanking sequence, and the yields are >27% in the 5\xe2\x80\xb2-d(GGGT) flanking sequence. The traceless involvement of dA\xe2\x80\xa2 in tandem lesion formation may be general for nitrogen centered radicals in nucleic acids, and presents a new pathway for forming a deleterious form of DNA damage.

traceless_tandem_lesion_formation_in_dna_from_a_nitrogen-centered_purine_radical

INTRODUCTION<!>Preliminary characterization and sequence dependence for tandem lesion formation from dA\xe2\x80\xa2<!>Structure determination of the major tandem lesion formed in 5\xe2\x80\xb2-d(GTA\xe2\x80\xa2) sequences<!>Mechanistic investigation of the formation of tandem lesion 12<!>CONCLUSIONS<!>Materials and methods<!>Oligonucleotide synthesis<!>Photolysis of oligonucleotides<!>Post-photolysis treatments<!>Enzymatic digestion of oligonucleotides to 2\xe2\x80\xb2-deoxynucleosides for UPLC analysis<!>UPLC-MS/MS analysis of oligonucleotides<!>

<p>DNA damage is deleterious to cells and can potentially lead to cell death or cancer. The cytotoxicity of DNA damage is exploited by cancer treatments such as ionizing radiation, which oxidizes DNA yielding modified DNA lesions.1,2 Advances in LC tandem mass spectrometry have provided insight on the structure of these DNA lesions and mechanism of their formation.3–5 In this product-based approach, the DNA lesions detected are considered to be the direct consequences of the corresponding nucleotides reacting with exogenous oxidizing agents. We recently reported tandem lesion formation, an important type of DNA damage produced by ionizing radiation, from a neutral purine radical, 2′-deoxyadenosin-N6-yl radical (dA•).6 dA• is produced via the direct effect of ionizing radiation via deprotonation of the radical cation (dA•+, Scheme 1). The indirect effect of ionizing radiation produces dA• via dehydration of the corresponding hydroxyl adduct, a process that is competitive with O2 trapping.7 Herein, we report the characterization and mechanism of formation of this tandem lesion. Interestingly, dA• is repaired during tandem lesion formation. Thus, the involvement of dA• is traceless by product analyses. The role of dA• in the chemical process is brought to light by its independent generation within chemically synthesized oligonucleotides, and reveals a possible general role of nucleobase nitrogen-centered radicals in DNA damage that was previously unrecognized.</p><p>Clustered lesions are defined as two or more damaged nucleotides within ~1.5-turns of duplex DNA.8–10 Although some antitumor agents (e.g. neocarzinostatin) produce clustered lesions, they are a hallmark of γ-radiolysis.11–13 Their formation by γ-radiolysis is significant because clustered lesions pose a more significant obstacle to base excision repair (BER) than isolated modified nucleotides, result in increased promutagenic events upon replication in cells, and are potential sources of double-strand breaks.14–16 Tandem lesions, which consist of contiguously damaged nucleotides, are a subset of clustered lesions that are unique to ionizing radiation.17 Some tandem lesions have been identified in cellular DNA following irradiation.18–20 In addition to posing a greater challenge to BER, tandem lesions can be more potent replication blocks and more highly mutagenic than the respective isolated lesions.18,20–22</p><p>Most tandem lesions have been attributed to pyrimidine nucleobase radicals,21,23–32 and have been investigated by product analysis. Independent generation of pyrimidine nucleobase radicals has provided a number of examples in which the radical and/or corresponding peroxyl radical adds to the adjacent nucleobases.23–25,32 Addition to the π-system is typically favored over hydrogen atom abstraction from the 2′-deoxyribose component of the 5′-adjacent nucleotide.27,28,33,34 Importantly, the involvement of pyrimidine radicals leads to formation of modified nucleotides at the site which the radical is generated. However, in one example, independent generation of a C3′-radical under aerobic conditions in single-stranded DNA resulted in C5′-hydrogen atom abstraction from the 3′-adjacent nucleotide by of the corresponding peroxyl radical.30</p><p>Neutral purine radicals are commonly observed in ionizing radiation, but there is a dearth of information concerning their role in tandem lesion formation. For instance, dA• produced by the direct and indirect effects of γ-radiolysis (Scheme 1).2,35 However, prior to our preliminary report on dA•, there was only one other example of a tandem lesion from a purine radical.29 Formation of dA• from 1 upon photolysis in 2 produced a tandem lesion in which damage at dG11 and T12 was detected by denaturing PAGE. That damage at these nucleotides was part of a tandem lesion, and arose from dependent chemical events, was based upon the variance of the relative amounts of cleavage depending on which terminus of the oligonucleotide was 32P-labeled (Figure 1). Cleavage was induced either by alkali (piperidine) treatment or incubation with the BER enzyme, formamidopyrimidine glycosylase (Fpg), which cleaves DNA at many purine lesions.6,36 8-Oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) was believed to be the major product at dG11, based upon its lability to Fpg and the ability of β-mercaptoethanol (BME) to prevent piperidine induced cleavage.37 However, the structure of the modification(s) at T12 and the final fate of dA• were undetermined. Structural and mechanistic studies on this novel tandem lesion, as well as the scope of its formation are described herein. The observations raise the possibility that traceless involvement (via product analysis) of nucleobase radicals in DNA damage induced by ionizing radiation may be a common process.</p><p> </p><!><p>Tandem lesions resulting from an independently generated reactive intermediate at a specific site within 32P-labeled DNA are readily detected by denaturing PAGE. Only the damaged nucleotide that is closest to the 32P-label in a particular molecule is detected (as a cleavage site) by denaturing PAGE. Hence, tandem lesion formation can be inferred by comparing the cleavage pattern in 5′-and 3′-32P-labeled substrates (Figure 1A). (The strand in which dA• is generated is radiolabeled in all experiments.) The ratio of cleavage detected at two positions within one DNA strand is independent of which terminus is labeled if the damage results from independent processes. However, the cleavage ratio at dG11 versus T12 in 2 is dependent on whether the 5′-or 3′-terminus is labeled (Figure 1B). This is indicative of a tandem lesion (Figure 1A).</p><p>Oxidative strand damage within a dGGG triplet is consistent with this sequence′s lowest ionization potential of any trinucleotide sequence.38 However, Fpg-induced strand damage at the corresponding 3′-terminal 2′-deoxyguanosine (dG12) in photolyzed 5′-32P-3 (2.9 ± 0.3 %, Figure S4) was ~10-fold less than in 5′-32P-2. Digestion of the photolysates of 2 and 3 suggests that the disparity in strand cleavage is not caused by the difference in the conversion of 1. The conversion of 1 is approximately 80% after 8 h photolysis in each duplex (Figure S6, and S7). The primary difference between duplexes 2 and 3 is the absence of a thymidine between dA• and the dGGG triplet in the latter. This suggests that despite the favorable thermodynamic driving force, dA• does not directly oxidize dG, such as by proton coupled electron transfer.39–42 This also indicates that the thymidine (T12) in 2 is critical for efficient damage at dG11, which is not adjacent to the original position at which dA• is produced.</p><p> </p><p>The spin density of dA• is mostly localized at N6,43 and conformational restrictions imposed by the right-handed helix were expected to have a significant effect on tandem lesion formation from dA• by controlling the juxtaposition of reactive species. Indeed, inverting the sequence from 5′-d(GGGT1) (2) to 5′-d(1T14G15GG) (4) results in no alkali-labile strand damage at T14 or dG15 upon photolysis in 4, and only a small amount of piperidine cleavage is observed at T12 (1.7 ± 0.1 %, Figure S8).6 This is attributed to the importance of the juxtaposition of reactive species.</p><p>Further illustration of the preference for a 5′-d(GT1) sequence is reflected by the absence of any alkali-labile damage at dA11 in 5′-32P-5. Damage is not detected at dA11 in 5, despite it being flanked on the 5′-side by a dGGG triplet. In addition, the alkali-induced cleavage at T12 in 5′-32P-5 (5.4 ± 0.2 %, Figure S10) is less than that observed at the corresponding thymidine in 5′-32P-2 (Figure 1B). The lack of tandem lesion formation in a 5′-d(AT1) sequence is attributed to the less favorable oxidation potential of dA compared to dG.41 Furthermore, the reduced damage at the thymidine that is 5′-adjacent to the site at which dA• is generated in 5 compared to 2, also suggests more distal sequence effects on the nucleotide′s reactivity.</p><p> </p><p>This possibility was examined further by comparing the strand damage detected in 2 with that in 6–8 (Figure 2). These substrates each contain a thymidine at T12, the nucleotide bonded to the 5′-phosphate of dA•. However, the oxidation potential of the sequence flanking the 5′-phosphate of T12 varies.38 Strand damage at dG11 and T12 in 5′-32P-8 was considerably lower than in 5′-32P-2 (Figure 2A), but analysis of 3′-32P-8 applying the aforementioned criteria indicates that damage at dG11 is attributable to tandem lesion formation (Figure S11 and S12). Comparing tandem lesion formation in a series of 3 substrates that differ by a single base at the position most remote from dA• in a 5-nucleotide span (5′-d(XGG11T121)) reveals a correlation with ionization potential (Figure 2B). The effect of the flanking sequence ionization potential is evident at T12 (Figure 2A) as well. Overall, these data show that the extended π-system affects the redox properties of DNA.</p><!><p>More detailed structural analysis was carried out using dodecamer 9 containing the 5′-d(GGGT1) sequence, and when possible the findings were corroborated using 5′-32P-2.</p><p> </p><p>Insight into the nature of the damaged nucleotide(s) at T12 was obtained by the observation that NaBH4 treatment of 5′-32P-2 photolyzate prior to piperidine cleavage practically eliminated strand scission at this position (0.4 ± 0.1 %, Figure S15). Reaction of photolyzed 5′-32P-9 (Tm = 35.8 °C) with aldehyde reactive probe (ARP) produced a product (8.1 ± 0.5 %, Figure S16) that migrated more slowly in a denaturing polyacrylamide gel. These observations are consistent with formation of a piperidine labile carbonyl-containing compound at the thymidine bonded to the 5′-phosphate of dA•. 5-Formyl-2′-deoxyuridine (fdU) was a good candidate for this product.44</p><p> </p><p>More definitive identification of the tandem lesion was obtained by LC-MS/MS analysis of photolyzed 9 (Figure 3). LC-MS/MS is a powerful method for identifying nucleic acid modifications. It is most frequently used to identify isolated lesions following enzyme digestion of the DNA. However, DNA lesion location is lost upon enzyme digestion. Tandem lesions have been identified in this manner as well by taking advantage of their resistance to the enzyme digestion conditions and/or covalent bonding between nucleotides. 3,4,21,23,26,32,45 Analysis of intact oligonucleotides using collision-induced dissociation (CID) enables identifying where a lesion is located, but is less common.46–48 We took advantage of the HFIP-TEA ion-pairing system, in conjunction with reverse-phase UPLC to provide product separation and reduced ion suppression to analyze the crude photolyzate of 9.49 The major product observed is that resulting from reduction of dA• (10), and is consistent with denaturing PAGE analysis of photolyzed 2. However, we also observed a product with m/z =3688.8921 that is consistent with the expected mass for the tandem lesion containing 5′-d(oxodGuo-fdU) and dA at the position where dA• (12) is generated (11, calculated m/z = 3688.6137) (Figure 3). Treatment of the photolysate with NaBH4 yielded a new product that was two mass units higher than 11 (m/z = 3690.9253, Figure S21). This is consistent with the presence of fdU. Finally, collision-induced dissociation of the ion corresponding to z = 3 of 11 (Figure 3, Table S1) was also consistent with the assigned structure.</p><p> </p><p>The LC-MS/MS and more inferential gel electrophoresis data all support 12 as the major tandem lesion upon generation of dA• flanked by 5′-d(GGGT). To our knowledge, this is the first example in which a nucleotide radical intermediate participates in tandem lesion formation but is ultimately repaired during the process. If one were to rely only upon product analysis of randomly damaged DNA, such as when the biopolymer is exposed to γ-radiolysis, the involvement of dA• would be traceless.</p><!><p>The formation of a tandem lesion composed of 8-oxodGuo and fdU suggested that dA• abstracts the hydrogen atom from the C5-methyl group of the 5′-adjacent thymidine (Scheme 2, step 1), and the resulting 5-(2′-deoxyuridinyl)methyl radical (T•) ultimately transfers damage to the 5′-adjacent dG. Based upon the approximate BDEs of the N6-H bond (~97 kcal/mol) in dA and the allylic C5-H bond (BDE ≤ 90 kcal/mol) of thymidine, this process should be exothermic.50,51 In addition, a molecular model suggests that the methyl group of the 5′-adjacent thymidine is approximately 2 Å closer than the corresponding hydrogen in the 3′-adjacent thymidine to the radical at N6 of dA• (Figure 4). Hence, hydrogen atom abstraction from the methyl group of thymidine would explain the directional preference for tandem lesion formation, as well as the requirement that dA• is repaired in 12.</p><p> </p><p>This possibility was explored by examining strand damage in duplexes in which the 5′-adjacent thymidine in 2 was substituted either by 5,6-dihydrothymidine (dHT, 13) or 2′-deoxyuridine (dU, 14). The C-H bond strength of C5-methyl group in dHT is higher than that in dT, and hydrogen atom abstraction from its methyl group by dA• should be disfavored. Since dHT is cleaved by piperidine or Fpg, photolyzates of 5′-32P-13 and 5′-32P-2 were treated with hOGG1, followed by NaOH to assay for damage at dG11 (Figure S23).52 Strand scission at dG11 was significantly reduced from 26.1 ± 0.2 % in 2 to 2.0 ± 0.6 % in the duplex containing dHT (13). The low level of strand damage that is still detected at dG11 in 5′-32P-13 could be attributed to hydrogen atom abstraction by dA• from the C5-methyl group in dHT, but this is uncertain. Replacement of the 5′-adjacent thymidine by dU has an even more definitive effect on tandem lesion. Strand damage at dG11 in 5′-32P-14 photolyzates is completely eliminated (Figure S23). Overall, these observations are consistent with the major pathway for tandem lesion formation from dA• involving initial C5-methyl hydrogen atom abstraction from the 5′-adjacent thymidine (Scheme 2, step 1).</p><p> </p><p>FdU formation and the O2 dependence of tandem lesion formation suggest that the corresponding peroxyl radical of T• (Tp•) oxidizes the 5′-adjacent dG (Scheme 2, steps 2, 3). The viability of this process was investigated by independently generating T• from 17 within DNA (15, 16).53–55 These duplexes were designed to replicate the environment in which T• was produced from dA• (above). Indeed, alkali-labile lesions were generated at dG11 in photolyzed 5′-32P-15 and 5′-32P-16 under aerobic conditions (Figure S24–26). Yields of alkali-labile lesion products were lower than when dA• was generated from 1 ( cleavages at G11 are 6.7 ± 0.7 % in 15 and 1.3 ± 0.2 % in 16). This is consistent with the generation of carbocation from 17 in addition to T•.55 The alkali-labile cleavage yield in the substrate containing the 5′-d(GGG) sequence (15) was considerably higher than in 16. This is consistent with the above experiments (Figure 2) in which the flanking sequence up to 4 nucleotides away affects dA• reactivity. Cleavage by Fpg, as well as the reduction in alkali-induced cleavage at dG11, when the photolyzate was treated with piperidine/BME was consistent with 8-oxodGuo formation at this site, as was observed when tandem lesion formation was initiated by dA•. In addition, the elimination of alkali-labile lesions at T12 following NaBH4 treatment is consistent with fdU formation at this position. Overall, independent generation of T• at T12 (15) within the otherwise identical sequence in which dA• is produced (2), supports that the T• generated by hydrogen atom abstraction is trapped by oxygen (Scheme 2, step 2), and that the resulting peroxyl radical (Tp•) oxidizes the 5′-adjacent dG (Scheme 2, step 3), ultimately resulting in a tandem lesion (12) consisting of 5′-oxodGuo-fdU.</p><p>Although peroxyl radicals have been suggested to oxidize dG via an outer sphere process,56 studies on related nucleobase peroxyl radicals indicate that addition to the purine ring is more likely.27 Outer sphere and inner sphere mechanisms are distinguishable by product analysis. The former involves formation of G•+ that gives rise to hole migration.57–59 Hole migration would result in preferential damage at the 5′-and middle-dG′s of a dGGG sequence. The absence of strand damage at dG9 and dG10 in 2 argue against this process.60 Furthermore, transformation of G•+ into 8-oxodGuo requires incorporation of oxygen from H2O.61 However, 18O-incorporation was not detected by LC-MS/MS following photolysis of 9 in H218O (Figure S27). These observations are also consistent with damage on dG11 arising by an inner sphere mechanism.</p><p>Inspection of molecular models suggested that Tp• was well positioned to add to C8 of the 5′-adjacent dG (18) (Figure 5). The second one electron oxidation can be achieved by addition of O2, followed by superoxide elimination (Scheme 2, step 4).62,63 We propose that 1,2-hydride migration (Scheme 2, step 5) in the carbocation (19), followed by deprotonation from 20 (Scheme 2, step 6) produces the tandem lesion (12).</p><p>The efficiency with which Tp• reacts with dG11 was explored by measuring the effect of BME on the yield of alkali-labile lesions at the purine position in photolyzed 5′-32P-2 (Figure 6). Assuming that the maximum rate constant for trapping T• by BME is 1 × 107 M−1s−1, even the highest concentration of BME (0.5 mM) employed would not compete with O2 (0.2 mM), which reacts with alkyl radicals at ~2 × 109 M−1s−1, for the alkyl radical.64 In addition, BME reacts inefficiently with dA•.36 Hence, we are confident that the reduction in cleavage at dG11 as a function of BME concentration is a reflection of the competition between the reaction of Tp• with the purine (kAdd) and the thiol (kRed). The amount of Tp• reduction product was calculated to be the difference in alkali-induced cleavage at dG11 in the absence of BME and the presence of a given thiol concentration. The slope of the line (4.8 × 103 M−1, Figure 6) is the ratio of rate constants (kRed / kAdd, Eqn. 1), and if we assume that kRed = 2 × 103 M−1s−1, then kAdd = 4.2 × 10−2 s−1.65 The estimated rate constant for addition of Tp• to the 5′-adjacent dG11 is slightly slower than that estimated for other nucleobase peroxyl radicals using competitive kinetics.27 The trapping of Tp• by BME is further corroborated by LC-MS (Figure S30, and 31). When 9 is photolyzed in the presence of 1 mM BME, the tandem lesion is replaced by a product with m/z =3674.8979, which is consistent with the reduction Tp• yielding 5-hydroxymethyl-2′-deoxyuridine (21, calculated m/z = 3674.6344).</p><!><p>Tandem lesions are an important type of DNA damage. 2′-Deoxyadenosin-N6-yl radical (dA•) is the first neutral purine nitrogen radical shown to unequivocally produce tandem lesions. The involvement of dA• in this process is only detectable because it is independently generated. To our knowledge, this is the first time that a radical has been shown to play a traceless role in tandem lesion formation.</p><p>The use of LC-MS/MS on intact oligonucleotides was also vitally important for elucidating the structure of the tandem lesion. Employing the more common approach of digesting the biopolymer would have provided significantly less information on tandem lesion (12) formation. In addition, utilizing relatively long hybridized oligonucleotides (25 bp) enabled us to examine this chemistry in duplex substrates, as well as to investigate the effects of extended sequences (e.g. 5′-d(NGG)) on the oxidation chemistry. Tandem lesions from dA• are formed relatively efficiently when flanked on the 5′-side by 5′-d(NGGT) sequences, which based upon statistics will appear >106 times in the human genome. Thus, this type of tandem lesion could be potentially relevant to human health.</p><p>The role of nitrogen-centered purine radicals in DNA damage has been overshadowed by other nucleoside reactive intermediates, carbon centered radicals and radical cations. Unlike radical cations, nitrogen-centered radicals do not react with water. Nitrogen radicals also do not react rapidly with O2, unlike carbon radicals. Consequently, other reaction pathways, including hydrogen atom abstraction by dA•, may be general for nitrogen-centered radicals in DNA. Considering the frequent formation of neutral purine radicals (Scheme 1), their heretofore minor role in DNA damage is surprising.29 The experiments described here raise the possibility that such radicals may play important, general roles in the formation of tandem lesions that are produced by γ-radiolysis.</p><!><p>All solvents were distilled before use. Dichloromethane, DIPEA, DMF and pyridine were distilled from CaH2. THF is distilled from sodium. T4 polynucleotide kinase (T4 PNK), human 8-oxoguanine DNA N-glycosylase 1 (hOGG1) and terminal transferase were obtained from New England Biolabs. DNA Degradase Plus was obtained from Zymo Research. γ-32P-ATP and α-32P-cordycepin 5'-triphosphate were purchased from Perkin Elmer. C18-Sep-Pak cartridges were obtained from Waters. 5,6-Dihydrothymidine phosphoramidite was synthesized as described in the literature.66 PBS buffer (0.1 M NaCl, 10 mM sodium phosphate, pH 7.2) and water were treated with Chelex® 100 resin (Bio-Rad). Oligonucleotides were synthesized on an Applied Biosystems Incorporated 394 oligonucleotide synthesizer. Oligonucleotide synthesis reagents were purchased from Glen Research (Sterling, VA). Commercially available fast deprotecting phosphoramidites were used for DNA synthesis of oligonucleotides containing 1. ESI-MS was carried out on a Thermoquest LCQDeca. UPLC-MS analyses were carried out on Waters Acquity/Xevo-G2 UPLC-MS system equipped with a ACQUITY UPLC HSS T3 Column (100 Å, 1.8 μm, 2.1 mm × 100 mm) or Oligonucleotide BEH C18 Column (130Å, 1.7 μm, 2.1 mm × 100 mm). Oligonucleotide masses were obtained via deconvolution using MassLynx 4.1 software. CID was processed using Microsoft Excel. MALDI-TOF analyses were carried out on a Bruker AutoFlex III Maldi-TOF. Quantification of radiolabeled oligonucleotides was carried out using a Molecular Dynamics Phosphorimager 860 equipped with ImageQuant Version TL software.</p><!><p>A 5 min coupling time was used for the previously reported modified phosphoramidite.6 Deprotection of synthesized oligonucleotides containing 1 was performed with concentrated aqueous ammonia for 16 h at room temperature, followed by concentration under reduced pressure. The oligonucleotides were purified using 20% denaturing PAGE. Oligonucleotides containing 17 were synthesized, deprotected, and purified according to previously reported procedures.67 Oligonucleotides were eluted from polyacrylamide gel and desalted using C18-Sep-Pak cartridges.</p><!><p>The strand (1 μM) containing the radical precursor was labeled at the 5' end with γ-32P-ATP using T4 PNK in T4 PNK buffer (70 mM Tris-HCl, pH 7.6, 10 mM MgCl2, 5 mM DTT, 45 min, 37 °C). The labeled strand was hybridized to the complementary strand (1.5 eq.) in PBS by heating at 90°C for 1 min and slowly cooling to room temperature. The hybridized duplexes were diluted to 0.1 μM in PBS before photolysis. All photolyses were carried out at 25 °C in Pyrex using a Rayonet photoreactor equipped with 16 lamps having a maximum output at 350 nm. All anaerobic photolyses were carried out in sealed Pyrex tubes, which were degassed by freeze-pump-thaw degassing (3 cycles). Samples were sealed while under vacuum at 77 K. Photolyses of DNA containing 1 and 17 were carried out for 8 h and 20 min respectively.</p><!><p>Aliquots from photolyzed solutions or unphotolyzed controls were treated with piperidine (1 M, 30 min, 90 °C), Fpg (1.25 μM, 1 μL, 10 mM Bis Tris-Propane HCl (pH 7), 10 mM MgCl2, 1 mM DTT, 100 μg/mL BSA, 1 h, 37 °C), or Ir4+ (0.1 mM of Na2IrCl6•6H2O) for 1 h at 25 °C, quenched (2 mM Hepes, 10 mM EDTA, pH 7), and treated with piperidine (1 M, 30 min, 90 °C). Alternatively, photolyzates were treated with in the presence of 0.25 M BME, NaBH4 (100 mM, 1 h, 4 °C) followed by piperidine (1 M, 30 min, 90 °C), or hOGG1 (8 units, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT, 100 μg/mL BSA) for 1 h at 37 °C, followed by NaOH (1 M, 30 min, 37 °C). NaOH treated samples were neutralized with HCl (1 equiv.). All samples treated with enzymes were precipitated (0.3 M NaOAc, pH 5.2, 0.1 g/mL calf thymus DNA) with ethanol. Piperidine treated samples were evaporated to dryness under vacuum, and washed with 2 × 10 μL water, which was also removed under vacuum. Samples were analyzed by dissolving in formamide loading buffer prior to analyzing by 20% denaturing PAGE. BME, piperidine, NaBH4, and Ir4+ solutions were prepared fresh on the day of the experiment.</p><!><p>Duplexes 2, 3, and the corresponding photolyzates (4 pmol) in 1.6 μL H2O, 10 × DNA degradase plus® buffer (5 μL) and dU (2.5 μL, 80 μM) as internal standard were incubated with DNA degradase plus® (2 μL, 5 U/μL) for 4 h at 37 °C. The reaction mixture was filtered through a nanosep® 3K filter by centrifuging the mixture for 5–10 min at 16000 × g. The filter was washed once with 25 μL H2O and the combined filtrate (50 μL) was analyzed using an Agilent 1290 infinity UPLC equipped with the ACQUITY UPLC HSS T3 Column (A, 10 mM ammonium formate; B, acetonitrile; 5% B from t = 0 to t = 2 min; 5–80% B linearly over 7.5 min; 80% B from t = 9.5 to t = 12.5 min; 80–5% B linearly over 1 min; 5% B from t = 13 to t = 16 min; flow rate, 0.3 mL/min.)</p><!><p>Photolysates (8 μL) containing 5 μM of duplex dodecamer were analyzed by UPLC-MS using the Oligonucleotide BEH C18 Column (A, 100 mM HFIP and 8.6 mM TEA; B, Methanol; 2% B from t = 0 to t = 5 min; 2–9% B linearly over 3 min; 9% B from t = 8 to t = 20 min; 9–30% B linearly over 5 min; 30% B from t = 25 to t = 30 min; 30–2% B linearly over 5 min; 2% B from t = 35 to t = 40 min; flow rate, 0.2 mL/min.). The column temperature was 60 °C. The collision energy was set to ramp from 10 to 45 V.</p><!><p> Supporting Information </p><p>Representative autoradiograms, expanded LC-MS/MS data analysis, MS characterization of oligonucleotides containing modified nucleotides. The Supporting Information is available free of charge on the ACS Publications website.</p>

PubMed Author Manuscript

Differential susceptibility of transgenic mice expressing human surfactant protein B genetic variants to Pseudomonas aeruginosa induced pneumonia

Surfactant protein B (SP-B) is essential for lung function. Previous studies have indicated that a SP-B 1580C/T polymorphism (SNP rs1130866) was associated with lung diseases including pneumonia. The SNP causes an altered N-linked glycosylation modification at Asn129 of proSP-B, e.g. the C allele with this glycosylation site but not in the T allele. This study aimed to generate humanized SP-B transgenic mice carrying either SP-B C or T allele without a mouse SP-B background and then examine functional susceptibility to bacterial pneumonia in vivo. A total of 18 transgenic mouse founders were generated by the DNA microinjection method. These founders were back-crossed with SP-B KO mice to eliminate mouse SP-B background. Four founder lines expressing similar SP-B levels to human lung were chosen for further investigation. After intratracheal infection with 50\xce\xbcl of P. aeruginosa solution (1\xc3\x97107 CFU/mouse) or saline in SP-B-C, SP-B-T mice the mice were sacrificed 24 hours post-infection and tissues were harvested. Analysis of surfactant activity revealed differential susceptibility between SP-B-C and SP-B-T mice to bacterial infection, e.g. higher minimum surface tension in infected SP-B-C versus infected SP-B-T mice. These results demonstrate for the first time that human SP-B C allele is more susceptible to bacterial pneumonia than SP-B T allele in vivo.

differential_susceptibility_of_transgenic_mice_expressing_human_surfactant_protein_b_genetic_variant

1. Introduction<!>2.1. Mice<!>2.2. Constructs<!>2.3. Generation of hTG SP-B mice<!>2.4. Western blotting analysis<!>2.5. Analysis of histology and immunohistochemistry (IHC)<!>2.6. Pseudomonas aeruginosa infection<!>2.7. Electron microscopy analysis<!>2.8. Surfactant large and small aggregates<!>2.9. Analysis of Surfactant activity<!>2.10. Statistical analysis<!>3.1. Generation of hTG SP-B-C and SP-B-T mice<!>3.2. Expression of transgene SP-B-T or SP-B-C in hTG mice<!>3.3. Bacterial pneumonia<!>3.4. Differential surfactant activity between SP-B-C and SP-B-T in response to bacterial infection<!>Discussion

<p>Surfactant protein B (SP-B), a hydrophobic protein, is essential for normal lung function, which is expressed by alveolar type II epithelial cells in the lung. SP-B protein is critical for the formation of the pulmonary surfactant film at the surface of alveoli that lowers the surface tension and prevents the collapse of alveoli in the lung [1]. Human SP-B (hSP-B) is encoded by sftpb gene (approximately 9.5 kb containing 11 exons) on chromosome 2 [2,3]. The mature SP-B product is an 8 kDa protein (79 residues) which is derived from a SP-B precursor (pro-SP-B) by a complex protein processing pathway [4,5].</p><p>hSP-B genetic variation is associated with various lung diseases, like respiratory distress syndrome in pre-term neonates (RDS), congenital alveolar protein deposition disease (CAP), bronchopulmonary dysplasia (BPD) [6], and the interstitial lung disease [7]. Critical mutations of hSP-B gene result in SP-B deficiency which is lethal for newborn infants [8]. For example, a two-base-insertion in codon 121 of hSP-B cDNA causes SP-B deficiency and neonatal alveolar proteinosis [9] and a 1-bp deletion (1553delT) in exon 4 causes a reading frame shift and the premature translational termination in exon 6. Infants carrying these homozygous mutants died shortly after birth because of a lack of mature SP-B protein [10].</p><p>A common SP-B single nucleotide polymorphism, SP-B 1580 C/T (SNP, rs11130866), causes a change in the amino acid residue from Threonine (Thr) for the C allele to Isoleucine (Ile) for the T allele at position 131 of SP-B precursor. This altered residue located at a glycosylation recognition sequence results in the C allele containing a glycosylation modification at Asn129 which is not present in the T allele [11]. Patients-based genotyping studies demonstrate that this SP-B SNP (rs11130866 C/T) is associated with several pulmonary diseases including pneumonia [12] and pneumonia-induced ARDS [13], but its effects on functional susceptibility to pulmonary pathogens induced pneumonia have never been tested. We hypothesized that the change of glycosylation status at the Asn129 in SP-B precursor has an impact on SP-B processing and physiological/pathophysiological function.</p><p>In the current study, to test our hypotheses we generated humanized SP-B transgenic (hTG) mice carrying either SP-B C or T allele but without a mouse SP-B (mSP-B) gene background, and then examine the functional susceptibility of SP-B variants on surfactant activity in response to P. aeruginosa pneumonia in the hTG SP-B-C and SP-B-T mice.</p><!><p>Wild type (WT) FVB/N mice used in the present study were purchased from the Jackson laboratory and maintained in the animal core facility at SUNY Upstate Medical University. The hSP-B transgenic mice carrying either hSP-B C or T allele without a mouse SP-B gene background were generated in this study. Mice were housed in pathogen-free conditions and the animal protocols (IACUC# 236 and 380) in this study were approved by Institutional Animal Care and Use Committee at SUNY Upstate Medical University, they also meet the National Institutes of Health and ARRIVE guidelines on the use of laboratory animals.</p><!><p>A 5.4-kb DNA fragment (Fig. 1) used for DNA microinjection was excised from a recombinant plasmid by restriction enzymes Nde I and Not I. The DNA fragment consisted of a human SP-C promoter (3.7-kb), a human SP-B cDNA (1.3-kb), and a SV40 small t-intron poly (A) sequence (0.4-kb). The basic 3.7-hSP-C/SV40 vector was kindly provided by Drs. Jeffrey A. Whitsett and Stephan W. Glasser (Cincinnati Children's Research Foundation, Cincinnati, OH) [14]. The recombinant DNA processes were performed using standard methods of molecular cloning. The cDNA of human SP-B was cloned into a basic 3.7-hSP-C/SV40 vector and recombinant construct was verified by DNA sequencing. A 5.4 kb DNA fragment was microinjected into fertilized FVB/N oocytes from WT mice.</p><!><p>Human SP-B positive transgenic founders carrying either hSP-B C or T allele (hSP-B+, mSP-B +/+) were bred with conditional SP-B KO mice (mSP-B −/−) for several generations to eliminate mSP-B gene background. Then homozygous hTG SP-B-C and SP-B-T mice (hSP-B +/+, mSP-B −/−) were generated by the self-breeding of hemizygous mice (hSP-B +/−, mSP-B −/−). All mice were genotyped using DNA from tail samples by PCR genotyping with Primer pair 1458/189 for hSP-B and primer pair 75/76 for mSP-B. To verify hSP-B in hTG mice, primer pair 1458/1401 was used to amplify the whole fragment of hSP-B gene and the PCR products were analyzed by DNA sequencing.</p><!><p>To examine the expression of hSP-B protein level in hTG SP-B mice, Bronchoalveolar lavage fluid (BALF) from hTG mice was analyzed by Western blotting with anti-SP-B antibody (Hycult Biotech, Plymouth Meeting, PA) at 1:200 dilutions as previously described [15,16].</p><!><p>Mouse lungs from hTG mice (8 to 12 weeks) were fixed by 10% formalin solution at about 25cm of water pressure for at least 24 hr, and then processed into paraffin blocks. The sections of lung tissue were approximately 5μm in thickness. Lung sections were stained with hematoxylin and eosin, or used for IHC analysis using the ABC kit (Vector Laboratories, Burlingame, CA) as described previously [15,16].</p><!><p>hTG SP-B-C and SP-B-T mice were infected by intratracheal inoculation of 50μl of P. aeruginosa PA01 (1×107 CFU/mouse) or sterile saline (sham control). Mice were sacrificed 24 hrs post infection. Tissues were harvested and prepared as described previously [15,16].</p><!><p>Lung tissues were prepared as previously described [17]. The samples were fixed by 4% glutaraldehyde, 2.5% paraformaldehyde for 24 h, then stained with osmium tetroxide, 1.5% potassium ferrocyanide and embedded in Embed812 resin (Electron microscopy science). The tissues were cut into ultrathin sections (90 nm) and stained with lead citrate and 2% aqueous uranyl acetate before electron microscopy analysis. Each sample was examined at the magnification varied from ×10, 000 to ×40, 000, and at least 100 fields were examined from several sections.</p><!><p>Mouse lungs were lavaged for 3 times each with 0.7 ml saline solution. BALFs from 6 mice of each group were prepared and centrifuged at 150xg, 4°C for 10 min. and the supernatant was then centrifuged for 15 min at 40,000xg, 4°C. After centrifugation, the pellet containing surfactant large aggregates was resuspended in 0.3 ml of saline for surface tension study as previous description [18,19]. The phospholipid concentration of the large aggregates was determined using phosphate assays to be around 1 mg/mL.</p><!><p>Surface activity of the mouse surfactants was determined with a constrained drop surfactometer (CDS; BioSurface Instruments, HI) [20]. A droplet of the mouse surfactant of ~10 μL was dispensed onto the CDS drop holder. After the equilibrium surface tension was established by rapid adsorption, the surfactant film was compressed and expanded at a rate of 3 seconds per cycle with a compression ratio controlled to be less than 40% of the initial surface area. At least five compression-expansion cycles were studied for each droplet. Cycles were quantified with the minimum surface tension (γmin) at the end of compression and the maximum surface tension (γmin) at the end of expansion. Drop images were taken at 10 frames per second. The surface tension and surface area were determined with Axisymmetric Drop Shape Analysis (ADSA) [21].</p><!><p>All the experiments were repeated at least 3 times in this study. Western blot bands were quantified by software Quantity One (version 4.6.1). Statistics were performed by SigmaStat version 3.5 software. Significant difference in statistics among groups was considered when p<0.05 by t-test or ANOVA.</p><!><p>To generate hTG SP-B-C and SP-B-T mice, the cDNA of hSP-B gene was cloned into a basic 3.7-hSP-C/SV40 vector (Fig. 1A) and A 5.4 kb DNA fragment was microinjected into fertilized FVB/N oocytes from WT mice. A total of 18 hSP-B positive founders were identified by PCR. To eliminate the mSP-B background, the conditional SP-B knockout (KO) mice by inserting Neomycin resistant gene into the gene (Fig. 1B) were used to breed with hSP-B positive founders. The hSP-B positive F1 were bred with conditional SP-B KO mice to generate F2 hSP-B positive mice (hSP-B +/mSP-B −/−) (Fig. 1C). Finally, four homozygous hTG SP-B-C and SP-B-T mouse lines, e.g. (hSP-B +/+, mSP-B −/−), were obtained by the self-breeding of hemizygous mice (hSP-B +/−, mSP-B −/−) and showed healthy status suggesting that hSP-B protein functions well in the transgenic mice.</p><!><p>The histology of lung in the hTG SP-B-c and SP-B-T mice from four founders was assessed using H/E staining sections (Fig. 2A). The results demonstrated hTG SP-B-C founder line and SP-B-T founder lines displayed normal alveolar structure and septal thickness. The chord length (mean linear intercepts: Lm) as a measure of the acinar air space complex was measured, no significant difference was found between hTG SP-B-C and SP-B-T mice. Furthermore, hSP-B expression was analyzed in the lung by IHC method (Fig. 2B) and western blotting (data shown in 3.4). The results indicated that only alveolar type II epithelial cells showed positive staining for SP-B expression in the lung tissue of both hTG SP-B-T and SP-B-C mice (Fig. 2B).</p><!><p>To examine the functional variation of hSP-B genetic variants under infectious conditions, a bacterial pneumonia model was developed and applied for the assessment of functional effects of hSP-B genetic variants in vivo. hTG mice were infected intratracheally by 50μl of P. aeruginosa PA01 (107 CFU/mouse) or same amount of saline solution (Sham). The infected mice were sick but survival for 24 hrs. The lung pathohistology displayed pneumonia pathogenic characteristics and lung injury in the infected mice but not in Sham control (Fig. 3A). A large amount of neutrophils were observed in the BALF of infected mice but not in the control. Ultrastructural analysis revealed significant decrease of lamellar bodies in the type II cells and remarkable less microvilli on the surface of type II cells in infected mice compared to Sham (Fig. 3B, p<0.05), suggesting bacterial infection decreased activation of type II cells and surfactant/protein expression.</p><!><p>Surfactant activity of large aggregates of BALF from infected and sham mice was analyzed by the Constrained Drop Surfactometer (CDS) [18,19,20]. The results indicated that uninfected hSP-B-T and hSP-B-C mice (sham) had similar surface activity, i.e., minimum surface tension (γmin) about 1.8–2.2 (mN/m) (Fig. 4A). After infection, the minimum surface tension in infected SP-B-C mice increased significantly by two times compared to uninfected control (Fig. 4A, p<0.05), suggesting that surfactant activation has been inhibited in the infected SP-B-C mice. But the minimum surface tension of infected SP-B-T mice (hSP-B-T) showed less change compared to uninfected SP-B-T mice (Fig. 4A). These data indicate that the SP-B-T mice had less susceptibility on surfactant activity to bacterial infection when compared to SP-B-C mice (p<0.05) (Fig. 4A). Analysis of Western blotting demonstrated decreased SP-B levels of both infected SP-B-C and SP-B-T mice compared to their uninfected mice (Fig. 4B, p<0.01). Infected SP-B-C mice had lower SP-B tendency vs. infected SP-B-T mice.</p><!><p>Pulmonary surfactant consists of about 90% phospholipids and 10% surfactant-associated proteins, including SP-B, an essential protein for normal lung function [1]. The SP-B precursor is about 42 kDa peptide containing three sapsin-like protein domains, e.g. domain N, M, and C. Mature 8-kDa SP-B protein is derived from sapsin-like domain M, which plays a key role in lowering surface tension in the alveolar space [22]. Decreased levels of mature SP-B significantly influence lung function and oxygenation [23] and cause disease exacerbation [24]. The SP-B SNP (rs11130866 C/T) was identified to be associated with several pulmonary diseases including pneumonia [12], in which the individuals carrying the C allele of SP-B are more susceptible than those with the T allele [13]. However, the detailed mechanisms are unclear. The humanized SP-B transgenic mouse model provides a powerful in vivo tool for studying the mechanistic role of human SP-B genetic variants in the pathogenesis of pulmonary diseases [25].</p><p>Previous studies found that the production of mature SP-B protein is completed by complex SP-B precursor processing and trafficking [26]. SP-B was secreted to alveoli or stored in the lamellar bodies in the alveolar type II cells of lung [26]. Several proteases are involved in the protein processing, including protease Napsin and Cathepsin H [26]. Differentially posttranslational modifications of SP-B precursor caused by genetic variation may have an impact on the efficiency of SP-B processing and trafficking in the type II cells. The C allele of human SP-B has an additional protein glycosylation site at the residue Asn129 compared to the T allele [11,27]. SP-B C and T alleles are two common genetic alleles in human population and no obviously abnormal SP-B expression was observed in the healthy individuals with either the C or T allele, indicating the additional Asn129 glycosylational modification in SP-B precursor may not significantly influence physiological function under healthy conditions. The similar levels of the SP-B expression in the hTG SP-B-C and SP-B-T mice in this study also confirmed the observation in human beings. However, it is unknown whether the additional glycosylation site at the As129 in the SP-B-C variant has an negative impact on pro-SP-B processing and trafficking under disease conditions, like bacterial pneumonia.</p><p>Previous studies demonstrate that there are decreased expressions of SP-B after a variety of infectious conditions [28]. In the present study we observed a significant decrease of SP-B level in the BALF of infected mice, and SP-B level in infected SP-B-C mice was lower than infected SP-B-T mice, suggesting that altered glycosylation modification at the Asn129 in the SP-B-C mice influenced SP-B processing and trafficking under bacterial pneumonia. Although decreased levels of other surfactant proteins (SP-A, SP-C and SP-D) in the BALF were also observed in the infected mice compared to uninfected mice (data not shown), but no differences of these protein expressions were detected in infected SP-B-C and SP-B-T mice in the infectious condition. Furthermore, ultrastructural analysis of the lung tissues indicated lower density of LB in type II cells suggesting decreased activation of alveolar type II cells in infected mice. These pathological changes in the cellular and molecular levels in the lung of infected SP-B-C and SP-B-T mice might cause lung dysfunction in mouse pneumonia.</p><p>The SNP (rs11130866 C/T) studied in the present work has been found to be associated with several pulmonary diseases in several independent groups [13] [7,12]. For instance, individual with C/C genotype was found more susceptible to RDS [29] while individual with T/T genotype was found to protect the patients with SSC against the development of ILD[7]. The individuals carrying one or more C allele had more severe lung injury and required MV compared to those with T/T genotype [30]. These patients-based genotyping analyses demonstrate that the C allele of SP-B is more susceptible to various pulmonary diseases. This hTG model may provide a unique tool to study the mechanisms of human SP-B genetic variants on functional variation in various pulmonary diseases. Indeed, the results observed in this work revealed differential susceptibility of human SP-B genetic variants in response to bacterial pneumonia. Decreased surfactant activity of BALF in the lung might lead to worse respiratory function and disease severity in pneumonia. However, it is warrant to explore detailed mechanisms how the altered glycosylational modification at the Asn129 influence pro-SP-B processing, folding and trafficking in the alveolar type II cells in vivo in various diseases in the future.</p>

PubMed Author Manuscript

Quantifying the Contribution of Grape Hexoses to Wine Volatiles by High-Precision [U13C]-Glucose Tracer Studies

Many fermentation volatiles important to wine aroma potentially arise from yeast metabolism of hexose sugars, but assessing the relative importance of these pathways is challenging due to high endogenous hexose substrate concentrations. To overcome this problem, gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) was used to measure high-precision 13C/12C isotope ratios of volatiles in wines produced from juices spiked with tracer levels (0.01\xe2\x80\x931 APE) of uniformly labeled [U-13C]-glucose. The contribution of hexose to individual volatiles was determined from the degree of 13C enrichment. As expected, straight-chain fatty acids and their corresponding ethyl esters were derived almost exclusively from hexoses. Most fusel alcohols and their acetate esters were also majority hexose-derived, indicating the importance of anabolic pathways for their formation. Only two compounds were not derived primarily from hexoses (hexanol and isobutyric acid). This approach can be extended to other food systems or substrates for studying precursor\xe2\x80\x93product relationships.

quantifying_the_contribution_of_grape_hexoses_to_wine_volatiles_by_high-precision_[u13c]-glucose_tra

INTRODUCTION<!>Fermentations<!>Sample Preparation<!>Quantification and Identification of Volatiles by GC-MS<!>Carbon Isotope Ratio Analysis of Wine Volatiles by GC-C-IRMS<!>Bulk Analysis of Grape Must Carbon Isotope Ratio by Elemental Analyzer-IRMS (EA-IRMS)<!>Statistical Analysis<!>Isotope Addition and APE Calculations<!>Calculating the Percentage of Carbon Derived from Hexose in Volatile Compounds<!>Volatile Composition of Fermentations<!>Native 13C/12 C Ratios<!>Determination of Hexose Contribution to Volatiles through Tracer Experiments<!>Recommended Enrichment Levels for GC-C-IRMS Studies of Fermentations<!>

<p>Hundreds of volatile compounds have been identified in wine, of which about 70 appear to be critical for wine aroma.1 A small subset of these key aroma compounds are so-called "primary" odorants that are detectable in the grape and extracted during fermentation, for example, rotundone ("black pepper" in Syrah) and methyl anthranilate ("grapey/foxy" in Concord).2,3 However, many important wine aroma compounds are "secondary"; that is, they appear during fermentation either due to de novo biosynthesis by yeast or bacteria or through metabolism of grape-derived precursor compounds such as glycoconjugates.4 "Tertiary" odorants may also arise during storage through several mechanisms, including acid-catalyzed transformations, oak contact, or microbial spoilage.</p><p>Identifying precursors or formation mechanisms for wine components during or after fermentation is of interest to wine researchers, as this knowledge can lead to improved strategies for predicting the concentrations of various compounds in finished wines. Frequently, this is accomplished by addition of a putative precursor to an appropriate media. For example, limonene and α-terpeniol were added to model wines to evaluate the potential of these compounds to serve as precursors for 1,8-cineole ("eucalyptus" odor).5 In another experiment, a glycoconjugate-enriched extract was added to a model juice medium prior to fermentation to quantify the contribution of yeast metabolic activity to the release of volatile aglycones.6 Such approaches are appropriate for tracking must precursors at low initial concentrations, but are less useful for compounds with high endogenous concentrations (e.g., sugars, amino acids) where additions large enough to effect measurable changes in volatiles create the risk of altering microbial physiology. For example, the fusel alcohols (fermentation-derived alcohols with more than two carbons, e.g., isoamyl alcohol) and their corresponding acetate esters are important wine odorants, as the alcohols are associated with off-aromas at high concentrations and the acetate esters are credited with enhancing fruity aromas common to young wines.7 Many of the fusel alcohols are thought to be formed during alcoholic fermentation by one of two pathways related to amino acid metabolism: (i) catabolism of grape amino acids, characterized by deamination of amino acids to α-keto acids followed by decarboxylation to an aldehyde and, finally, reduction to a fusel alcohol (the Ehrlich pathway);8 or (ii) production of α-keto acids during amino acid biosynthesis from sugars, which may then be degraded to fusel alcohols as described above (the anabolic pathway.)7 Changes in must nitrogen composition can alter fusel alcohol production,9–11 but it is not always evident if changes in the Ehrlich or anabolic pathways, or both, are responsible for this outcome. To complicate matters further, some fusel alcohols (e.g., β-phenylethanol) exist as glycoconjugates in grapes and can serve as additional sources of these compounds in wine.6</p><p>Stable isotope tracers can be used as an alternative to adding unlabeled substrates in the evaluation of precursor–product relationships. Tracer studies have been widely used in food chemistry, for example, in studies of precursors of Maillard reaction products,12 and have occasionally been extended to studies of the fate and origin of wine components. In one work, deuterated isobutyric acid and ethyl isobutyrate spikes were used to study their formation in wine.13 However, to yield detectable changes using a typical GC-MS system, stable isotope tracers must be added at a concentration of at least >0.5% (and generally higher) of the endogenous concentration,14 which may be prohibitively expensive and, as mentioned above, create the risk of affecting fermentation physiology. Radioisotopes can be employed at lower concentrations due to their low natural abundance, with labeled compounds typically detected following off-line separation via a scintillation counter. In wine, [3H]-malvidin glucoside has been used as a model to track the fate of anthocyanins before and after fermentation,15 and [U-14C]-glucose, -leucine, and -isoleucine have been used to follow volatile production in grain mash fermentations.16 In practice, radioisotopes are rarely used as tracers in food chemistry studies because of associated hazards.</p><p>An alternative to conventional tracer experiments with GCMS is high-precision isotope ratio measurement by gas chromatography combustion isotope ratio mass spectroscopy (GC-C-IRMS). In GC-C-IRMS, organic compounds are combusted to CO2 following their elution from a GC, and the isotopomers of CO2 (m/z 44, 45, 46) are measured by IRMS for each peak.14 GC-C-IRMS has been used for natural abundance studies of wine, primarily for studies on authenticity and adulteration. For example, 13C/12C ratios can be used to detect illegal addition of cane sugar (derived from a C4 plant) to grape juice (derived from a C3 plant) prior to fermentation and, in combination with D/H and 18O/16O ratios, can serve as geographical fingerprints for wine provenance.17 The major advantage of GC-C-IRMS for tracer studies is that it can achieve much higher precison than conventional GC-MS; in practice, 13C enrichments as low as ~0.1 atom percent excess (APE) can be utilized for tracer studies.18 GC-C-IRMS has been used for tracer studies in a number of disciplines where a sizable endogenous pool of a potential precursor exists and large degrees of enrichment are not possible for economic or other practical reasons.14 For example, GC-C-IRMS has been used to monitor the fate and metabolism of dietary omega-3 fatty acids in animal studies.19 GC-C-IRMS has also been used in conjunction with 13CO2 labeling studies of chamber-grown grapevines to determine the timing of biosynthesis of glycoconjugate precursors,20 but to our knowledge GC-C-IRMS has not been used for tracer studies of wine fermentations or in related areas of food processing.</p><p>In this work, we report the use of GC-C-IRMS as a novel approach to follow the path of a stable isotope tracer during the alcoholic fermentation of hexose. Uniformly labeled [U-13C]-glucose was added to a grape must prior to fermentation at trace levels (0.01, 0.1, and 1 APE), and its contribution to a diverse range of fermentation volatiles (i.e., fusel alcohols, fatty acids, and their associated esters) was assessed. This technique can be used as a more general approach for studying the origin and fate of compounds in food systems while minimizing tracer concentration.</p><!><p>Fermentations were conducted using Riesling juice sourced from the Cornell research vineyards in Lansing, NY, USA, during the 2010 harvest. Fruit (450 kg) was received by the Cornell Vinification and Brewing Technology Laboratory, where it was crushed, destemmed, basket pressed, and settled overnight at ambient conditions. Approximately 30 L was siphoned into two 20 L Nalgene containers (Thermo Fisher Scientific, Waltham, MA, USA) and frozen at −20 °C. Before use, the juice was thawed in a 40 °C water bath for 1 h and mixed by inversion. Glucose, fructose, and yeast assimilable nitrogen (YAN) were quantified by enzymatic colorimetric methods using a Chemwell 2910 Multianalyzer (Unitech Scientific, Hawaiian Gardens, CA, USA). To supplement the initial juice YAN content of 40 mg N/L, diammonium hydrogen phosphate (674 mg/L) and Fermaid K (Lallemand, Santa Rosa, CA, USA) (250 mg/L) were added to yield a final YAN concentration of 214 mg N/L. Yeast (EC1118, Lallemand) was rehydrated in 40 °C spring water (Crystal Rock, Watertown, CT, USA) with 40 g/L GoFerm (Scott Laboratories, Petaluma, CA, USA), and the juice (5 L) was inoculated at a rate of 0.3 g/L. Aliquots of 541 g (500 mL) of inoculated must were then added to 1 L Pyrex media bottles (Corning Inc., Tewksbury, MA, USA). One treatment served as a control (no [U-13C]-glucose added), and uniformly labeled [U-13C]-glucose (Cambridge Isotope Laboratories, Andover, MA, USA) was added at one of three levels: 1.0 APE (1.0150 g/500 mL), 0.1 APE (0.1015 g/500 mL), or 0.01 APE (0.0102 g/500 mL). Fermentations were carried out in triplicate for each of the four treatments (three tracer levels + one control). The fermenters were topped with a three-piece airlock with a floating bubbler (Buon Vino Manufacturing, Cambridge, ON, Canada). Fermentations were conducted over a 10 day period at ambient temperatures in a dark cabinet and were measured for residual sugar after all fermenters had stopped producing CO2. At completion, all fermentations had residual sugar (glucose, fructose) values of <0.5 g/ L. The wine was stabilized by the addition of 50 mg/L of SO2 and then frozen and stored at −20 °C until needed for further analysis.</p><!><p>Volatile components were extracted from wine using the method described by Ortega and others.21 To a 15 mL screw-capped borosilicate glass centrifuge tube were added 4.5 g of (NH4)2SO4, 3 mL of wine, 7 mL of water, and 0.2 mL of dichloromethane (DCM). For GC-MS quantification 15 μL of a 140 μg/mL solution of 2-octanol was added to the sample as in internal sample; this step was omitted for the GC-C-IRMS analysis. The samples were mixed for 1 h using a carousel rotating at 20 rpm to invert the tubes and then centrifuged at 2500g for 10 min. Approximately 1.5 mL of the emulsified bottom layer was recovered with a glass Pasteur pipet, transferred to a 2.2 mL microfuge tube, and centrifuged for 4 min at 14000g. Finally, 100 μL of the DCM layer was transferred to an Agilent GC autosampler vial with a 250 μL microvolume insert.</p><!><p>Compounds in the DCM extracts were identified and quantified by gas chromatography–mass spectrometry (GC-MS). An HP 6890 GC with a split/splitless inlet (Agilent Technologies, Palo Alto, CA, USA) and autoinjector was coupled to an Agilent 5795 quadrupole mass selective detector (MSD). A 60 m × 0.32 mm × 0.25 μm Aglient HP-INNOWax column (polyethylene glycol) was used. The GC conditions were as follows: initial head pressure at 8.57 psi with a column flow of 1.6 mL/min in constant flow mode, inlet at 250 °C, 2 μL splitless injection, and total flow through the injector of 84.8 mL/ min. The oven parameters were as follows: initial temperature 35 °C (held for 2 min) ramped to 40 °C at 5 °C/min (held 5 for min), ramped to 200 °C at 3 °C/min (held for 5 min), and ramped to 250 °C at 25 °C/min (held for 20 min). The MSD was operated with an ionization energy of 70 eV with the electron multiplier voltage of 1700 V. The MSD was scanned over the mass range of m/z 33–300. Chemstation MSD (Agilent Technologies) was used for data processing. Compound identification was performed by matching retention times and mass spectra of peaks to authentic standards, and peak areas were normalized to that of the 2-octanol internal standard. Calibration curves were generated from the analysis of known concentrations of pure standards spiked into a model wine. Calibration curves for all 26 compounds had an r2 > 0.99. Quantifier ions and retention times for each compound are listed in Table 1.</p><p>Due to its coelution with the extraction solvent DCM, ethanol was quantified separately via a direct injection method.22 Wine was filtered through a 0.25 μm filter and 100 μL diluted with 900 μL of water; 1 μL of this sample was then injected at a 50:1 split into a 250 °C inlet. The oven parameters were as follows: initial 35 °C (held for 2 min) ramped to 40 °C at 5 °C/min (held for 5 min), ramped to 70 °C at 5 °C/min (held for 5 min), and ramped to 250 °C at 25 °C/min (held for 7 min).</p><!><p>The design and operation of the GC-C-IRMS system are described in more detail elsewhere.23 Briefly, the HP 6890 GC-MS described above was coupled to a Thermo MAT 253 IRMS (Bremen, Germany) via a specially designed combustion interface. The IRMS was tuned for high linearity with the conduction limit open three full turns and operated at a source pressure of 9.3 × 10−7 Torr and an accelerating potential of 9.5 kV. The measured absolute sensitivity was ~1600 molecules/ion. Data were collected and analyzed using ISODAT 3.0 (Thermo Scientific). The GC column was connected to an online microcombustion reactor via a four-way rotary valve, which permitted solvent diversion to the MSD and sample introduction into IRMS. The microcombustion reactor was constructed from a continuous fused silica capillary (50 cm × 0.53 mm i.d.) hand packed with three 15 cm × 0.1 mm wires (one Cu, one Pt, and one Ni wire) in the center of the capillary. The capillary portion containing the wires was mechanically retained in a 30 cm × 0.8 mm i.d. alumina tube with metal fittings (Valco Instruments, Houston, TX, USA) on both ends. The exposed ends of the capillary were connected to the interface with press-tight fittings. The alumina tube was maintained at 950 °C using a 30 cm Thermcraft tube furnace (Winston-Salem, NC, USA), which had a 15 cm heated zone. Water generated due to combustion was removed from the system using a Nafion water trap (dimensions = 10 cm × 0.8 mm i.d.) immediately following the combustion reactor. The open-split consisted of the 0.6 m × 0.075 mm capillary connected to the IRMS inlet at one end, with the other end directly inserted into the postwater trap transfer line. For analysis of wine volatiles, excluding ethanol, a 60 m × 0.32 mm × 0.25 μm Agilent HP-INNOWax column described above was used. The GC conditions were as follows: initial head pressure at 8.57 psi with a column flow of 1.6 mL/min in constant flow mode, inlet at 250 °C, 2 μL splitless injection, and total flow through the injector of 84.8 mL/min. The oven parameters were as follows: initial 35 °C (held for 2 min) ramped to 40 °C at 5 °C/min (held for 5 min), ramped to 200 °C at 3 °C/min (held for 5 min), and ramped to 250 °C at 25 °C/min (held for 20 min).</p><p>Because of ethanol's coelution with the extraction solvent, DCM, it was characterized by GC-C-IRMS separately from the other measured compounds. Sample preparation, injection technique, and GC conditions were the same as described for GC-MS.</p><p>ISODAT 3.0 was used to calculate isotope ratios in delta notation; 17O corrections were made using the Santrock and Hayes method.24 Background was determined using the individual background determination with a 5 s moving linear regression, whereas peak detection was set to a slope of 0.5 mV/s for peak start and 0.4 mV/s for peak end. For calibration, CO2 gas pulses were admitted directly to the IRMS source from a Conflo III device (Thermo Scientific). CO2 was supplied by a pressurized tank during each GC-C-IRMS run for isotope ratio standardization, with three 30 s pulses at the beginning and three at the end of each chromatogram. The isotope ratio of the CO2 standard gas was traceable to the international standard Vienna Pee Dee Belemnite (VPDB) (RVPDB = 0.011180) by running calibration standards containing fatty acid methyl ester (FAME) 17:0 and FAME 21:0 with known isotope ratios determined by off-line dual-inlet IRMS.25 This FAME standard was run at the beginning and end of each day's runs to determine the "apparent" isotope ratio of the CO2 gas pulses, which in turn could be used to calculate the isotope ratios of wine components during analytical runs. This calibration approach addresses fractionation in the GC-C-IRMS system because the FAME standards go through the same path as the wine components (i.e., GC, online combustion, water trap, open split, IRMS).25 Isotope ratios are reported as δ13CVPDB in units of "per mil" (‰) and based on the International System of Units (SI), where δ13CVPDB is defined as (1)δCVPDB13=RX−RVPDBRVPDB Rx and RVPDB represent the 13C/12C isotope ratio of an analyte compound X and the VPDB standard, respectively.</p><!><p>A 2 mL aliquot of the frozen Riesling juice (no [U-13C]-glucose added) was submitted to the Cornell Stable Isotope Laboratory (COIL) for EA-IRMS analysis. Five microliters of juice taken from a 2 mL frozen aliquot was used for bulk stable isotope analysis using a continuous flow system.26 The sample was combusted at 900 °C in a Carlo Erba NC2500 elemental analyzer, and resultant CO2 was transferred via a Conflo IV interface (Thermo Scientific) to a Delta V isotope ratio mass spectrometer (Thermo Scientific) for carbon isotope analysis. Isotope ratios are reported in delta notation, as described above.</p><!><p>All linear regression analysis and ANOVA calculations were carried out using MiniTab (Minitab, Reading, MA, USA) statistical analysis software.</p><!><p>APE is defined in eq 2 (2)APE=100(FHEXe−FHEXn) where FHEXe is the atom fraction of the enriched hexose and FHEXn is the atom fraction of the native hexose pool. FHEXe is calculated by the amount of [U-13C]-glucose added to the system. The mass balance equation (eq 3) can be rearranged to eq 4, where FULG is the atom fraction of uniformly labeled glucose and mULG is the mass. (3)FHEXemHEXe=FULGmULG+FHEXnmHEXn(4)FHEXe=FULGmULGmHEXe+FHEXnmHEXnmHEXe For tracer level additions of [U-13C]-glucose, the atom fraction of uniformly labeled glucose (FULG) = 1, the mass fraction (mHEXn/mHEXe) will be ~1, and eq 4 simplifies to (5)FHEXe=mULGmHEXe+FHEXn Combining eqs 2 and 5 yields eq 6 and provides a good approximation for the APE of the enriched hexose pool (APEHEX) as a function of the amount of [U-13C]-glucose added to the fermentation and the original hexose concentration. (6)APEHEX=100(mULGmHEXe)</p><!><p>The observed APE of each volatile (APEobsd) was calculated from experimental data for each volatile in each tracer fermentation, where RE is the 13C/12C ratio in the enriched sample and RN is the mean 13C/12C ratio of the corresponding compound in the natural abundance fermentations:14 (7)APEobsd=RE−RN1+(RE−RN) To determine the percent of each volatile compound that originated from hexose substrate, (APEobsd) was assumed to be related to the APE of the hexose pool (APEhex) by eq 8. (8)APEobsd=α(%from hexose)×(APEHEX) APEhex is a factor of the tracer level (eq 6). The apparent fractionation factor, α, can arise from kinetic isotope effects and can potentially vary among compounds. However, in most biochemical processes, α is expected to range from between 0.98 and 1.02, and assuming α = 1 will introduce negligible error. Equation 8 can be simplified and rearranged to solve for the percent of carbon in a given volatile derived from hexose sugars (eq 9). (9)%fom hexose=APEobsdAPEHEX</p><!><p>A summary of the average concentration of volatile compounds by level of enrichment is given in Table 1. Mean concentrations of each compound were within the range of values reported previously in the analysis of wine samples.21 Individual ANOVAs were calculated for each of the 28 compounds by level of enrichment. To account for potential type 1 error due to the number of comparisons, the Bonferroni correction was applied using a family error rate of α = 0.05. Whereas most compounds did not show significant differences, ethyl lactate, ethyl decanoate, and decanoic acid show significant differences between the control fermentation and the enriched fermentations. These differences may have arisen because the control fermentation was conducted at a different time from the enriched fermentations, rather than as an effect of enrichment treatment. In particular, production of midchain fatty acids such as decanoic acid and their corresponding ethyl esters is known to be affected by variation in oxygen status during fermentation.27 However, for all other compounds, the lack of difference indicates that labeling does not perturb the system.</p><!><p>Table 2 reports the native isotope ratios of 19 volatile compounds in the experimental Riesling wine, expressed as δ13C values. Literature reports on compound-specific isotope ratios of wine volatiles other than ethanol are relatively rare,28 and to our knowledge our work represents the largest number reported to date. Significant differences were observed in natural abundance isotope ratios among volatile compounds, and δ13C values ranged from −13 to −35‰. Fewer compounds were characterized by GC-CIRMS than were quantified by GC-MS because the combustion step of GC-C-IRMS converts all compounds to CO2; thus, coeluting peaks cannot be resolved as they can be in GC-MS through use of a unique m/z ion to quantify each compound.14 Precision, calculated as the standard deviation (SD) of δ13C values (‰) from fermentation replicates, ranged from ±0.1‰ (ethanol) to ±7.5‰ (hexyl acetate), with a mean value of ±1.9‰. This precision is lower than that for benchmark values for high-precision GC-C-IRMS, SD (δ13C) < 0.4‰,26 because it reflects biological variability among the fermentation replicates rather than analytical variability alone. We observed much better precision, SD (δ13C) < 1.4‰, for analytical replicates of the same fermentation samples. The higher precision observed for ethanol likely reflects the fact that it is the major fermentation product throughout alcoholic fermentation, and, thus, should be less prone to fractionation. Furthermore, the sample preparation did not require extraction. By comparison, many of the other volatiles in Table 2 account for <0.1% of the initial hexose substrate and thus may be more sensitive to changes in conditions among fermentation replicates.</p><p>The isotope ratio of ethanol (δ13C = −27.47‰) was within the range typically observed for wine29 and was depleted with respect to the isotope ratio for the bulk grape juice (δ13C = −26.57‰). The depletion of ethanol and concurrent enrichment of CO2 with respect to the sugar substrate has been previously reported and is believed to occur because ethanol is derived from the C-1, C-2, C-5, and C-6 positions of hexoses, which are depleted with respect to the C-3 and C-4 positions.30 Several short- and mid-chain fatty acids (acetic, isobutyric, and decanoic) were enriched with respect to ethanol, as has been previously reported.28 These volatile fatty acids are produced by yeast toward the end of fermentation.31 Because lighter isotopes of sugars are metabolized preferentially during fermentation,32 the observed effect may be due to enrichment of the hexose pool by the time the majority of these fatty acids are formed. However, as mentioned under Materials and Methods, this fractionation effect is small and constant and thus will not compromise results from labeling studies. Most other volatiles, including fusel alcohols, ethyl esters, and several acetate esters, did not differ significantly in isotope ratio from ethanol. The most depleted compound measured was 1-hexanol (δ13C = −34.19‰). As discussed in the next section, this is likely because hexanol is primarily derived from the 13C-depleted grape lipid fraction29 as opposed to being synthesized by yeast from sugars. Phenylethyl acetate was also significantly depleted with respect to ethanol, although the reason for this was unclear.</p><!><p>Typical chromatograms obtained at different levels of [U-13C]-glucose enrichment are shown in Figure 1. With increasing enrichment of the hexose pool the m/z 45 trace increased relative to the m/z 44 trace for compounds such as ethyl lactate that derive most of their carbon from sugar. The 45/44 ratio for compounds that are derived primarily from grape compounds other than sugars, such as hexanol, did not change significantly. APEobsd of volatile compounds in each fermentation experiment were calculated using eq 7, and the percent of carbon derived from the hexose pool was calculated by plotting APEobsd versus APEhex (eq 9). Representative plots are shown in Figure 2 for octanoic acid, ethyl octanoate, ethanol, β-phenylethanol, and 1-hexanol, where the slope represents the percent of carbon within a compound derived from the hexose pool. Figure 3 and Supplementary Table 1 in the Supporting Information report the fraction of carbon derived from hexose in each volatile compound. The error associated with measurements of percent hexose in individual compounds follows a similar pattern to what was observed in natural abundance studies.16 The contribution of the hexose pool to ethanol, which is present in large quantities and represents a major fermentation product, can be determined with high confidence (94 ± 0.5%), whereas the error associated with low-concentration volatiles such as ethyl 3-hydroxybutyrate increases by an order of magnitude or greater.</p><p>The contribution of hexoses to ethanol (94%) was somewhat surprising, as it indicated that a small amount of non-hexose substrate contributed to ethanol formation. Excluding the possibility of impure standards or experimental error, one likely explanation is that fermentable sugars other than hexoses contributed to ethanol. In particular, sucrose is reported to be present in musts at concentrations of 2–10 g/kg,33 or up to 5% of the hexose concentration in our work. Unfortunately, sucrose was not measured, and it was not possible to evaluate this hypothesis.</p><p>Like ethanol, nearly all wine volatiles measured in our studies were derived primarily from hexoses (Figure 3 and Supporting Information Supplementary Table 1). Notably, fusel alcohols associated with amino acid metabolism (e.g., methionol, β-phenylethanol, isoamyl alcohol, isobutyl alcohol) were >75% hexose derived. The acetate esters (isoamyl acetate, phenylethyl acetate) are formed by acetylation of fusel alcohols and were also derived primarily from hexoses. This indicates that the major contributor to these fusel alcohols is the anabolic pathway, in which carbon skeletons are synthesized de novo from hexoses,7 as opposed to catabolism of amino acids via the Ehrlich pathway.8 A third potential source for β-phenylethanol is from juice in either a free or glycosylated form, although, based on data from a previous study, this could account for only 3% of the total β-phenylethanol observed.34 To our knowledge, this is the first time that the relative contributions of different pathways to fusel alcohols in a wine fermentation have been evaluated. Whereas the values observed here are likely dependent on fermentation conditions such as yeast strain, our observation that the anabolic pathway is dominant is consistent with the amounts of fusel alcohols produced and the typical concentrations of amino acids in must available for Ehrlich degradation. For example, we observed ca. 100 mg/L of isoamyl alcohol in wine, whereas typical leucine concentrations in juice are reported to be <25 mg/L.35 Although glucose labeling studies have not, to our knowledge, been previously performed on wine fermentations, [U-14C]-glucose has been used to track production of fusel alcohols in grain mash fermentations.16 In the earlier work, 25–45% of fusel alcohols were derived from glucose (no replicates), substantially less than in our current study. The discrepancy may be because the mash fermentation was performed at lower gravity, which would have decreased the relative importance of the anabolic pathway once amino acid sources were exhausted.</p><p>The high contribution (>90%) of the hexose pool to straight-chain fatty acids (hexanoic, octanoic, and decanoic) and their corresponding ethyl esters (ethyl hexanoate and ethyl octanoate) was as expected; these compounds are known to be produced by yeast fatty acid metabolism and, in the case of ethyl esters, through subsequent esterification with ethanol.7 Interestingly, two of the fatty acids (octanoic and hexanoic) and ethyl 3-hydroxybutyrate had apparent hexose contributions slightly but significantly greater than 100% (103−121%). Because these semipolar compounds demonstrated slight tailing and because GC-C-IRMS peak tails are 13C depleted, hexose contributions of more than 100% may be an analytical artifact. Unlike the straight-chain fatty acids, only about half of the branched-chain isobutyric acid was derived from the hexose pool (Supporting Information Supplementary Table 1). Branched-chain fatty acids have been shown to decrease with supplementation of must with ammonia salts,36 suggesting that they may arise from catabolism of amino acids.37 This result may indicate that a significant portion of isobutyric acid arises from valine catabolism with the fermentation conditions employed.</p><p>Only one compound, 1-hexanol, was derived primarily from non-hexose sources. Although hexanol is generally present at concentrations of ≥1 mg/L in wine, alcoholic fermentation of juice-like media in the absence of non-sugar-grape derived compounds results in no detectable hexanol.6 Hexanol is detectable in grape must and can also be produced during fermentation by the reduction of C6 aldehydes and unsaturated C6 alcohols.38 These C6 compounds are largely derived from enzymatic oxidation of unsaturated fatty acids during grape crushing.39 Unsaturated fatty acids, along with other lipids, are known to be 13C-depleted in plants,40 a result which correlates with our previous observation that native hexanol is depleted as compared to other wine volatiles (Table 2). Hexyl acetate, formed by enzymatic esterification of hexanol,41 is enriched to the level predicted by the mass balance of acetic acid and hexanol (Table 2).</p><!><p>The precision of GC-C-IRMS depends not only on the precision of the analytical instrumentation but also on the concentration of the compound of interest, the 13C enrichment level, and the degree of biological variability. The fermentations were enriched with 0.01, 0.1, and 1.0% [U-13C]-glucose, corresponding to roughly +11, +110, and +1100‰ maximal possible enrichments in volatiles, respectively. The average SD values for the three enrichment treatments were 1.7, 6.4, and 53.4‰ across increasing enrichment treatments. Defining the limit of detection as 3 times the noise, the smallest contribution of hexose to a volatile compound would be APEobsd = 46, 17, and 15 for the 0.01, 0.1, and 1.0 APE treatments, although this will vary among compounds. Because of the marginal improvements associated with using 1.0 APE and the high costs of [U-13C]-glucose, using 0.1 APE provides the best balance of cost and variability. For 500 mL fermentations containing 100 g of hexose per 500 mL of must, generating 0.1 APE requires 0.1 g of [U-13C]-glucose, or $15 per fermentation at current prices of $150 per gram of [U-13C]-glucose. Lower tracer levels may be possible, but will require limiting variability across replicates.</p><p>In conclusion, this study is the first evaluation of a method utilizing GC-C-IRMS to trace the origin of volatile compounds during the fermentation of grape juice into wine by enriching the hexose pool with 13C. Under the conditions studied, the majority of fermentation volatiles studied, including most esters, fatty acids, and alcohols, derive at least 50% of their carbon from hexoses. Hexanol was the only compound observed to be majority (>90%) derived from grape compounds other than sugars. Using 0.1 APE of [U-13C]-glucose provides the optimal balance between precision and cost. In future studies, the effects of varying physiological conditions, such as temperature, soluble solids concentration, and nutrient status, on the relative importance of different pathways could be quantified by this approach, assuming adequate analytical performance. This method could also be applied to other complex food systems, such as Maillard reactions, to clarify the contributions of different pathways while minimizing costs.</p><!><p>ASSOCIATED CONTENT</p><p>Supporting Information</p><p>Supplementary Table 1. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>The authors declare no competing financial interest.</p><p>IRMS chromatograms of wine extract at different levels of 13C enrichment: (A) control; (B) 0.01% [U-13C]-glucose; (C) 0.1% [U-13C]-glucose; (D) 1.0% [U-13C]-glucose. The m/z 44 and 45 signals represent CO2 ions containing 12C and 13C, respectively, from aroma compounds after separation and combustion. The top trace of each chromatogram depicts the 45/44 ratio. Peak identification: 1, hexyl acetate; 2, ethyl lactate; 3, hexanol; 4, ethyl octanoate; 5, acetic acid.</p><p>Linear regression of observed APE versus hexose APE. Shaded areas represent the 95% confidence interval.</p><p>Contribution of hexose-derived carbon to each compound. The mean is indicated by the circles, and the error bars represent the 95% confidence interval.</p><p>Volatile Composition of Wines, in Milligrams per Liter, Produced by Fermentations with Different Levels of [U-13C]-Glucose Enrichmenta</p><p>Letters within a row indicate significant difference using the Bonferroni family error rate α = 0.05; if no letters are present, then no significant differences were observed for that compound.</p><p>RT = retention time in minutes.</p><p>Qion = quantifier ion by GC-MS.</p><p>Measured using direct injection method.</p><p>δ13C Values in Natural Abundance Control Fermentationsa</p><p>Results are for means and standard deviations of fermentation replicates.</p><p>Compounds with different letters differ at p < 0.05 level (Tukey's test).</p>

PubMed Author Manuscript

Evolution of a Strategy for the Enantioselective Total Synthesis of (+)-Psiguadial B

(+)-Psiguadial B is a diformyl phloroglucinol meroterpenoid that exhibits anti-proliferative activity against the HepG2 human hepatoma cancer cell line. This full account details the evolution of a strategy that culminated in the first enantioselective total synthesis of (+)-psiguadial B. A key feature of the synthesis is the construction of the trans-cyclobutane motif by a Wolff rearrangement with in situ catalytic, asymmetric trapping of the ketene. An investigation of the substrate scope of this method to prepare enantioenriched 8-aminoquinolinamides is disclosed. Three routes toward (+)-psiguadial B were evaluated that featured the following key steps: 1) an ortho-quinone methide hetero\xe2\x80\x93Diels\xe2\x80\x93Alder cycloaddition to prepare the chroman framework; 2) a Prins cyclization to form the bridging bicyclo[4.3.1]decane system, and 3) a modified Norrish\xe2\x80\x93Yang cyclization to generate the chroman. Ultimately, the successful strategy employed a ring-closing metathesis to form the seven-membered ring and an intramolecular O-arylation reaction to complete the polycyclic framework of the natural product.

evolution_of_a_strategy_for_the_enantioselective_total_synthesis_of_(+)-psiguadial_b

INTRODUCTION<!>DESIGN PLAN: FIRST GENERATION STRATEGY<!>SECOND GENERATION STRATEGY<!>THIRD GENERATION STRATEGY<!>CONCLUSION<!>General Procedures<!>Preparation of diazoketone 20:21,65<!>(38)<!>(39)<!>Small-scale screening protocol for enantioenriched amides 18, 42\xe2\x80\x9345<!>Large-scale protocol for enantioenriched amide 18:65<!>(42)<!>(43)<!>(44)<!>(45)<!>(50)<!>(49)<!>(51)<!>(17)<!>(55)<!>(16)<!>(57\xe2\x80\x9360, thermal reaction)<!>Data for 57 (peak 2)<!>Data for 58 (peak 1)<!>Data for 59 (peak 3)<!>Data for 60 (peak 4)<!>(57\xe2\x80\x9360, Cu-mediated reaction)<!>(62)<!>(63)<!>(64\xe2\x80\x9365)<!>Data for 65<!>Data for 64<!>(66\xe2\x80\x9367)<!>Data for 66<!>Data for 67<!>(78)<!>(70)<!>(79)<!>(68)<!>(80)<!>(+)-psiguadial B (3):76

<p>Plant extracts used in traditional folk medicine have long served as rich sources of structurally complex, bioactive compounds. For example, the bark, leaves, and fruit of the Psidium guajava plant are known for their medicinal properties, and have been used to treat ailments such as diabetes and hypertension.1 Efforts to isolate and characterize the bioactive constituents have identified a variety of diformyl phloroglucinol meroterpenoids with interesting structures,2 including 1 and 2 (Figure 1), which inhibit phosphodiesterase-4 (PDE4D2), a drug target for inflammatory and respiratory diseases.3 In 2010, Shao and coworkers reported the discovery of four new meroterpenoids, psiguadials A–D (3–6),4,5 which exhibit potent cytotoxicity against the HepG2 human hepatoma cancer cell line (IC50 = 46–128 nM). The most potent antiproliferative agent in this family, (+)-psiguadial B (3), is unique from a structural standpoint in that it possesses a strained bicyclo[4.3.1]decane terpene core, fused to a trans-cyclobutane ring.</p><p>Biosynthetically, this motif is proposed to arise via a mixed terpene-polyketide pathway.5 Intramolecular cyclization of farnesyl pyrophosphate (7) generates humulyl cation 8,6 which undergoes stereoselective ring closure guided by caryophyllene synthase to produce β-caryophyllene (9, Scheme 1). Michael reaction of 9 with ortho-quinone methide (o-QM) 10—likely derived from the known P. guajava metabolite 3,5-dimethyl-2,4,6-trihydroxybenzophenone7—is proposed to afford tertiary carbocation 11,8 which can cyclize to give (−)-guajadial (1)1g and (+)-psidial A (12),9 isomeric natural products that have also been isolated from P. guajava. Alternatively, carbocation 11 can isomerize through proton transfer processes to form tertiary carbocation 13,5 which can undergo transannular ring closure to generate bridgehead cation 14. Finally, this species can be trapped by the pendant phenol to furnish (+)-psiguadial B (3). Cramer8,10 and Lee11 have validated this biosynthetic hypothesis by semi-syntheses of 3, 1, and 11, from β-caryophyllene (9).</p><p>While semi-synthetic approaches to phloroglucinol meroterpenoids provide direct access to β-caryophyllene-derived natural products, we viewed an abiotic approach to 3 as an opportunity to develop new chemistry and strategy concepts that could be applicable in broader synthetic contexts. Here, we describe a full account of our efforts to develop an enantioselective total synthesis of (+)-psiguadial B (3),12,13 which was enabled by an asymmetric Wolff-rearrangement to construct the trans-fused cyclobutane.</p><!><p>As disclosed in our prior communication,12 the construction of the central bicyclo[4.3.1]decane, which is trans-fused to a cyclobutane, was recognized as the primary synthetic challenge posed by 3. Closer analysis identified the C1−C2 bond (Figure 2), which links the A and C rings through vicinal stereogenic centers, as a strategic disconnection. On the basis of this analysis, we were interested in forming this bond by a Pd-catalyzed C(sp3)−H alkenylation reaction between cyclobutane 18 and vinyl iodide 19.</p><p>Having identified a tactic to join the A and C rings, a retrosynthesis of 3 was conceived in which the 7-membered B ring would be generated via a late-stage intramolecular Prins cyclization, thus allowing simplification of 3 to 15. Although the ring closure to form this strained system was expected to be challenging, the Prins reaction has been previously used for the preparation of bridging polycycles.14 Tricycle 15 could be assembled through a bioinspired ortho-quinone methide hetero-Diels–Alder (o-QMHDA) reaction between enol ether 17 and an o-QM generated from 16.15 Although o-QMHDA reactions are widely used to construct chroman frameworks, simple acyclic enol ethers or styrenes are typically employed as dienophiles, and are used in excess to avoid o-QM dimerization.16 In contrast, the proposed strategy necessitates use of a functionalized cyclic enol ether, ideally as the limiting reagent. At the outset of these studies, we were unaware of any reported examples in which cyclohexanone-derived enol ethers were employed as dienophiles in o-QMHDA cycloadditions; thus, the proposed studies could potentially contribute a new substrate class for o-QMHDA reactions.17 Based on stereochemical analysis of reported o-QMHDA cycloadditions, we anticipated that the reaction would favor the desired anti-relationship between C1′ and C9, however, whether the stereochemistry of 17 would impart the desired facial selectivity in the approach of the heterodiene was less clear.15,18</p><p>Enol ether 17 was envisioned to be accessible in short order from the product of the directed C(sp3)−H alkenylation reaction, joining fragments 18 and 19. Although the direct product of this reaction would be a cis-cyclobutane, the thermodynamically more stable trans-cyclobutane was anticipated to be accessible through an epimerization process. While C(sp3)−H functionalization using 8-aminoquinoline as a directing group was well established as a powerful strategy in the context of total synthesis,19,20 it was uncertain whether the proximal methyl C−H bonds might intervene unproductively. Finally, the required cyclobutane, 18, could be easily prepared from known diazoketone 20 via photochemical Wolff rearrangement.21</p><p>A key question presented by the proposed retrosynthesis was how best to synthesize cyclobutane 18 in enantioenriched form. Elegant studies by Fu and coworkers had demonstrated that N-acylpyrroles can be prepared with excellent enantioselectivity from the reaction between aryl ketenes (e.g. 21) and 2-cyanopyrrole (22) using chiral DMAP catalyst 23 (Scheme 2a).22 We hypothesized that a similar transformation could be used to prepare 18 directly from 20 by using 8-aminoquinoline (29) as a nucleophile in the presence of an appropriate catalyst. While there were no examples from Fu's work in which the ketene was generated in situ photochemically, a single example from Lectka showed that a ketene could be generated in situ by a Wolff rearrangement, and engage in an enantioselective reaction (Scheme 2b).23</p><p>Following a survey of chiral nucleophilic catalysts known to engage with ketenes, 22–24 it was discovered that irradiation of a mixture of 20 and 3 equiv 2925 in the presence of 50 mol % (+)-cinchonine (30) produced 18 in 61% yield, and 79% ee (Table 1, entry 1). Investigation of various solvents revealed that THF provided the highest levels of enantioselectivity.26 More concentrated reaction mixtures led to lower yields, presumably due to poor light penetration as a result of the sparing solubility of 30 in THF. When scaling the reaction to quantities relevant for total synthesis (30 mmol), the catalyst loading of 30 could be reduced to 10 mol %, which provided 18 in 62% yield and 79% ee (see Scheme 3). Moreover, enantiomerically pure 18 was obtained after a single recrystallization by layer diffusion.</p><p>Although our total synthesis efforts focused on the preparation of 18, we wondered if this tandem Wolff rearrangement/enantioselective addition reaction could be applied to other α-diazoketone substrates. Unfortunately, substantially lower levels of enantioinduction (9–64% ee) were observed using 30 as a catalyst with these substrates (Table 1, entries 9, 17, 25, and 33). Evaluation of alternative cinchona derivatives 31–37 revealed that synthetically useful levels of enantioselectivity could be achieved for each substrate, depending on the catalyst. For instance, while 31–37 produced 18 with lower enantioinduction (16–64% ee, entries 2–8), catalysts 34 and 33 proved optimal for the 6- and 7-membered analogs of 20, providing amides 42 and 43 each in 71% ee (entries 13 and 20). When these reactions were conducted on preparative scale, the catalyst loading could be dropped to 20 mol %, providing cyclopentyl amide 42 (n = 2) in 80% yield and 67% ee and cyclohexyl amide 43 (n = 3) in 67% yield and 67% ee.26 On the other hand, benzo-fused diazoketones, 40 and 41, performed best in the presence of dimeric cinchona catalysts 37 and 36 (entries 32 and 39). At present, a general catalyst for the tandem Wolff rearrangement/enantioselective addition of 8-aminoquinoline has not been identified, though further mechanistic investigations may inform future efforts to improve the generality of this reaction.</p><p>Having identified conditions to prepare multigram quantities of 18 in enantiopure form, we were pleased to find that treatment of 18 with Pd(OAc)2 (15 mol %), Ag2CO3, and 19 in TBME at 90 °C smoothly effected the C(sp3)–H alkenylation reaction to give 46 in 75% yield on gram scale (Scheme 3). Exposure of 46 to DBU furnished the requisite trans-cyclobutane via selective epimerization at C2, as determined by deuterium-labeling studies.26 It was at this stage that single crystals of trans-cyclobutane 47 suitable for X-ray diffraction were obtained. Unfortunately, 47 was found to be in the incorrect enantiomeric series for elaboration to natural 3. To our dismay, this problem could not be circumvented by simply employing (−)-cinchonidine (32) in the tandem Wolff rearrangement/asymmetric ketene addition, as this pseudoenantiomeric catalyst afforded (+)-18 in only 57% ee (Table 1, entry 3). Nevertheless, we elected to advance (–)-47 in the interest of validating the key reactions in our retrosynthetic analysis as soon as possible.</p><p>To this end, attention turned to formation of the C1 quaternary center (Scheme 3). Subjection of cis-cyclobutane 46 to a number of standard conjugate addition conditions provided only trace yields of the corresponding product (not shown), presumably due to steric encumbrance by the proximal large aminoquinoline group. On the other hand, treatment of trans-cyclobutane 47 with excess Gilman's reagent smoothly furnished 49 and 50 in near quantitative yield as a 2.5:1 mixture of diastereomers, respectively. Separation of the diastereomers by HPLC allowed single crystals of 50 to be obtained, and X-ray analysis unambiguously confirmed that the major diastereomer (49) possessed the desired (R) configuration of the methyl group at the C1 quaternary center.</p><p>In an effort to improve the diastereoselectivity of this transformation, we turned to asymmetric catalysis. Fortunately, application of the conditions developed by Alexakis and coworkers for copper-catalyzed conjugate addition27 provided 49 in 62% yield and 30:1 dr, albeit using 50 mol % [Cu(OTf)2]•PhMe and a stoichiometric equivalent of phosphoramidite ligand 48. Presumably, the high catalyst loading is required due to the presence of the highly-coordinating 8-aminoquinolinamide, which can deactivate the catalyst or inhibit turnover.</p><p>With the quaternary center secured, ketone 49 was converted to the corresponding dimethyl ketal 51 (Scheme 4a), a precursor to the dienophile for the o-QMHDA reaction (vide infra). While phenolic aldol conditions28 failed to produce 16, this acid-labile o-QM precursor was prepared from phloroglucinol 5429 via the morpholine adduct (55, Scheme 4b).30 A control experiment determined that heating of 51 to 170 °C in toluene results in thermal extrusion of methanol to afford a 1:1 mixture of enol ethers 17 (Scheme 5).31 When a mixture of 51 and 16 was heated to 170 °C for 21 h, the cycloadduct was obtained in 68% yield, albeit as a complex mixture of diastereomers.</p><p>Analytically pure samples of the four highest abundance diastereomers (57–60) were obtained by HPLC purification. Spectroscopic analysis by 2D NMR led to the assignment of 57 and 58 as the two major diastereomers, which bear the expected relative anti relationship between C9 and C1′. The formation of these products in a ~1:1 ratio indicates that 17 does not exert significant facial selectivity in the o-QMHDA reaction. The trans-fused isomer, 60, presumably results from thermal equilibration of the ketal under the reaction conditions.</p><p>In considering how to improve the selectivity for desired diastereomer 57, we drew inspiration from Evans' highly enantioselective inverse-demand hetero-Diels–Alder chemistry, which proceeds via bidentate coordination of heterodienes such as 61 to a chiral Cu(II)-BOX Lewis acid catalyst (Scheme 6a).32 We envisioned that chelation of the aminoquinoline in 17 to a Cu complex could engage 56 as depicted in Scheme 6b, thereby directing the o-QM to the top face of enol ether 17 (Scheme 6b). Formation of 56 could be induced by the equivalent of triflic acid generated via complexation of Cu(OTf)2 with aminoquinoline.17,3333</p><p>To test this hypothesis, enol ether 17 was prepared by heating in PhMe,34 and after exchanging the solvent for CH2Cl2, Cu(OTf)2 and 16 were added. Analysis of the crude reaction mixture by 1H NMR revealed that although the ratio of 57:58 had improved relative to the thermal reaction, significant quantities of the undesired isomers, 59 and 60, were still formed. Moreover, this reaction suffered from lower overall yields due to rapid hydrolysis of 17 and reversion of 16 to phloroglucinol 54. At this stage, it was clear that implementation of this strategy would require a significant investment in reaction optimization and we felt that such an effort would only be warranted if the proposed late-stage Prins reaction were proved feasible. Thus, attention turned to assessing this key reaction in a model system.</p><p>To this end, the aminoquinoline auxiliary in 51 was reductively cleaved by treatment with Schwartz's reagent to furnish aldehyde 62, which was homologated to alkyne 63 using the Ohira–Bestmann reagent (Scheme 7). Nickel-catalyzed hydrothiolation35 proceeded with good regioselectivity to give vinyl sulfide 65 in low yield, mainly due to the facile conversion of this intermediate to a mixture of enol ethers 64 under the reaction conditions.</p><p>Unfortunately, exposure of ketal 65 to a variety of Lewis acids led to hydrolysis, yielding ketone 67 in nearly all cases. The use of InCl3,36 however, delivered the desired Prins product 66 in 11% yield. Formation of the 7-membered ring was confirmed by a key HMBC correlation between the C12 axial proton and the distinct sp2 C7 signal at δ 140 ppm. Although the formation of the seven-membered ring through a Prins cyclization was promising, our excitement was tempered by the fact that 66 was obtained in poor yield and challenges were encountered with reproducibility. Taken together with the significant diastereoselectivity issues plaguing the o-QMHDA reaction, we revised our retrosynthetic analysis.</p><!><p>In our revised retrosynthesis, we envisioned that the chroman substructure could be constructed via a modified Norrish–Yang cyclization,37 revealing 68 as a key intermediate (Figure 3a). Benzophenones such as 68 are known to undergo photoexcitation upon irradiation with UV light38 to give triplet species (i.e. 68*) that can engage in Norrish type-II 1,5-hydrogen atom abstraction and subsequent radical recombination.37,39 In the absence of any available γ or δ-hydrogens, it was hypothesized that 68* could abstract a hydrogen atom from C9 to generate diradical 72.40 Recombination of the carbon-centered radicals would furnish the core of 3. We recognized that achieving the desired regioselectivity could prove challenging since the C7 and C12 methylenes in 68* were also within range for 1,7-H–atom abstraction (Figure 3b). Although the outcome of this transformation was uncertain, conformational analysis suggested that the product resulting from hydrogen atom abstraction at C9 would produce the least sterically encumbered chroman product. Moreover, this strategy was particularly appealing since it was expected that 68 could be assembled in an expedient and convergent fashion. Benzophenone 68 was envisioned to be accessible from tertiary alcohol 70 via an intermolecular O-arylation reaction with aryl bromide 69.41 We reasoned that the strained 7-membered ring in 70 could be formed by ring-closing metathesis,42 leading back to vinyl ketone 71, which could in turn be synthesized from known intermediates prepared during our studies of the C(sp3)–H alkenylation/asymmetric Wolff rearrangement.</p><p>With this revised retrosynthetic plan, we set out to prepare vinyl ketone 71, and to also address two key challenges identified in the first generation approach: 1) to lower the catalyst loading in the conjugate addition reaction used to set the C1 quaternary center, and 2) to develop an epimerization sequence to prepare vinyl ketone 71 in the correct enantiomeric series from quinolinamide (–)-18. In terms of the latter challenge, we anticipated that the desired enantiomeric series could be accessed by epimerization of compounds derived from 18 (e.g. 46) at C5 instead of C2 (Scheme 8). A straightforward approach would involve disfavoring γ-deprotonation at C2 by masking the ketone of 46 in order to advance to a C5 epimerization substrate. Unfortunately, these efforts proved unfruitful, as ketalization of 46 under a variety of conditions always resulted in rapid epimerization at C2 to furnish trans-cyclobutane 77 in low yields.43</p><p>Instead, it was recognized that 74 could be accessed directly by coupling 18 with vinyl iodide 73.44 To our delight, the Pd-catalyzed coupling with vinyl iodide 73 performed even better than its enone counterpart (19), requiring only 2 equiv of 73 to furnish 74 in 72% yield on a gram scale. Exposure of 74 to Schwartz's reagent effected reduction to the corresponding cis-aldehyde, which was epimerized at C5 by treatment with KOH in methanol to give trans-aldehyde 75 in 70% yield over the two steps. Gratifyingly, Wittig methylenation and hydrolysis provided (+)-76, the required enantiomer for synthesis of natural psiguadial B (3). In addition, cross-coupling of 73 eliminated a linear protection step and substantially improved the material throughput.45</p><p>To demonstrate that either enantiomer of 76 can be prepared using a single enantiomer of organocatalyst, an alternative sequence was also developed. Epimerization of 46 to the trans-cyclobutane under the previously developed conditions, followed by ketalization provided 77. Reductive cleavage of the aminoquinoline auxiliary gave the corresponding aldehyde (ent-75), which was telescoped through a Wittig olefination and hydrolysis as before to afford vinyl enone (−)-76 in 58% yield over the two steps.</p><p>With the desired enantiomer of enone 76 in hand, attention turned to the installation of the C1 quaternary center using a catalytic asymmetric conjugate addition. In the absence of the amino-quinoline auxiliary, we were pleased to find that use of CuTC (15 mol %) in conjunction with ligand ent-48 (30 mol %) provided 71 in 94% yield and 19:1 dr (Scheme 9). Addition of vinyl Grignard to ketone 71 proceeded uneventfully, providing alcohol 78 in excellent yield and diastereoselectivity. Gratifyingly, exposure of 78 to second-generation Hoveyda–Grubbs catalyst at elevated temperature delivered bridged bicycle 70 in 93% yield. Subsequent hydrogenation under standard conditions led to tertiary alcohol 79. After some experimentation, we found that the combination of Pd(OAc)2 and dppf catalyzed the intermolecular O-arylation between 79 and aryl bromide 69, affording aryl ether 68 in 45% yield. Unfortunately, the reproducibility of this transformation proved capricious, and attempts to improve the yield through further optimization were unsuccessful. Nevertheless, a sufficient amount of 68 was obtained to evaluate the key Norrish–Yang cyclization.</p><p>In the event, irradiation of 68 with 254 nm light in rigorously deoxygenated dioxane led to complete consumption of starting material within 1 hour and produced a complex mixture of several new products.46 The formation of the undesired Norrish–Yang product 80 was confirmed by 2D NMR spectroscopy; a prominent HMBC correlation was apparent between C5 and the newly formed methine proton at C7, and several key NOE signals were consistent with the stereochemical assignment (Scheme 10). Thus, while the anticipated reactivity was observed, 80 results from the wrong regioselectivity, and was isolated in a mere 6.5% yield—a result that would likely be difficult to substantially improve through reaction optimization.</p><p>Notably, the major compound isolated from this reaction is phenol 83,47 which was obtained in 28% yield. This side product presumably arises by fragmentation of diradical species 72 (or the diradical resulting from H-atom abstraction at C7), wherein C–O bond cleavage expels enol tautomer 82; the resulting terpene-based fragment likely undergoes further decomposition, as alkene 84 or related compounds were not isolated.48 In an effort to investigate whether this competing pathway could be suppressed, we examined a number of different solvents and irradiation wavelengths in a model system, but observed rapid formation of phenol 83 in all cases. Having determined that the late-stage Norrish–Yang cyclization was an untenable strategy to complete the chroman core of 3, an alternative synthetic route was devised.</p><!><p>In our final revision of the retrosynthesis, we simplified 3 to 85 and elected to construct the C9–C1′ bond at an earlier stage (Figure 4). Invoking a similar disconnection through the C–O aryl bond as in our second-generation route, it was anticipated that an intramolecular ring closure would prove more reliable than the challenging intermolecular arylation employed previously (see Scheme 9). This bond scission revealed aryl bromide 86, which could be accessed using the established ring-closing metathesis, while the arene functionality at C9 could be installed via aldol condensation with vinyl ketone 71.</p><p>In the forward sense, a methanolic solution of vinyl ketone 71 and aldehyde 87 was treated with potassium hydroxide at elevated temperature to afford exo-enone 88 in 92% yield (Scheme 11). Attempts to incorporate the C1′ phenyl group at this stage via conjugate addition were met with limited success, yielding only trace amounts of 90 as an inseparable mixture of diastereomers at C9 and C1′. An alternative aldol condensation between 71 and benzophenone 69 (see Figure 3 for structure) failed to produce 89 under otherwise identical conditions or Mukaiyama aldol conditions. Given the inability to introduce the C1′ phenyl substituent at this point, we elected to advanced 88 and attempt to install this group at a later stage in the synthesis.</p><p>In contrast to the previous system lacking substitution at C9 (i.e. 71), 1,2-addition into this more sterically hindered ketone proved challenging. Treatment of 88 with vinyl magnesium bromide under the conditions used previously led to incomplete conversion—presumably due to competitive α-deprotonation— affording 91 (Scheme 12) in low yields and moderate diastereoselectivity.26 Attempts to improve conversion using Lewis acid activators gave higher yields of 91, but resulted in lower levels of diastereoselectivity (<2:1). Ultimately, the desired allylic alcohol was obtained in good yield with serviceable dr by employing vinyllithium in THF at −78 °C, allowing isolation of 91 as a single diastereomer in 54% yield. The ring-closing metathesis proceeded with equal efficiency on this new substrate to furnish 86 in 93% yield. With the strained sesquiterpene framework secured, both the di- and trisubstituted olefins in 86 were hydrogenated in the presence of Crabtree's catalyst, which engaged in a hydroxyl-directed reduction49 to establish the C9 stereogenic center with 16:1 dr, providing 93 in excellent yield. The final ring of the psiguadial framework was formed by a Cu-catalyzed intramolecular O-arylation reaction, which furnished pentacycle 94 in 75% yield.50</p><p>With the successful development of a scalable and high-yielding route to 94, the task of appending the C1′ phenyl group was now at hand. Ideally, the electron rich arene in 94 would be engaged directly in a benzylic arylation reaction; a possible mechanism would involve benzylic oxidation at C1′ followed by trapping with a phenyl nucleophile. Whereas a number of laboratories have shown that electron rich arenes can trap benzylic cations in simple systems,51 it was unclear whether an electronically neutral, unsubstituted phenyl group would be sufficiently reactive to engage as the nucleophile in this type of transformation. Nonetheless, we investigated this possibility with reagents commonly used in flavonoid chemistry (e.g. DDQ,51a,d,52 Chloranil, Pb3O4,53 Oxone/CuSO4,54 and NOBF455), followed by trapping with benzene, PhMgBr, or PhB(OH)2, all without success. Efforts to apply Shi's FeCl2-catalyzed benzylic dehydrogenative arylation,56 or Muramatsu's C(sp3)–H arylation using DDQ and PIFA57 were also unfruitful.</p><p>Having failed to achieve a direct arylation, a stepwise protocol was employed. Oxidation with DDQ in the presence of ethoxyethanol52 afforded 95—a relatively stable product—which could be isolated in modest yields. The remaining mass balance of the reaction consisted of side products suspected to result from over oxidation and elimination of the benzylic ether. A survey of reaction parameters revealed that adding acetonitrile as a co-solvent led to cleaner reaction profiles, albeit at the expense of conversion. Presumably, the acetonitrile solvent helps to stabilize the intermediate benzylic cation (i.e. 96), favoring more efficient trapping with ethoxyethanol over unproductive side reactions. Synthetically useful yields of 95 were obtained under these conditions by re-subjecting recovered 94 to the reaction conditions a second time.</p><p>With respect to the stereochemistry at C1′, 95 was isolated as a 4.8:1 mixture of diastereomers, favoring the α-disposed ether. Conformational analysis of 94 indicates that the 7-membered ring protrudes from the bottom face of the molecule, suggesting that C–O bond formation appears to proceed with contrasteric selectivity. The observed stereochemical outcome might result from an overall double inversion process that proceeds by initial association of DDQ from the less hindered top face of 94 to form a tightly bound charge-transfer complex (i.e. 96).58 If this complex remains closely associated, ethoxyethanol would then attack from the bottom face, thus leading to α-ether 95 as the major diastereomer.</p><p>With a functional handle installed at C1′, TMSOTf was initially investigated as a Lewis acid to activate the ethoxyethyl benzyl ether, however, no phenylated product was obtained using PhB(OH)2, or PhMgBr as nucleophiles (Table 2, entries 1 and 2).52b Simple heating59 or nickel-catalyzed Kumada coupling with PhMgBr in PhMe60 yielded only eliminated products and complex reaction profiles (entries 3–5). Likewise, Bode's conditions for the addition of aryl trifluoroborates to oxonium ions, which use BF3•OEt2 as the Lewis acid, failed to produce 85 (entries 6 and 7).61 We were therefore delighted to obtain a near quantitative yield of 85 (in 1.7:1 dr) by treating a mixture of 95 and lithium diphenylcyanocuprate with BF3•OEt2 (entry 8).62 After some experimentation, it was found that the diastereoselectivity could be slightly improved to 2:1 by holding the reaction at –45 °C (entry 9). Although colder temperatures led to a further improvement in dr, this was accompanied by a lower yield (entry 10).</p><p>As the C1′ diastereomers of 85 were inseparable by silica gel chromatography, the mixture was subjected to pyridine hydrochloride at 200 °C, which afforded the corresponding demethylated products in 92% combined yield (Scheme 13). At this stage, the diastereomeric resorcinols were readily separable by column chromatography, providing 97 as a single diastereomer in 62% yield. Finally, the remaining two aryl aldehydes were simultaneously installed using Rieche formylation conditions,63 delivering (+)-psiguadial B (3) in 50% yield. Synthetic 3 was found to be spectroscopically identical in all respects to the natural sample reported by Shao et al.4</p><!><p>In summary, the first enantioselective total synthesis of the cytotoxic natural product, (+)-psiguadial B (3), was achieved in 15 steps from diazoketone 20. The successful synthetic strategy was enabled by the implementation of a tandem photochemical Wolff rearrangement/asymmetric ketene addition reaction. Having developed a novel protocol for the enantioselective preparation of quinolinamide 18, a variety of substrates were evaluated and conditions were identified to prepare the corresponding 5- and 6-membered ring products. De novo construction of the trans-fused cyclobutane ring in 3 was accomplished using a strategic Pd-catalyzed C(sp3)−H alkenylation reaction, followed by one of two distinct epimerization strategies, which permit access to both enantiomers of the natural product from a single enantiomer of organocatalyst.</p><p>In the course of this work, three different synthetic routes toward (+)-psiguadial B were investigated. These studies have led to the evaluation of several challenging transformations, including 1) an o-QMHDA cycloaddition between a highly functionalized enol ether and a phloroglucinol-derived o-QM; 2) a seven-membered ring-forming Prins cyclization; and 3) a modified Norrish–Yang cyclization. Ultimately, the strained sesquiterpene core was built using a remarkably efficient ring-closing metathesis, and elaborated through a short sequence to afford the natural product in 1.3% overall yield. We believe that the development of this route to 3 may enable the synthesis of unnatural analogs of 3, that would be difficult to access through semi-synthetic methods. Application of the key strategy concepts described herein to the synthesis of other trans-cyclobutane-containing natural products are currently ongoing in our laboratory.</p><!><p>Unless otherwise stated, reactions were performed under a nitrogen atmosphere using freshly dried solvents. Tetrahydrofuran (THF), methylene chloride (CH2Cl2), acetonitrile (MeCN), benzene (PhH), 1,4-dioxane, and toluene (PhMe) were dried by passing through activated alumina columns. Triethylamine (Et3N) and methanol (MeOH) were distilled over calcium hydride prior to use. Unless otherwise stated, chemicals and reagents were used as received. All reactions were monitored by thin-layer chromatography (TLC) using EMD/Merck silica gel 60 F254 pre-coated plates (0.25 mm) and were visualized by UV, p-anisaldehyde, or 2,4-dinitrophenylhydrazine staining. Flash column chromatography was performed either as described by Still et al.64 using silica gel (particle size 0.032-0.063) purchased from Silicycle or using prepackaged RediSep®Rf columns on a CombiFlash Rf system (Teledyne ISCO Inc.). Optical rotations were measured on a Jasco P-2000 polarimeter using a 100 mm path-length cell at 589 nm. 1H and 13C NMR spectra were recorded on a Bruker Avance III HD with Prodigy cryoprobe (at 400 MHz and 101 MHz respectively), a Varian 400 MR (at 400 MHz and 101 MHz, respectively), a Varian Inova 500 (at 500 MHz and 126 MHz, respectively), or a Varian Inova 600 (at 600 MHz and 150 MHz, respectively), and are reported relative to internal CHCl3 (1H, δ = 7.26) and CDCl3 (13C, δ = 77.1), C6H5 (1H, δ = 7.16) and C6D6 (13C, δ = 128), or d8-THF (1H, δ = 3.58) and (13C, δ = 67.6). Data for 1H NMR spectra are reported as follows: chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicity and qualifier abbreviations are as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, app = apparent. IR spectra were recorded on a Perkin Elmer Paragon 1000 spectrometer and are reported in frequency of absorption (cm–1). HRMS were acquired using an Agilent 6200 Series TOF with an Agilent G1978A Multimode source in electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), or mixed (MM) ionization mode. Analytical SFC was performed with a Mettler SFC supercritical CO2 analytical chromatography system with a Chiralcel AD-H column (4.6 mm × 25 cm).</p><p>Procedures and characterization data for compounds 3, 18, 20, 46, 47, 71, 73, 74, 75, 76, 77, 85, 86, 88, 91, 93, 94, 95, 97 were reported previously.12</p><!><p>To each of two flame-dried 1 L round-bottom flasks was added NaH (60% dispersion in mineral oil, 3.17 g, 79.2 mmol, 1.20 equiv) and the atmosphere was exchanged for N2 one time. Dry Et2O (30.0 mL) was then added via syringe and the suspension cooled to 0 °C. Ethyl formate (12.4 mL, 152 mmol, 2.30 equiv) was then added, followed by 2,2-dimethylcyclopentanone (7.40 g, 66.0 mmol) either neat, or as a 3.0 M solution in Et2O. A catalytic amount of wet methanol (~100 μL) was then added and the reaction left to stir at 0 °C.66 Upon completion, the reaction solidifies to a chunky, white solid that dissolved readily upon the addition of DI H2O. At this point, both reaction mixtures were combined for workup: after dilution with Et2O, the layers were separated and the aqueous layer was washed with Et2O 3x to remove organic impurities and a small amount of unreacted starting material. The aqueous layer was then cooled to 0 °C and acidified to pH = 3 using 5 M HCl. Et2O was then added and the acidified aqueous layer was extracted 6x. The combined organics were then dried over Mg2SO4, filtered, and concentrated in vacuo into a 500 mL round-bottom flask.</p><p>The crude α-formyl ketone was taken up in CH2Cl2 (132 mL) and the solution cooled to –10 °C. Triethylamine (55.2 mL, 396 mmol, 5.00 equiv) was added, followed by solid p-ABSA67 (31.8 g, 132 mmol, 1.00 equiv) in three portions. The reaction was stirred for 3 hours and allowed to gradually reach 10 °C, at which point an aqueous solution of KOH (55.0 mL, 4 M) was added. Additional CH2Cl2 and H2O were added, the layers were separated and the aqueous layer extracted with CH2Cl2 until no product remains by TLC. The combined organics were dried over Mg2SO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (20–30% Et2O/pentane) to afford 20 (17.4 g, 95% yield) as a bright yellow oil. 1H NMR (400 MHz, CDCl3) δ 2.88 (t, J = 7.0 Hz, 2H), 1.77 (t, J = 7.2 Hz, 2H), 1.04 (d, J = 1.0 Hz, 6H). 1H NMR (400 MHz, d8-THF) δ 2.94 (t, J = 7.0 Hz, 2H), 1.79 (t, J = 7.2 Hz, 2H), 1.04 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 204.8, 56.6, 46.3, 35.7, 24.1, 21.2. 13C NMR (101 MHz, d8-THF) δ 203.6, 56.1, 46.9, 36.6, 24.5, 21.9. FTIR (NaCl, thin film) 3754, 3414, 3332, 2962, 2934, 2892, 2869, 2672, 2642, 2578, 2510, 2080, 1981, 1673, 1581, 1471, 1460, 1382, 1362, 1339, 1309, 1267, 1245, 1204, 1133, 1110, 1058, 1030, 994, 977, 948, 919, 893, 780, 726, 697 cm.−1</p><p>Diazoketones 38–41 were prepared according to the procedure developed for 20. Spectroscopic data for 40 and 41 are consistent with that reported in the literature.68</p><!><p>Yellow Oil, (1.76 g, 36% yield over 2 steps). 1H NMR (400 MHz, CDCl3) δ 2.71 (t, J = 6.5 Hz, 2H), 1.82 – 1.73 (m, 2H), 1.68 –1.61 (m, 2H), 1.15 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 200.1, 62.6, 42.0, 37.5, 26.7, 22.9, 18.5. FTIR (NaCl, thin film) 2943, 2864, 2082, 1626, 1472, 1449, 1381, 1342, 1317, 1275, 1261, 1220, 1201, 1162, 1122, 1044, 1011, 910, 853, 738, 658 cm.−1 HRMS (EI) calc'd for C8H12N2O [M]+ 152.0950, found 152.0956.</p><!><p>Yellow Oil, (400.0 mg, 26% yield over 2 steps) 1H NMR (400 MHz, CDCl3) δ 2.55 (ddt, J = 7.0, 4.8, 2.3 Hz, 2H), 1.75 (dt, J = 4.4, 2.8 Hz, 4H), 1.57 (ddt, J = 6.3, 3.4, 1.7 Hz, 2H), 1.17 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 202.2, 68.3, 47.0, 37.9, 29.5, 25.8, 25.7, 25.6. FTIR (NaCl, thin film) 2981, 2966, 2927, 2858, 2083, 1704, 1617, 1474. 1448, 1387. 1364, 1350, 1324, 1272, 1251, 1231, 1203, 1147, 1113, 1057, 1020, 980, 953, 871, 845, 736, 656 cm.−1 HRMS (EI) calc'd for C9H14N2O [M]+ 166.1106, found 166.1095.</p><!><p>Oven-dried quartz tubes were each charged with aminoquinoline (21.6 mg, 0.150 mmol, 3.00 equiv) and catalyst (50 mol %). Inside a N2-filled glovebox, diazoketones 20, 38–40 (0.05 mmol) were then added to each as a solution in 0.500 mL THF (excluding diazoketone 41, which was added as a solid outside of the glovebox). The reactions were then sealed with a 19/38 rubber septum around the outside of each tube and sealed with electrical tape. The reactions were then brought out of the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp. The reactions were irradiated with stirring at room temperature for 18 hours. The reactions were then concentrated in vacuo, and the crude reaction mixtures were analyzed by 1H NMR with an added internal standard to determine % yield. The crude residues were purified by silica gel preparative TLC (2% Et2O/CH2Cl2) to provide 18, 42–45 in varying yields and enantiopurities.</p><!><p>To a flame-dried, 1 L quartz flask was added 8-aminoquinoline (29) (12.9 g, 89.5 mmol, 3.00 equiv) and (+)-cinchonine (30) (879 mg, 2.99 mmol, 0.100 equiv). The flask was evacuated and backfilled with N2 three times and dry THF (600 mL) was then added via cannula. Diazoketone 20 (4.12 g, 29.8 mmol, 1.00 equiv) was added last via syringe and the reaction was irradiated with stirring using a Honeywell 254 nm lamp at room temperature. Reaction progress was monitored by TLC (72-168 hours are typically required for complete conversion on this scale, and rotation of the flask every day provided faster conversion).69 Upon completion, the reaction mixture was concentrated in vacuo, the solids were taken up in CH2Cl2, and the suspension filtered. The filter cake was washed with CH2Cl2 three times and the filtrate was concentrated in vacuo to give a crude residue that was purified by silica gel flash chromatography (isocratic: 6% EtOAc/hexane) to provide 18 (4.69 g, 62%) as a pale-yellow solid. The enantiomeric excess was determined to be 79% by chiral SFC analysis (AD-H, 2.5 mL/min, 20% IPA in CO2, λ = 254 nm): tR (major) = 4.23 min, tR (minor) = 5.64 min. [α]D25.0=−66.0° (c = 0.560, CHCl3). Enantioenriched cyclobutane 18 was dissolved in a minimal amount of CH2Cl2 in a 100 mL round-bottom flask. An equal amount of hexanes was carefully layered on top of the CH2Cl2 to form a biphasic mixture. The layers were allowed to diffuse overnight to provide 18 as white, crystalline needles (mp: 66–68 °C). The supernatant was concentrated under reduced pressure and this process was repeated again to provide additional 18 (3.50 g total, 83% recovery of theoretical total of the desired enantiomer, 46% overall from 20): [α]D25.0=−109° (c = 0.720, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.68 (s, 1H), 8.80 (t, J = 1.8 Hz, 1H), 8.79 (dd, J = 13.6, 1.6 Hz, 1H), 8.15 (dd, J = 8.3, 1.7 Hz, 1H), 7.52 (q, J = 8.2, 7.5 Hz, 1H), 7.48 (dd, J = 8.3, 1.6 Hz, 1H), 7.45 (dd, J = 8.3, 4.2 Hz, 1H), 3.07 (ddd, J = 9.1, 8.2, 0.9 Hz, 1H), 2.48 (dq, J = 11.4, 9.4 Hz, 1H), 2.06 (dtd, J = 11.6, 8.6, 3.3 Hz, 1H), 1.85 (dt, J = 10.8, 9.1 Hz, 1H), 1.74 (dddd, J = 10.7, 9.5, 3.3, 0.9 Hz, 1H), 1.39 (s, 3H), 1.14 (s, 3H). 13C NMR δ 171.8, 148.3, 138.6, 136.4, 134.7, 128.1, 127.6, 121.7, 121.3, 116.4, 51.0, 40.4, 32.3, 30.9, 23.4, 17.4. FTIR (NaCl, thin film) 3353, 3047, 2952, 2861, 1685, 1595, 1577, 1526, 1485, 1460, 1424, 1385, 1324, 1261, 1239, 1187, 1169, 1153, 825, 791,756 cm.−1 HRMS (MM) calc'd for C16H19N2O [M+H]+ 255.1492, found 255.1501.</p><!><p>0.2 mmol scale: An oven-dried quartz tube was charged with aminoquinoline (29) (86.5 mg, 0.600 mmol, 3.00 equiv) and (34) (32.5 mg, 0.100 mmol, 0.500 equiv) and brought into a N2 filled glovebox. Diazoketone (38) (33.2 mg, 0.200 mmol, 1.00 equiv) was added as a solution in 2.00 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (6% EtOAc/hexanes) to afford 42 (37.5 mg, 77% yield) as a brown oil. The enantiomeric excess was determined to be 71% by chiral SFC analysis (AD-H, 2.5 mL/min, 20% IPA in CO2, λ = 254 nm): tR (major) = 4.28 min, tR (minor) = 5.41 min.</p><p>1 mmol scale: An oven-dried quartz tube was charged with aminoquinoline (29) (433 mg, 3.00 mmol, 3.00 equiv) and (34) (64.9 mg, 0.200 mmol, 0.200 equiv) and brought into a N2 filled glovebox. Diazoketone (38) (166 mg, 1.00 mmol, 1.00 equiv) was added as a solution in 10.0 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (6% EtOAc/hexanes) to afford 42 (215 mg, 80% yield) as a brown oil. The enantiomeric excess was determined to be 67% by chiral SFC analysis (AD-H, 2.5 mL/min, 20% IPA in CO2, λ = 254 nm): tR (major) = 4.28 min, tR (minor) = 5.41 min. [α]D25.0=−32.5° (c = 2.075, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.80 (s, 1H), 8.81 (d, J = 1.7 Hz, 1H), 8.80 (dd, J = 3.0, 1.7 Hz, 1H), 8.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.57 – 7.47 (m, 2H), 7.45 (dd, J = 8.3, 4.2 Hz, 1H), 2.61 (t, J = 8.4 Hz, 1H), 2.38 – 2.22 (m, 1H), 2.02 (dtd, J = 13.2, 8.5, 4.4 Hz, 1H), 1.95 – 1.82 (m, 1H), 1.79 – 1.65 (m, 2H), 1.63 – 1.57 (m, 1H), 1.31 (s, 3H), 1.01 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 173.1, 148.3, 138.6, 136.5, 134.8, 128.1, 127.6, 121.7, 121.3, 116.4, 58.1, 43.2, 42.1, 29.7, 27.9, 24.0, 22.5. FTIR (NaCl, thin film) 3362, 2957, 2924, 2854, 1729, 1690, 1525, 1486, 1464, 1424, 1381, 1325, 1262, 1164, 1145, 1132, 1072, 825, 791, 720 cm.−1 HRMS (MM) calc'd for C17H21N2O [M+H]+ 269.1648, found 269.1645.</p><!><p>0.2 mmol scale: An oven-dried quartz tube was charged with 8-aminoquinoline (29) (86.5 mg, 0.600 mmol, 3.00 equiv) and (33) (32.5 mg, 0.100 mmol, 0.500 equiv) and brought into a N2 filled glovebox. Diazoketone (39) (31.0 mg, 0.200 mmol, 1.00 equiv) was added as a solution in 2.00 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (6% EtOAc/hexanes) to afford 43 (33.3 mg, 59% yield) as a brown oil. The enantiomeric excess was determined to be 71% by chiral SFC analysis (AD-H, 2.5 mL/min, 12% IPA in CO2, λ = 254 nm): tR (major) = 9.67 min, tR (minor) = 10.34 min.</p><p>1 mmol scale: An oven-dried quartz tube was charged with 8-aminoquinoline (29) (433 mg, 3.00 mmol, 3.00 equiv) and (33) (64.9 mg, 0.200 mmol, 0.200 equiv) and brought into a N2 filled glovebox. Diazoketone (39) (152 mg, 1.00 mmol, 1.00 equiv) was added as a solution in 10.0 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (6% EtOAc/hexanes) to afford 43 (189 mg, 67% yield) as a brown oil. The enantiomeric excess was determined to be 67% by chiral SFC analysis (AD-H, 2.5 mL/min, 12% IPA in CO2, λ = 254 nm): tR (major) = 9.67 min, tR (minor) = 10.34 min. [α]D25.0=−17.3° (c = 1.68, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 8.82 (d, J = 1.7 Hz, 1H), 8.80 (dd, J = 2.7, 1.7 Hz, 1H), 8.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.57 – 7.47 (m, 2H), 7.45 (dd, J = 8.3, 4.2 Hz, 1H), 2.30 (dd, J = 11.8, 3.5 Hz, 1H), 1.99 – 1.78 (m, 3H), 1.55 – 1.47 (m, 2H), 1.39 – 1.27 (m, 3H), 1.13 (s, 3H), 1.10 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 173.6, 148.3, 138.6, 136.5, 134.7, 128.1, 127.6, 121.7, 121.3, 116.5, 56.5, 41.6, 33.4, 31.5, 25.7, 25.7, 22.1, 21.2. FTIR (NaCl, thin film) 3364, 2956, 2923, 2852, 1729, 1691, 1523, 1486, 1462, 1424, 1378, 1326, 1273, 1129, 1072, 825, 790 cm.−1 HRMS (MM) calc'd for C18H23N2O [M+H]+ 283.1805, found 283.1796.</p><!><p>An oven-dried quartz tube was charged with aminoquinoline (29) (86.5 mg, 0.600 mmol, 3.00 equiv) and (37) (85.7 mg, 0.100 mmol, 0.500 equiv) and brought into a N2 filled glovebox. Diazoketone (40) (31.6 mg, 0.200 mmol, 1.00 equiv) was added as a solution in 2.00 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (5–50 % EtOAc/hexanes followed by 0–1% Et2O/CH2Cl2) to afford 44 (16.8 mg, 31% yield). The enantiomeric excess was determined to be 34% by chiral SFC analysis (AD-H, 2.5 mL/min, 30% IPA in CO2, λ = 254 nm): tR (major) = 5.06 min, tR (minor) = 6.89 min. [α]D25.0=−4.1° (c = 0.565, CHCl3). 1H NMR (400 MHz, CDCl3) δ 10.21 (s, 1H), 8.79 (dd, J = 11.5, 1.7 Hz, 1H), δ 8.78 (d, J = 1.7 Hz, 1H), 8.15 (dd, J = 8.3, 1.7 Hz, 1H), 7.54 (dd, J = 8.3, 7.2 Hz, 1H), 7.50 (dd, J = 8.3, 1.8 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.38 – 7.29 (m, 2H), 7.22 – 7.16 (m, 1H), 4.56 (ddt, J = 5.8, 2.9, 0.8 Hz, 1H), 3.69 (ddd, J = 14.2, 5.7, 0.7 Hz, 1H), 3.60 (ddd, J = 14.2, 2.9, 0.8 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 170.6, 148.4, 144.7, 142.9, 138.7, 136.4, 134.5, 128.6, 128.0, 127.8, 127.5, 123.5, 122.7, 121.7, 121.7, 116.5, 49.3, 35.2. FTIR (NaCl, thin film) 3347, 3066, 2928, 2851, 1680, 1596, 1578, 1526, 1485, 1458, 1424, 1386, 1328, 1262, 1240, 1202, 1162, 1132, 869, 826, 791, 759, 734, 707, 679 cm.−1 HRMS (MM) calc'd for C18H15N2O [M+H]+ 275.1179, found 275.1178.</p><!><p>An oven-dried quartz tube was charged with diazoketone (41) (34.4 mg, 0.200 mmol, 1.00 equiv), aminoquinoline (29) (86.5 mg, 0.600 mmol, 3.00 equiv), and diazoketone (36) (88.1 mg, 0.100 mmol, 0.500 equiv) and brought into a N2 filled glovebox. The mixture was suspended in 2.00 mL THF and the tube was sealed with a 19/38 rubber septum and secured with electrical tape. The reaction was removed from the glovebox and placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp for 48 hours. The reaction mixture was then concentrated in vacuo. The crude residue was purified via silica gel flash chromatography (5–10% EtOAc/hexanes) to afford 45 (24.1 mg, 42% yield) as a brown oil. The enantiomeric excess was determined to be 75% by chiral SFC analysis (AD-H, 2.5 mL/min, 30% IPA in CO2, λ = 254 nm): tR (major) = 5.73 min, tR (minor) = 4.86 min. [α]D25.0=65.0° (c = 0.91, CHCl3). 1H NMR (400 MHz, CDCl3) δ 10.06 (s, 1H), 8.79 (dd, J = 7.1, 1.9 Hz, 1H), 8.75 (dd, J = 4.2, 1.7 Hz, 1H), 8.15 (dd, J = 8.3, 1.7 Hz, 1H), 7.56 – 7.46 (m, 3H), 7.44 (dd, J = 8.3, 4.2 Hz, 1H), 7.33 (d, J = 7.2 Hz, 1H), 7.31 – 7.18 (m, 2H), 4.27 (dd, J = 8.4, 6.1 Hz, 1H), 3.23 (dt, J = 15.2, 7.4 Hz, 1H), 3.09 – 2.95 (m, 1H), 2.69 – 2.48 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 172.7, 148.4, 144.8, 141.5, 138.7, 136.4, 134.7, 128.0, 127.9, 127.53, 126.9, 125.1, 125.0, 121.7, 121.7, 116.6, 54.0, 32.1, 30.4. FTIR (NaCl, thin film) 3347, 2957, 2923, 2852, 1728, 1689, 1524, 1484, 1461, 1424, 1380, 1325, 1272, 1163, 1132, 1072, 826, 791, 743 cm.−1 HRMS (MM) calc'd for C19H17N2O [M+H]+ 289.1335, found 289.1334.</p><!><p>To a flame-dried 100 mL flask was added copper (I) iodide (1.48 g, 7.75 mmol, 5.00 equiv) and Et2O (15.5 mL). The resulting suspension was cooled to –40 °C and methyllithium (1.6 M in Et2O; 9.68 mL, 15.5 mmol, 10 equiv) was added dropwise. The reaction mixture was stirred at −40 °C for 2 hours before 47 (540 mg, 1.55 mmol) was added dropwise as a solution in 5:2 CH2Cl2/Et2O. The reaction mixture was gradually warmed to 0 °C over 4 hours, then quenched with saturated aqueous NH4Cl (10 mL) and diluted with EtOAc. NH4OH was added until all of the solid copper salts were sequestered and two homogenous layers remained. The aqueous layer was extracted with EtOAc (3 × 20 mL) and the combined organics dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 20% EtOAc/Hexane) to afford a 2.5:1 mixture of 49 and 50 (543 mg, 96% yield), respectively as a white amorphous solid. Subsequent purification by reverse-phase HPLC using two Agilent Eclipse XDB-C8 5um 9.4 × 250 mm columns connected in series (gradient: 77–85%MeCN/H2O) afforded analytically pure samples of each diastereomer, from which 50 was crystallized for X-Ray analysis26 (mp: 80–83 °C). Data for minor diastereomer 50: [α]D25.0=−25.5° (c = 1.50, CHCl3). 1H NMR (500 MHz, CDCl3) δ 9.64 (s, 1H), 8.82 (dd, J = 4.2, 1.7 Hz, 1H), 8.75 (dd, J = 7.4, 1.6 Hz, 1H), 8.17 (dd, J = 8.3, 1.7 Hz, 1H), 7.56 – 7.48 (m, 2H), 7.46 (dd, J = 8.2, 4.2 Hz, 1H), 2.89 – 2.77 (m, 2H), 2.35 – 2.26 (m, 2H), 2.24 (d, J = 13.3 Hz, 1H), 2.09 (d, J = 13.4 Hz, 1H), 2.07 – 1.99 (m, 1H), 1.88 – 1.77 (m, 1H), 1.72 – 1.61 (m, 3H), 1.55 – 1.48 (m, 1H), 1.35 (s, 3H), 1.13 (s, 3H), 0.92 (s, 3H). 13C NMR (126 MHz, CDCl3) δ 212.4, 170.6, 148.4, 138.5, 136.5, 134.6, 128.1, 127.6, 121.7, 121.4, 116.4, 51.5, 50.8, 41.2, 39.8, 39.6, 35.2, 34.1, 33.0, 30.8, 23.7, 22.2, 21.3. FTIR (NaCl, thin film) 3349, 3044, 2952, 2863, 1706, 1687, 1595, 1577, 1523, 1484, 1460, 1424, 1383, 1325, 1238, 1228, 1163, 827, 792 cm.−1 HRMS (MM) calc'd for C23H29N2O2 [M+H]+ 365.2224, found 365.2261. XRCD: A suitable crystal of C23H28N2O2 (50) was selected for analysis. Low-temperature diffraction data (φ- and ω-scans) were collected on a Bruker AXS D8 VENTURE KAPPA diffractometer coupled to a PHOTON 100 CMOS detector with Cu-Kα radiation (λ = 1.54178 Å) from a IμS HB micro-focus sealed X-ray tube. All diffractometer manipulations, including data collection, integration, and scaling were carried out using the Bruker APEXII software.70</p><!><p>Inside a N2-filled glovebox, [Cu(OTf)]2•PhMe (72.4 mg, 0.140 mmol, 0.25 equiv) and (S,R,R) ligand 4871 (302 mg, 0.560 mmol, 1.00 equiv) were added to a 25 mL flask. The reagents were suspended in Et2O (5.60 mL) and stirred at room temperature for 30 mins before trans-cyclobutane 47 (195 mg, 0.560 mmol) was added as a solid, in one portion. The reaction was sealed under N2, removed from the glovebox and cooled to –30 °C under argon using a cryocool unit to control the temperature. Me3Al (2.0 M in heptane; 560 μL, 1.12 mmol, 2.00 equiv) was then added dropwise, taking care to avoid an exotherm and the reaction mixture stirred vigorously at –30 °C for 16 hours. MeOH (1.00 mL) was then added to quench excess Me3Al and then the reaction was warmed to room temperature. The mixture was diluted with EtOAc and H2O, then the organic layer was separated. The aqueous layer was extracted with EtOAc (3 × 5 mL) and the combined organics dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (2% Et2O/CH2Cl2 until ligand/impurities elute, then 4% Et2O/CH2Cl2) to afford a 30:1 mixture of 49 and 50 (126 mg, 62% yield), respectively as a white solid: [α]D25.0=−84.7° (c = 0.600, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.64 (s, 1H), 8.81 (dd, J = 4.2, 1.7 Hz, 1H), 8.75 (dd, J = 7.2, 1.8 Hz, 1H), 8.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.56 – 7.47 (m, 2H), 7.45 (dd, J = 8.3, 4.2 Hz, 1H), 2.89 – 2.76 (m, 2H), 2.36 – 2.28 (m, 2H), 2.25 (ddd, J = 12.5, 6.6, 1.1 Hz, 1H), 2.04 (dt, J = 13.4, 2.0 Hz, 1H), 1.96 (ddq, J = 13.7, 7.0, 3.6 Hz, 1H), 1.81 (dtt, J = 13.7, 12.0, 5.0 Hz, 1H), 1.68 – 1.62 (m, 2H), 1.62 – 1.51 (m, 2H), 1.35 (s, 3H), 1.13 (s, 3H), 0.89 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 212.4, 170.6, 148.3, 138.5, 136.5, 134.6, 128.1, 127.5, 121.7, 121.4, 116.4, 51.5, 50.4, 41.3, 40.9, 39.5, 35.2, 33.8, 32.6, 30.8, 23.7, 22.1, 20.8. FTIR (NaCl, thin film) 3351, 3047, 2954, 2870, 1708, 1688, 1524, 1485, 1460, 1424, 1384, 1325, 1281, 1259, 1240, 1228, 1163, 919, 827, 792, 757, 732 cm.−1 HRMS (MM) calc'd for C23H29N2O2 [M+H]+ 365.2224, found 365.2228.</p><!><p>To a flame-dried 15 mL flask was added ketone 49 (100 mg, 0.274 mmol) and dissolved in freshly distilled MeOH (2.7 mL). Trimethylorthoformate (150 μL, 1.37 mmol, 5.00 equiv) was then added, followed by p-toluenesulfonic acid monohydrate (2.60 mg, 0.014 mmol, 0.05 equiv). The reaction was topped with a reflux condenser and heated to 65 °C for 1 hour, then quenched with saturated aqueous NaHCO3. The aqueous layer was extracted with EtOAc (3 × 5 mL), and the combined organics were dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by Florisil® flash chromatography (isocratic: 10% EtOAc/Hexane) to afford 51 (106 mg, 94% yield) as a white, foamy solid: [α]D25.0=−83.3° (c = 1.60, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.66 (s, 1H), 8.81 (dd, J = 4.3, 1.7 Hz, 1H), 8.78 (dd, J = 7.4, 1.6 Hz, 1H), 8.14 (dd, J = 8.3, 1.7 Hz, 1H), 7.56 – 7.46 (m, 2H), 7.44 (dd, J = 8.3, 4.2 Hz, 1H), 3.16 (s, 3H), 3.13 (s, 3H), 2.80 (d, J = 10.0 Hz, 1H), 2.69 (q, J = 9.7 Hz, 1H), 2.01 (ddd, J = 13.2, 3.5, 1.6 Hz, 1H), 1.74 (dt, J = 14.0, 2.4 Hz, 1H), 1.70 – 1.50 (m, 4H), 1.31 (s, 3H), 1.28 – 1.13 (m, 4H), 1.11 (s, 3H), 1.01 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.0, 148.3, 138.5, 136.4, 134.7, 128.0, 127.6, 121.6, 121.2, 116.4, 100.8, 51.3, 47.9, 47.3, 42.3, 38.6, 34.8, 34.7, 34.0, 33.3, 32.5, 30.7, 23.9, 21.4, 18.8. FTIR (NaCl, thin film) 3356, 3048, 2950, 2867, 2828, 1690, 1525, 1485, 1460, 1424, 1384, 1368, 1325, 1288, 1276, 1261, 1242, 1155, 1108, 1096, 1048, 946, 927, 826, 792, 756, 690, 666 cm.−1 HRMS (MM) calc'd for C24H31N2O2 [M–OCH3]+ 379.2380, found 379.2376.</p><!><p>To a 15 mL thick-walled, screw top pressure vessel were added dimethyl ketal 51 (59.8 mg, 0.146 mmol) and PhMe (5.0 mL). The tube sealed under a stream of N2. The reaction was heated to 170 °C in a preheated oil bath for 3.5 hours. The reaction was then cooled to room temperature and concentrated in vacuo to afford 17 (55.1 mg, quantitative yield), an inseparable ~1:1 mixture of enol ether isomers, as a foamy colorless gum: [α]D25.0=−78.8° (c = 1.25, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.70 (s, 1H), 8.90 – 8.72 (m, 2H), 8.15 (dd, J = 8.2, 1.5 Hz, 1H), 7.57 – 7.40 (m, 3H), 4.48 (s, 1H), 3.48 (s, 3H), 2.87 – 2.74 (m, 2H), 2.12 – 1.93 (m, 2H), 1.74 – 1.57 (m, 4H), 1.48 – 1.36 (m, 1H), 1.33 (s, 3H), 1.31 – 1.27 (m, 1H), 1.12 (s, 3H), 0.97 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.6, 171.0, 156.3, 154.3, 148.3, 148.3, 138.6, 138.5, 136.4, 136.4, 134.8, 134.7, 128.5, 128.1, 128.1, 127.6, 126.9, 126.8, 121.7, 121.6, 121.2, 121.2, 116.4, 116.3, 99.4, 92.1, 54.1, 53.9, 52.6, 51.5, 40.9, 40.1, 36.4, 35.4, 35.2, 34.0, 33.5, 33.0, 32.6, 30.9, 30.8, 30.7, 29.9, 28.2, 26.1, 25.1, 24.1, 23.9, 21.2, 20.7, 19.5. FTIR (NaCl, thin film) 3354, 3051, 2949, 2930, 2862, 1690, 1668, 1524, 1484, 1461, 1424, 1384, 1368, 1326, 1238, 1215, 1162, 1147, 1026, 826, 791, 756, 694 cm.−1 HRMS (MM) calc'd for C24H31N2O2 [M+H]+ 379.2380, found 379.2395.</p><!><p>To a flame-dried 100 mL round-bottom flask were added phloroglucinol 5472 (1.00g, 4.13 mmol), followed by freshly distilled MeOH (41.0 mL). Benzaldehyde (421 μL, 4.13 mmol, 1.00 equiv), morpholine (361 μL, 4.13 mmol, 1.00 equiv), and triethylamine (576 μL, 4.13 mmol, 1.00 equiv) were then added successively via syringe and the reaction stirred at room temperature for 24 hours. The precipitate thus formed was collected by vacuum filtration and washed with MeOH (20 mL) and dried under high vacuum to afford analytically pure 55 (1.19 g, 69% yield) as a white powder. 1H NMR (400 MHz, CDCl3) δ 15.34 (s, 1H), 13.16 (s, 1H), 12.53 (s, 1H), 7.45 (d, J = 7.2 Hz, 2H), 7.34 – 7.20 (m, 3H), 4.88 (s, 1H), 3.99 (s, 3H), 3.91 (s, 3H), 3.90 – 3.40 (br m, 4H), 3.08 (br s, 1H), 2.46 (ddd, J = 11.9, 6.2, 3.1 Hz, 2H), 2.18 (br s, 1H). 13C NMR (101 MHz, CDCl3) δ 171.7, 166.2, 165.6, 165.1, 138.2, 128.9, 128.4, 103.8, 96.5, 94.2, 69.0, 66.6, 52.7, 52.6. FTIR (NaCl, thin film) 3404 (br), 3062, 3030, 2955, 2894, 2854, 2716, 2562 (br), 2252, 1953 (br), 1731, 1654, 1603, 1494, 1454, 1431, 1403, 1326, 1290, 1250, 1205, 1169, 1121, 1080, 1029, 1006, 986, 942, 915, 878, 843, 825, 808, 761, 732, 700, 648 cm.−1HRMS (MM) calc'd for C21H24NO8 [M+H]+418.1496, found 418.1515.</p><!><p>To a 50 mL round-bottom flask was added benzhydryl morpholine 55 (200 mg, 0.479 mmol), followed by a 1:1 mixture of THF/H2O (9.6 mL). p-Toluenesulfonic acid monohydrate (91.1 mg, 0.479 mmol, 1.00 equiv) was then added in one portion and the reaction was heated to 60 °C for 4 hours. Note: it is best to monitor this reaction closely by TLC to mitigate degradation of the product to 54, presumably via acid-mediated retro aldol. Upon completion, the reaction was cooled to room temperature and quenched with saturated aqueous NaHCO3. The reaction was diluted with EtOAc and the organic layer separated. The aqueous layer was extracted with EtOAc (2 × 5 mL) and the combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 5% EtOAc/CH2Cl2 + 0.5% AcOH, necessary to avoid streaking on the column). Fractions containing pure product were combined, washed with saturated aqueous NaHCO3, dried over MgSO4, filtered, and concentrated in vacuo to afford 16 (82.0 mg, 49% yield) as a white solid. 1H NMR (400 MHz, CDCl3) δ 11.89 (s, 2H), 11.70 (s, 1H), 7.46 – 7.39 (m, 2H), 7.31 (t, J = 7.4 Hz, 2H), 7.26 – 7.19 (m, 1H), 6.38 – 6.23 (m, 1H), 4.09 (d, J = 11.6 Hz, 1H), 4.02 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 170.9, 165.0, 164.7, 143.9, 128.2, 127.0, 125.6, 110.2, 94.5, 68.1, 53.2. FTIR (NaCl, thin film) 3563 (br), 3357 (br), 3085, 3058, 3028, 3006, 2956, 2851, 2749 (br), 1727, 1655, 1623, 1599, 1492, 1434, 1333, 1318, 1245, 1201, 1170, 1129, 1039, 1026, 972, 909, 836, 816, 733, 698, 622 cm.−1 HRMS (MM) calc'd for C17H15O7 [M–OH]+331.0812, found 331.0825.</p><!><p>To a 15 mL thick-walled, screw top pressure vessel were added dimethyl ketal 51 (105 mg, 0.256 mmol) and o-QM precursor 16 (98.0 mg, 0.281 mmol, 1.10 equiv). PhMe (4.3 mL) was then added and the tube sealed under a stream of argon. The reaction was heated to 170 °C in a preheated oil bath for 21 hours. The reaction was then cooled to room temperature and concentrated in vacuo. The crude residue was first purified by silica gel flash chromatography to remove separable impurities (4% EtOAc/CH2Cl2 + 0.5% AcOH) to afford a complex mixture of diastereomers, including 57–60 (109 mg, 68% yield). Analytically pure samples of the four diastereomers produced in greatest abundance (i.e. 57–60) were obtained by subsequent reverse-phase HPLC purification using an Agilent XDB-C18 5 μm 30 × 250 mm column (gradient: 83–100% MeCN/H2O).</p><!><p> [α]D25.0=−32.2° (c = 0.360, CHCl3) White Solid. 1H NMR (400 MHz, CDCl3) δ 12.81 (s, 1H), 12.08 (s, 1H), 9.65 (s, 1H), 8.78 – 8.74 (m, 2H), 8.15 (dd, J = 8.3, 1.6 Hz, 1H), 7.53 – 7.46 (m, 2H), 7.43 (dd, J = 8.3, 4.2 Hz, 1H), 7.22 (d, J = 7.5 Hz, 2H), 7.14 – 7.07 (m, 3H), 3.93 (s, 3H), 3.93 (s, 3H), 3.91 (d, J = 7.8 Hz, 1H), 3.39 (s, 3H), 2.82 – 2.76 (m, 2H), 2.12 (s, 1H), 1.86 – 1.73 (m, 2H), 1.69 – 1.49 (m, 5H), 1.33 (s, 3H), 1.25 (d, J = 9.6 Hz, 1H), 1.10 (s, 3H), 1.05 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.4, 170.8, 169.9, 166.0, 164.7, 158.9, 148.3, 145.9, 138.5, 136.5, 134.7, 128.1, 128.1, 127.8, 127.6, 126.0, 121.7, 121.3, 116.3, 104.2, 104.1, 97.1, 95.7, 52.7, 52.7, 52.2, 49.0, 44.2, 41.7, 39.9, 37.7, 35.1, 35.1, 33.9, 30.8, 28.9, 24.0, 23.5, 22.8. FTIR (NaCl, thin film) 3412 (br), 3354 (br), 3059, 3022, 3006, 2951, 2928, 2864, 1731, 1686, 1654, 1648, 1643, 1594, 1524, 1484, 1459, 1426, 1384, 1338, 1325, 1249, 1222, 1201, 1157, 1122, 1081, 1092, 1028, 976, 945, 936, 847, 826, 792, 755, 700, 667 cm.−1 HRMS (MM) calc'd for C41H45N2O9 [M+H]+ 709.3120, found 709.3141.</p><!><p> [α]D25.0=−13.8° (c = 0.420, CHCl3) White Solid. 1H NMR (400 MHz, CDCl3) δ 12.22 (s, 1H), 11.68 (s, 1H), 9.65 (s, 1H), 8.81 (dd, J = 4.2, 1.7 Hz, 1H), 8.77 (dd, J = 7.3, 1.7 Hz, 1H), 8.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.56 – 7.48 (m, 2H), 7.46 (dd, J = 8.3, 4.2 Hz, 1H), 7.24 – 7.16 (m, 2H), 7.16 – 7.09 (m, 1H), 7.09 – 7.02 (m, 2H), 3.96 (s, 3H), 3.93 (s, 3H), 3.91 (s, 1H), 3.05 (s, 3H), 2.82 – 2.67 (m, 2H), 2.16 (dd, J = 12.4, 3.5 Hz, 1H), 1.98 (d, J = 13.8 Hz, 1H), 1.76 (dd, J = 13.3, 4.1 Hz, 1H), 1.70 – 1.39 (m, 6H), 1.32 (s, 3H), 1.12 (s, 3H), 0.96 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 171.0, 170.8, 169.9, 165.0, 163.1, 157.2, 148.3, 145.6, 138.5, 136.5, 134.7, 128.1, 127.8, 127.6, 127.3, 125.6, 121.7, 121.3, 116.4, 102.4, 102.1, 99.0, 95.3, 52.8, 52.5, 51.3, 47.8, 45.3, 41.9, 41.4, 40.1, 34.7, 34.6, 32.7, 32.6, 30.8, 27.4, 23.8, 22.3. FTIR (NaCl, thin film) 3410 (br), 3355 (br), 3055, 3021, 3000, 2950, 2864, 1734, 1686, 1654, 1643, 1599, 1524, 1484, 1460, 1426, 1384, 1336, 1326, 1279, 1247, 1225, 1163, 1142, 1093, 1063, 988, 973, 949, 841, 826, 791, 754, 698, 667 cm.−1 HRMS (MM) calc'd for C41H45N2O9 [M+H]+ 709.3120, found 709.3119.</p><!><p> [α]D25.0=−98.4° (c = 0.206, CHCl3) White Solid. 1H NMR (400 MHz, CDCl3) δ 12.11 (s, 1H), 11.61 (s, 1H), 9.64 (s, 1H), 8.82 (dd, J = 4.3, 1.7 Hz, 1H), 8.76 (dd, J = 7.3, 1.7 Hz, 1H), 8.16 (dd, J = 8.2, 1.7 Hz, 1H), 7.57 – 7.43 (m, 3H), 7.30 (dd, J = 8.6, 5.1 Hz, 2H), 7.17 (s, 2H), 6.81 (s, 1H), 4.54 (d, J = 7.3 Hz, 1H), 3.94 (s, 3H), 3.92 (s, 3H), 3.21 (s, 3H), 2.74 (q, J = 9.8 Hz, 2H), 2.10 (d, J = 13.7 Hz, 1H), 1.97 – 1.83 (m, 1H), 1.62 (d, J = 8.9 Hz, 2H), 1.45 (d, J = 13.8 Hz, 1H), 1.32 (s, 3H), 1.29 – 1.24 (m, 1H), 1.18 (d, J = 13.2 Hz, 1H), 1.11 (s, 3H), 1.10 – 1.06 (m, 1H), 1.04 (s, 3H), 0.76 (dd, J = 13.1, 3.9 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 171.0, 170.9, 169.7, 164.9, 162.7, 158.0, 148.3, 142.2, 138.5, 136.5, 134.7, 128.5, 128.1, 127.7, 127.6, 125.9, 121.7, 121.3, 116.4, 104.2, 102.2, 99.3, 95.5, 52.8, 52.5, 51.3, 49.0, 43.7, 42.2, 40.3, 38.5, 34.8, 34.4, 33.0, 32.6, 30.8, 23.8, 22.1, 21.7. FTIR (NaCl, thin film) 3408 (br), 3354 (br), 3059, 3022, 3009, 2952, 2868, 1738, 1732, 1682, 1658, 1652, 1645, 1599, 1525, 1485, 1462, 1455, 1426, 1385, 1327, 1281, 1251, 1225, 1165, 1133, 1090, 1077, 1031, 991, 946, 872, 826, 792, 755, 703 cm.−1 HRMS (MM) calc'd for C41H45N2O9 [M+H]+ 709.3120, found 709.3133.</p><!><p> [α]D25.0=−13.4° (c = 0.226, CHCl3) White Solid. 1H NMR (400 MHz, CDCl3) δ 11.95 (s, 1H), 11.23 (s, 1H), 9.66 (s, 1H), 8.81 (dd, J = 4.2, 1.7 Hz, 1H), 8.76 (dd, J = 7.3, 1.7 Hz, 1H), 8.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.55 – 7.42 (m, 3H), 7.22 (dd, J = 7.9, 6.5 Hz, 2H), 7.18 – 7.13 (m, 1H), 7.10 (d, J = 7.4 Hz, 2H), 3.90 (s, 3H), 3.87 (s, 3H), 3.67 (d, J = 11.0 Hz, 1H), 3.16 (s, 3H), 2.81 – 2.66 (m, 2H), 2.10 (dd, J = 14.2, 1.6 Hz, 1H), 1.71 (td, J = 10.8, 5.3 Hz, 1H), 1.64 (dd, J = 9.2, 2.6 Hz, 2H), 1.56 – 1.48 (m, 2H), 1.45 (d, J = 14.3 Hz, 1H), 1.38 – 1.32 (m, 1H), 1.31 (s, 3H), 1.21 – 1.14 (m, 1H), 1.12 (s, 3H), 1.08 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 170.8, 170.8, 169.5, 164.0, 162.4, 157.4, 148.3, 145.4, 138.5, 136.5, 134.7, 128.1, 128.1, 127.6, 125.9, 121.7, 121.3, 116.4, 108.0, 101.7, 99.6, 95.5, 52.7, 52.5, 51.2, 49.4, 49.0, 42.0, 41.1, 36.2, 35.3, 34.8, 33.3, 32.6, 30.8, 23.8, 22.5, 21.1. FTIR (NaCl, thin film) 3412 (br), 3354 (br), 3055, 3023, 3003, 2950, 2866, 1732, 1688, 1656, 1598, 1524, 1484, 1453, 1426, 1384, 1327, 1277, 1248, 1225, 1165, 1062, 993, 954, 925, 826, 792, 755, 702 cm.−1 HRMS (MM) calc'd for C41H45N2O9 [M+H]+ 709.3120, found 709.3139.</p><!><p>Inside a N2-filled glovebox, methyl enol ether 17 (17.0 mg, 0.045 mmol) and o-QM precursor 16 (16.4 mg, 0.047 mmol, 1.05 equiv) were added to a 1 dram vial and dissolved in CH2Cl2 (400 μL). Cu(OTf)2 was then added as a solid in one portion and the reaction immediately turns a light green color, then yellow-brown within the first 5 minutes. The reaction was stirred at room temperature for 1 hour, then quenched with saturated aqueous NaHCO3 and diluted with CHCl3. The reaction mixture was extracted with CHCl3 (3 × 1 mL) and the organics filtered through a plug of Na2SO4 and concentrated in vacuo. The crude residue was analyzed by 1H NMR and determined to contain 57, 58, 59, and 60 in an approximate ratio of 2:1:3:3, respectively.</p><!><p>Inside a N2-filled glovebox, Schwartz's reagent (119 mg, 0.462 mmol, 2.00 equiv) was added to a 10 mL flask and sealed under N2. The flask was removed from the glovebox and THF (1.2 mL) was added via syringe. To the milky-white suspension was added ketal 51 (94.8 mg, 0.231mmol) as a solution in THF (1.2 mL) in a quick drip. The reaction immediately beings to turn yellow, eventually becoming a darker orange color over 1 hour, at which time the reaction was quenched by the addition of saturated aqueous NaHCO3. The reaction was diluted with EtOAc and the organic layer separated. The aqueous layer was extracted with EtOAc (2 × 5 mL) and the combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 5% EtOAc/hexane + 1% Et3N) to afford 62 (36.9 mg, 63% yield) as a pale yellow oil: [α]D25.0=−33.1° (c = 0.500, CHCl3). 1H NMR (400 MHz, CDCl3) δ 9.70 (d, J = 3.0 Hz, 1H), 3.14 (s, 3H), 3.09 (s, 3H), 2.64 (td, J = 9.7, 8.5 Hz, 1H), 2.58 (dd, J = 9.9, 3.0 Hz, 1H), 1.86 (ddt, J = 13.6, 4.2, 2.6 Hz, 1H), 1.61 (t, J = 10.3 Hz, 1H), 1.57 – 1.44 (m, 4H), 1.34 – 1.18 (m, 2H), 1.16 (s, 3H), 1.14 (s, 3H), 1.13 – 1.05 (m, 2H), 0.89 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 204.7, 100.5, 55.2, 47.6, 47.1, 39.8, 39.3, 35.7, 34.4, 33.9, 33.0, 32.7, 31.1, 24.3, 21.7, 18.6. FTIR (NaCl, thin film) 2952, 2868, 2828, 2705, 1713, 1461, 1383, 1368, 1341, 1288, 1262, 1246, 1180, 1166, 1110, 1098, 1048, 1009, 945, 924, 823, 828 cm.−1 HRMS (FAB) calc'd for C15H25O2 [M–OCH3]+ 237.1849, found 237.1855.</p><!><p>To a 10 mL round bottom flask were added aldehyde 62 (36.0 mg, 0.134 mmol) and K2CO3 (37.0 mg, 0.268 mmol, 2.00 equiv). The flask was fitted with a septum and the atmosphere exchanged 2x for N2. Freshly distilled MeOH (1.5 mL) was then added via syringe and the solution cooled to 0 °C. Dimethyl-1-diazo-2-oxopropylphosphonate73 (38.6 mg, 0.201 mmol, 1.50 equiv) was weighed into a tared syringe and added dropwise to the reaction, neat. The reaction was allowed to gradually warm to room temperature and stirred for 12 hours. The reaction was then diluted with Et2O, saturated aqueous NaHCO3 was added, and the organic layer separated. The aqueous layer was extracted with Et2O (3 × 5 mL) and the combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The crude residue was purified by Florisil® flash chromatography (isocratic: 5% Et2O/pentane) to afford 63 (32.9 mg, 93% yield) as a pale yellow oil: [α]D25.0=−43.6° (c = 0.355, CHCl3). 1H NMR (400 MHz, CDCl3) δ 3.17 (s, 3H), 3.13 (s, 3H), 2.43 (dd, J = 10.1, 2.4 Hz, 1H), 2.16 – 2.05 (m, 2H), 2.04 – 1.93 (m, 1H), 1.69 (ddd, J = 13.9, 2.8, 1.8 Hz, 1H), 1.59 – 1.50 (m, 2H), 1.48 (d, J = 9.6 Hz, 2H), 1.29 – 1.17 (m, 5H), 1.16 (d, J = 2.7 Hz, 4H), 1.03 (s, 3H), 0.97 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 100.7, 85.8, 70.5, 49.1, 47.9, 47.3, 39.1, 35.2, 35.1, 34.0, 33.5, 33.2, 33.2, 29.9, 24.8, 21.1, 18.8. FTIR (NaCl, thin film) 3310, 3263, 2953, 2866, 2828, 1459, 1383, 1364, 1342, 1323, 1288, 1266, 1243, 1180, 1157, 1106, 1094, 1048, 945, 926, 858, 830, 655, 621 cm.−1 HRMS (MM) calc'd for C16H25O2 [M–OCH3]+233.1900, found 233.1887.</p><!><p>Inside a N2-filled glovebox, THF (400 μL) was added to a 1 dram vial containing alkyne 63 (12.4 mg, 0.047 mmol), followed by Ni(acac)2 as a stock solution in THF (0.10 M, 70 μL, 0.007 mmol, 0.15 equiv). The reaction was stirred for 10 minutes at room temperature before thiophenol (10 μL, 0.094 mmol, 2.00 equiv) was added neat. The reaction was sealed with a Teflon cap and heated to 60 ° C in a preheated aluminum block inside the glovebox. After 3 hours, the reaction was cooled to room temperature and diluted with CH2Cl2. The reaction mixture was filtered over a small pad of celite, washed with CH2Cl2 until the filtrate runs colorless, and concentrated in vacuo. The crude residue was taken up in EtOAc and shaken with 5M NaOH (to remove excess thiophenol). The organic layer was then filtered through a plug of Na2SO4, concentrated, and purified by silica gel preparative TLC (5% EtOAc/hexane + 1% Et3N) to afford 64 (8.50 mg, 53% yield) and 65 (2.7 mg, 15% yield) each as colorless oils.</p><!><p> [α]D25.0=+12.3° (c = 0.115, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.42 (dd, J = 8.1, 1.6 Hz, 2H), 7.36 – 7.28 (m, 3H), 5.17 (d, J = 1.3 Hz, 1H), 4.96 (s, 1H), 3.17 (s, 3H), 3.13 (s, 3H), 2.51 (d, J = 10.2 Hz, 1H), 2.26 (q, J = 9.7 Hz, 1H), 2.04 – 1.92 (m, 1H), 1.65 (ddd, J = 13.8, 2.8, 1.6 Hz, 1H), 1.50 (ddd, J = 9.6, 7.0, 3.7 Hz, 2H), 1.45 – 1.39 (m, 2H), 1.21 – 1.12 (m, 4H), 1.11 (s, 3H), 0.98 (s, 3H), 0.91 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 145.9, 133.8, 133.4, 129.2, 127.9, 111.6, 100.8, 49.4, 47.9, 47.3, 45.1, 39.7, 35.6, 34.8, 34.6, 33.2, 32.4, 30.6, 23.2, 21.7, 18.9. FTIR (NaCl, thin film) 2950, 2863, 2827, 1610, 1583, 1476, 1459, 1439, 1379, 1364, 1322, 1274, 1260, 1247, 1178, 1145, 1130, 1100, 1083, 1049, 1024, 946, 926, 856, 831, 822, 747, 691 cm.−1 HRMS (MM) calc'd for C22H31OS [M–OCH3]+343.2090, found 343.2073.</p><!><p> [α]D25.0=−11.0° (c = 0.982, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.46 – 7.36 (m, 2H), 7.36 – 7.27 (m, 3H), 5.23 – 4.84 (m, 2H), 4.63 – 4.29 (m, 1H), 3.40 (s, 3H), 2.58 – 2.50 (m, 1H), 2.40 (dq, J = 34.9, 9.5 Hz, 1H), 2.11 – 1.91 (m, 3H), 1.64 (ddd, J = 15.0, 5.9, 2.4 Hz, 2H), 1.48 – 1.40 (m, 2H), 1.39 – 1.30 (m, 1H), 1.11 (d, J = 9.9 Hz, 3H), 1.00 (d, J = 2.4 Hz, 3H), 0.83 (d, J = 21.0 Hz, 4H). 13C NMR (101 MHz, CDCl3) δ 155.3, 154.4, 146.0, 145.9, 133.8, 133.5, 133.4, 133.3, 129.2, 129.1, 127.9, 127.7, 112.3, 111.1, 100.6, 92.0, 54.1, 53.9, 50.2, 49.5, 43.5, 40.9, 37.7, 36.1, 35.1, 35.1, 34.5, 34.1, 32.9, 32.6, 31.6, 30.6, 30.5, 28.2, 25.8, 23.3, 23.2, 22.0, 20.8, 19.4. FTIR (NaCl, thin film) 3061, 2991, 2950, 2930, 2862, 2843, 1667, 1609, 1583, 1476, 1460, 1453, 1440, 1380, 1366, 1251, 1215, 1148, 1066, 1024, 940, 817, 747, 691 cm.−1 HRMS (MM) calc'd for C22H31OS [M+H]+ 343.2090, found 343.2087.</p><!><p>Inside a N2-filled glovebox, CH2Cl2 was added to a 1 dram vial containing 65 (9.30 mg, 0.025 mmol), followed by InCl3 (5.49 mg, 0.025 mmol, 1.00 equiv). The reaction was stirred at room temperature for 2 hours, then quenched with saturated aqueous NaHCO3 and diluted with CH2Cl2. The reaction was extracted with CH2Cl2 (3 × 500 μL), the combined organics filtered a plug of Na2SO4, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (40–60% CH2Cl2/hexane) to afford 66 (0.900 mg, 11% yield) as a colorless oil, with the remaining mass balance accounted for by ketone 67, as determined by crude 1H NMR.</p><!><p> [α]D25.0=+58.2° (c = 0.053, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.40 – 7.36 (m, 2H), 7.30 (ddd, J = 8.3, 7.1, 0.8 Hz, 2H), 7.24 – 7.18 (m, 1H), 5.52 (dd, J = 2.8, 1.7 Hz, 1H), 3.18 (s, 3H), 2.91 – 2.81 (m, 1H), 2.00 – 1.72 (m, 5H), 1.66 (ddd, J = 11.6, 6.8, 3.5 Hz, 1H), 1.44 – 1.37 (m, 2H), 1.35 (dd, J = 12.3, 1.7 Hz, 1H), 1.29 (m, 1H), 1.28 (s, 3H), 1.19 (dd, J = 14.1, 7.0 Hz, 1H), 1.00 (s, 3H), 0.80 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 139.7, 139.6, 135.0, 131.7, 129.2, 127.2, 80.6, 52.4, 50.2, 49.2, 48.6, 40.1, 38.3, 36.2, 33.7, 31.3, 31.2, 28.9, 22.4, 21.2. FTIR (NaCl, thin film) 3062, 2945, 2927, 2860, 2820, 1734, 1718, 1701, 1654, 1583, 1560, 1476, 1458, 1438, 1370, 1294, 1254, 1232, 1151, 1086, 1066, 1024, 950, 870, 840, 800, 743, 690 cm.−1 HRMS (MM) calc'd for C22H31OS [M+H]+343.2090, found 343.2077.</p><!><p> [α]D25.0=+31.6° (c = 0.100, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.46 (dd, J = 8.1, 1.6 Hz, 2H), 7.40 – 7.32 (m, 3H), 5.13 (d, J = 1.3 Hz, 1H), 4.98 (s, 1H), 2.53 (d, J = 10.1 Hz, 1H), 2.38 (td, J = 10.0, 8.9 Hz, 1H), 2.32 – 2.23 (m, 2H), 2.17 (d, J = 13.6 Hz, 1H), 2.02 (dt, J = 13.4, 1.9 Hz, 1H), 1.93 – 1.90 (m, 1H), 1.82 (dddd, J = 9.7, 8.1, 3.9, 2.3 Hz, 1H), 1.57 (q, J = 4.4 Hz, 1H), 1.49 – 1.44 (m, 1H), 1.44 – 1.33 (m, 2H), 1.16 (s, 3H), 1.03 (s, 3H), 0.82 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 212.6, 145.5, 134.1, 133.0, 129.3, 128.2, 111.4, 51.1, 49.8, 43.1, 41.3, 40.3, 35.1, 34.2, 32.5, 30.6, 23.1, 22.1, 21.8. FTIR (NaCl, thin film) 3059, 2953, 2927, 2860, 1711, 1680, 1611, 1583, 1476, 1461, 1440, 1381, 1364, 1347, 1311, 1283, 1253, 1228, 1151, 1087, 1067, 1024, 890, 855, 749, 692 cm.−1 HRMS (MM) calc'd for C21H29OS [M+H]+ 329.1934, found 329.1943.</p><!><p>To a 15 mL round-bottom flask was added vinyl ketone 71 (91.0 mg, 0.413 mmol) and the atmosphere was exchanged 3x for N2. Dry THF (4.10 mL) was then added via syringe and the reaction cooled to –30 °C using a closely monitored acetone/CO2 bath. Vinylmagnesium bromide (2.06 mL, 1.0 M in THF, 2.06 mmol, 5.00 equiv) was then added dropwise. The reaction was maintained at –30 °C for 30 minutes, then quenched at that temperature with saturated aqueous NaH2PO4. The reaction mixture was diluted with Et2O and the layers separated. The aqueous layer was extracted with Et2O (2 × 5 mL) and the combined organics were dried over Mg2SO4, filtered, and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (10% EtOAc/hexane) to afford 78 (92.7 mg, 91% yield) as a colorless oil: [α]D25.0=+54.4° (c = 1.75, CHCl3). 1H NMR (400 MHz, CDCl3) δ 5.88 (dd, J = 17.3, 10.6 Hz, 1H), 5.75 (ddd, J = 17.1, 10.2, 8.7 Hz, 1H), 5.18 (dd, J = 17.3, 1.3 Hz, 1H), 5.01 – 4.85 (m, 3H), 2.32 (t, J = 9.3 Hz, 1H), 1.92 (q, J = 9.6 Hz, 1H), 1.82 (qt, J = 13.5, 3.4 Hz, 1H), 1.55 (dddd, J = 14.0, 5.3, 3.5, 1.9 Hz, 1H), 1.48 (dq, J = 13.8, 3.5 Hz, 1H), 1.45 – 1.39 (m, 2H), 1.35 (dd, J = 13.5, 4.0 Hz, 1H), 1.31 – 1.22 (m, 3H), 1.16 – 1.11 (m, 1H), 1.11 (s, 1H), 1.06 (s, 3H), 0.97 (s, 3H), 0.97 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 148.1, 140.6, 114.6, 110.5, 73.1, 49.0, 48.0, 45.0, 37.6, 34.6, 34.3, 33.9, 32.8, 30.1, 23.8, 22.7, 17.8. FTIR (NaCl, thin film) 3601, 3452 (br), 3077, 2996, 2950, 2932, 2865, 1635, 1459, 1441, 1413, 1380, 1367, 1343, 1291, 1275, 1250, 1200, 1170, 1081, 1058, 994, 974, 909, 858, 846, 666 cm.−1 HRMS (ESI) calc'd for C17H27 [M–OH]+ 231.2107, found 231.2101.</p><!><p>A 50 mL round-bottom flask containing divinyl alcohol 78 (88.0 mg, 0.355 mmol) was pumped into a N2-filled glovebox where Hoveyda–Grubbs second-generation catalyst (22.2 mg, 0.035 mmol, 0.100 equiv) was added. The flask was sealed under nitrogen, removed from the glovebox and dry PhH (17.7 mL) was added via syringe. The green reaction mixture was heated to 80 °C for 3.5 hours, then cooled to room temperature. Ethyl vinyl ether was added to inactivate the catalyst and stirred for 15 minutes before the reaction mixture was concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 30% Et2O/hexane) to afford allylic alcohol 70 (72.5 mg, 93% yield) as a pale yellow oil and a single diastereomer at C1: [α]D25.0=−62.9° (c = 2.67, CHCl3). 1H NMR (400 MHz, CDCl3) δ 5.84 (dd, J = 10.9, 2.5 Hz, 1H), 5.15 (ddd, J = 11.0, 2.9, 2.2 Hz, 1H), 2.41 (dt, J = 11.6, 2.7 Hz, 1H), 2.09 (td, J = 11.5, 10.7, 7.9 Hz, 2H), 1.69 (ddd, J = 13.0, 3.2, 1.1 Hz, 1H), 1.64 (s, 1H), 1.63 – 1.56 (m, 2H), 1.54 – 1.41 (m, 2H), 1.34 – 1.25 (m, 2H), 1.15 (dd, J = 12.8, 2.2 Hz, 1H), 1.12 – 1.06 (m, 1H), 1.05 (s, 3H), 1.03 (s, 3H), 0.87 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 134.1, 132.5, 75.1, 49.9, 45.3, 43.9, 39.0, 38.1, 37.8, 35.0, 32.6, 30.9, 26.9, 21.3, 20.2. FTIR (NaCl, thin film) 3350 (br), 3004, 2948, 2930, 2866, 1460, 1443, 1369, 1380, 1366, 1329, 1270, 1256, 1238, 1175, 1106, 1044, 1030, 999, 973, 958, 925, 875, 864, 766, 723 cm.−1 HRMS (MM) calc'd for C15H23 [M–OH]+ 203.1794, found 203.1790.</p><!><p>To a 100 mL round-bottom flask were added allylic alcohol 70 (107 mg, 0.486 mmol) and Pd/C (103 mg, 10% by weight, 0.097 mmol, 0.200 equiv). The flask was fitted with a septum and the atmosphere exchanged 1x for N2. MeOH (9.7 mL) was then added via syringe and the reaction placed under a balloon atmosphere of H2 (purged through a needle for 30 seconds). The reaction was stirred vigorously at room temperature for 2.5 hours, at which time the atmosphere was purged with argon. The reaction mixture was filtered over celite, washed thoroughly with Et2O, and the filtrate concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 40% Et2O/pentane) to afford 79 (101 mg, 94% yield) as a colorless oil: [α]D25.0=+6.37° (c = 0.800, CHCl3). 1H NMR (400 MHz, CDCl3) δ 1.97 (ddd, J = 11.8, 10.7, 7.9 Hz, 1H), 1.86 – 1.78 (m, 1H), 1.78 – 1.68 (m, 3H), 1.67 (d, J = 0.8 Hz, 3H), 1.51 – 1.39 (m, 2H), 1.34 (dt, J = 3.5, 2.0 Hz, 1H), 1.33 – 1.22 (m, 4H), 1.15 – 1.04 (m, 1H), 1.02 (d, J = 12.8 Hz, 1H), 0.97 (s, 3H), 0.96 (s, 3H), 0.80 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 74.0, 50.2, 46.3, 40.3, 40.1, 39.7, 38.2, 36.4, 34.6, 32.8, 30.7, 27.1, 22.7, 20.9, 20.7. FTIR (NaCl, thin film) 3368 (br), 2948, 2927, 2863, 1460, 1443, 1384, 1364, 1332, 1288, 1249, 1217, 1183, 1124, 1102, 1050, 1022, 993, 976, 936, 918, 873, 862 cm.−1 HRMS (ESI) calc'd for C15H25 [M–OH]+ 205.1951, found 205.1951.</p><!><p>Inside a N2-filled glovebox, to a 1 dram vial containing tertiary alcohol 79 (14.4 mg, 0.065 mmol) were added Pd(OAc)2 (4.36 mg, 0.019 mmol, 0.300 equiv), dppf (21.6 mg, 0.039 mmol, 0.600 equiv), and NaH (95%, 3.11 mg, 0.130 mmol, 2.00 equiv). PhMe (650 μL) was then added and the orange reaction mixture stirred at room temperature for 5 minutes before aryl bromide 6974 (22.8 mg, 0.071 mmol, 1.10 equiv) was added as a solid in one portion. The reaction was sealed with a Teflon cap and heated to 110 °C in a preheated aluminum block inside the glovebox. After 13.5 hours, the reaction was cooled to room temperature, diluted with EtOAc and saturated aqueous Na2HPO4 was added. The layers were separated and the aqueous layer was extracted with EtOAc until the organic layer was colorless. The combined organics were filtered over a plug of celite and Na2SO4. The filtrate was concentrated in vacuo and the crude residue purified by silica gel flash chromatography (isocratic: 30% hexane/CH2Cl2 + 1% EtOAc) to afford 68 (13.4 mg, 45% yield) as a milky white gum: [α]D25.0=+1.27° (c = 0.345, CHCl3). 1H NMR (400 MHz, CDCl3) δ 7.86 – 7.76 (m, 2H), 7.51 (tt, J = 7.5, 2.7 Hz, 1H), 7.44 – 7.35 (m, 2H), 6.29 (d, J = 2.1 Hz, 1H), 6.23 (d, J = 2.1 Hz, 1H), 3.83 (s, 3H), 3.70 (s, 3H), 1.90 (ddd, J = 12.0, 10.7, 7.9 Hz, 1H), 1.78 (d, J = 2.3 Hz, 1H), 1.73 (t, J = 6.5 Hz, 2H), 1.68 (dt, J = 13.0, 2.3 Hz, 1H), 1.65 – 1.57 (m, 1H), 1.55 – 1.45 (m, 2H), 1.45 – 1.35 (m, 3H), 1.27 – 1.23 (m, 2H), 1.22 – 1.11 (m, 2H), 1.04 (d, J = 12.9 Hz, 1H), 0.92 (s, 3H), 0.91 (s, 3H), 0.69 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 196.0, 161.3, 158.7, 155.2, 138.8, 132.8, 129.6, 128.3, 116.8, 100.3, 92.5, 86.9, 55.9, 55.6, 47.6, 45.6, 39.7, 37.5, 36.8, 36.2, 36.1, 34.6, 32.7, 30.7, 27.1, 22.5, 20.9, 20.5. FTIR (NaCl, thin film) 3059, 2948, 2930, 2861, 1671, 1601, 1582, 1458, 1451, 1438, 1420, 1364, 1335, 1312, 1266, 1216, 1199, 1157, 1138, 1107, 1052, 1015, 998, 948, 917, 843, 819, 802, 721, 702, 689 cm.−1 HRMS (MM) calc'd for C30H38NaO4 [M+Na]+ 485.2662, found 485.2672.</p><!><p>To a 13 × 100 quartz test tube was added benzophenone 68 (15.5 mg, 0.034 mmol). The tube was fitted with a 19/38 rubber septum and the atmosphere was exchanged 3 × for N2. Rigorously degassed dioxane (4.70 mL, freeze-pump-thawed 3x) was then added via syringe and the tube was sealed with electrical tape. The reaction was then placed in a bottomless test tube rack in front of a Honeywell 254 nm lamp and irradiated for 1 hour at room temperature. The reaction mixture was transferred to a cone-bottom flask and concentrated in vacuo. The crude residue was purified by silica gel preparative TLC (30% hexane/CH2Cl2 + 1% EtOAc) to afford 8375 (2.4 mg, 28% yield) as a white solid and 80 (1.00 mg, 6.5% yield) as a colorless oil: [α]D25.0=+13.8° (c = 0.050, CHCl3). Note: an additional ~18% yield of a complex mixture of products is also isolated as a single band. Although this mixture generally appears similar to 80 by 1H NMR, definitive characterization was not achieved. 1H NMR (400 MHz, CDCl3) δ 7.34 – 7.26 (m, 1H), 7.24 – 7.09 (m, 4H), 6.11 (dd, J = 2.5, 1.1 Hz, 1H), 6.01 (dd, J = 2.4, 1.2 Hz, 1H), 3.95 (d, J = 1.1 Hz, 1H), 3.78 (d, J = 1.2 Hz, 3H), 3.35 (d, J = 1.1 Hz, 3H), 2.65 (dd, J = 12.7, 3.5 Hz, 1H), 2.62 – 2.52 (m, 1H), 2.40 (t, J = 14.4 Hz, 1H), 2.26 (q, J = 10.4 Hz, 1H), 2.11 – 1.90 (m, 1H), 1.86 (d, J = 13.0 Hz, 1H), 1.83 – 1.72 (m, 1H), 1.69 – 1.57 (m, 1H), 1.43 – 1.34 (m, 2H), 1.30 – 1.09 (m, 4H), 0.80 (s, 3H), 0.78 (s, 3H), 0.75 (s, 3H), 0.50 (dt, J = 14.5, 4.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 160.5, 158.8, 154.3, 149.3, 127.4, 126.1, 125.8, 111.4, 94.2, 93.5, 80.6, 74.6, 55.6, 55.4, 48.5, 48.1, 44.0, 37.3, 36.8, 35.7, 35.5, 34.6, 33.2, 30.5, 26.4, 25.0, 20.6, 20.4. FTIR (NaCl, thin film) 3542 (br), 3312, 3187 (br), 2960, 2924, 2854, 1738, 1726, 1710, 1666, 1614, 1592, 1492, 1462, 1453, 1445, 1423, 1376, 1366, 1351, 1332, 1261, 1215, 1203, 1150, 1112, 1045, 1020, 865, 800, 736, 702, 664 cm.−1 HRMS (MM) calc'd for C30H37O3 [M–OH]+ 445.2737, found 445.2729.</p><!><p>To a 2-dram vial was added resorcinol 97 (15.4 mg, 0.037 mmol) and the atmosphere exchanged three times for N2. CH2Cl2 (1.30 mL) was then added via syringe, followed by dichloromethyl methyl ether (0.083 mL, 0.920 mmol, 25.0 equiv). The solution was cooled to –78 °C and a freshly prepared stock solution of TiCl4 (0.190 mL, 0.912 M in CH2Cl2, 0.173 mmol, 4.68 equiv) was added dropwise. The reaction immediately turns dark red. The reaction was stirred at –78 °C for 5 minutes, then warmed to room temperature and stirred for an additional 3 hours and 40 minutes. DI H2O (2.00 mL) was then added via syringe and the reaction stirred vigorously for 15 minutes before the layers were separated. The aqueous layer was extracted five times with CH2Cl2 and the combined organic layers were filtered over a plug of Na2SO4 and concentrated in vacuo. The crude residue was purified by silica gel flash chromatography (isocratic: 2% EtOAc/hexane + 1% AcOH) to afford (+)-psiguadial B (3) (8.7 mg, 50%) as an ivory solid. Note: 3 is streaky on SiO2 and after an initial concentrated band elutes, approximately 12% of the product is contained in the following very dilute fractions. The natural product is weakly UV active, but can also be visualized by TLC using 2,4-dinitrophenylhydrazine stain. [α]D25.0=+94.0° (c = 0.265, CHCl3). 1H NMR (400 MHz, CDCl3) δ 13.51 (s, 1H), 13.04 (s, 1H), 10.07 (s, 2H), 7.26 (dd, J = 14.6, 1.5 Hz, 2H), 7.23 – 7.17 (m, 1H), 7.10 (br s, 2H), 3.49 (d, J = 11.5 Hz, 1H), 2.20 – 2.12 (m, 1H), 2.09 (dd, J = 12.7, 2.4 Hz, 1H), 1.92 (ddd, J = 14.9, 12.8, 4.2 Hz, 1H), 1.82 (ddd, J = 12.3, 8.8, 5.6 Hz, 1H), 1.73 – 1.59 (m, 3H), 1.53 – 1.44 (m, 1H), 1.49 (ddd, J = 11.6, 8.1, 2.9 Hz, 2H), 1.44 – 1.29 (m, 4H), 1.05 (dd, J = 7.6, 5.8 Hz, 1H), 1.02 (s, 3H), 1.00 (s, 3H), 0.85 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 192.3, 191.5, 169.6, 168.5, 163.5, 143.4, 128.2, 126.2, 105.7, 104.6, 104.1, 84.1, 50.0, 47.4, 44.0, 40.4, 37.6, 36.9, 35.4, 35.1, 33.4, 30.6, 29.3, 26.1, 23.9, 20.7, 20.1. FTIR (NaCl, thin film) 3026, 2945, 2926, 2864, 2720, 1633, 1603, 1493, 1437, 1382, 1363, 1300, 1270, 1251, 1231, 1184, 1154, 1143, 1031, 1006, 976, 926, 917, 875, 851, 840, 824, 768, 701, 636, 618, 606, 564 cm.−1 HRMS (MM) calc'd for C30H35O5 [M+H]+ 475.2479, found 475.2487.</p>

PubMed Author Manuscript

Flexibility Coexists with Shape Persistence in Cyanostar Macrocycles

Shape-persistent macrocycles are attractive functional targets for synthesis, molecular recognition, and hierarchical self-assembly. Such macrocycles are non-collapsible and geometrically well-defined, and they are traditionally characterized by having repeat units and low conformational flexibility. Here, we find it necessary to refine these ideas in the face of a highly flexible yet shape-persistent macrocycles. A molecule is shape-persistent if it has a small change in shape when perturbed by external stimuli (e.g., heat, light, and redox chemistry). In support of this idea we provide the first examination of the relationships between a macrocycle\xe2\x80\x99s shape persistence, its conformational space, and the resulting functions. We do this with a star-shaped macrocycle called cyanostar that is flexible as well as being shape-persistent. We employed molecular dynamics (MD), density functional theory (DFT), and NMR experiments. Considering a thermal bath as a stimulus we found a single macrocycle has 332 accessible conformers with olefins undergoing rapid interconversion by up-down and in-out motions on short time scales (0.2 ns). These many interconverting conformations classify single cyanostars as flexible. To determine and confirm that cyanostars are shape-persistent, we show that it has a high 87% shape similarity across these conformations. To further test the idea, we use the binding of diglyme to the single macrocycle as guest-induced stimulation. This guest has almost no effect on the conformational space. However, formation of a 2:1 sandwich complex involving two macrocycles enhances rigidity and dramatically shifts the conformer distribution towards perfect bowls. Overall, the present study expands the scope of shape-persistent macrocycles to include flexible macrocycles if and only if their conformers have similar shapes.

flexibility_coexists_with_shape_persistence_in_cyanostar_macrocycles

INTRODUCTION<!>A. Delineating Cyanostar\xe2\x80\x99s Conformational Landscape<!>Up-and-down Conformations of Cyanostar Determine Local and Global Bowl Chirality<!>In-and-out Conformations of Cyanostar Are Connected by Olefin Rock and Roll Rotations<!>A Landscape of 332 Thermally Accessible Conformers<!>Rapidly Inverting Bowl Chirality in Flexible Cyanostars<!>B. Shape persistence of Single Cyanostars<!>Quantitative Definition of Shape Persistence for Flexible Cyanostar Monomers<!>Quantitative NOE Verifies the Bowl Shape of Single Cyanostars as Predicted by Simulations<!>C. Shape Persistence under the Perturbation of Diglyme Binding and Molecular Self-association<!>The Conformational Landscape changes to Favor Perfect Bowls in Dimers of Cyanostars stabilized by Complexation with Diglyme<!>Steric Hindrance, Not Binding, Rigidifies Cyanostar in the 2:1 Complex with Diglyme<!>CONCLUSIONS

<p>Shape-persistent macrocycles are traditionally defined as cyclic compounds with low conformational flexibility to ensure they are non-collapsible and geometrically well-defined.1 Shape persistence is advantageous for many applications: synthesis of macrocycles,2 preorganization,3 in silico design,4 catalysis,5 configurationally stable stereochemistry,6 and hierarchical self-assembly7,8,9 of multi-functional architectures,3a,8a,10 To design shape-persistent macrocycles, Moore has suggested that they have limited degrees of conformational freedom, i.e., they are rigid.11 Yet, through the results obtained in the present study, we were confronted with an apparent contradiction involving cyanostar macrocycles (Figure 1a).12 Cyanostars appear to be flexible based on their many conformations and yet geometrically well-defined based on their pre-organization towards anionic guests.</p><p>To resolve the contradiction, we propose a more robust definition of shape persistence: A molecule is shape-persistent if it undergoes small change in shape when perturbed by external stimuli (e.g., heat, light, redox chemistry, etc.). This definition retains Moore's original ideas while providing a broader context by introducing the ability to assess the extent of shape changes under any type of perturbation. This refined definition subsumes the concept of preorganization in which guest binding is considered as just one type of perturbation from among many. Here, we present a close examination of the shape persistence of flexible cyanostar macrocycles, and how the shape is largely unchanged with temperature and guest association. We show that, with the new definition, flexible molecules can be shape-persistent if the differences in shape between their conformers are quantified to be small.</p><p>The conformational space of several macrocycles had been studied in the past13-17 but, to the best of our knowledge, their shape persistence had not been assessed. Considering Moore's phenylacetylene macrocycles, they were shown13 to have very few conformations, with only one unique conformation in the planar hexamer and two in the octamer (boat and chair). Similarly, prior work with triazolophane macrocycles showed only four low-lying, semi-planar conformations.14 These macrocycles clearly follow the structural definition of shape-persistence by displaying little conformational freedom. In these cases, shape persistence is self-evident. Other examples of macrocycles include the classic calixarenes,15 cyclic peptides,16 and crown ethers17 of host-guest chemistry. The last, crown ethers, are seen to collapse upon themselves thus not conforming to being shape-persistent, despite being considered pre-organized18 relative to their linear counterparts, podands. For the case of cyanostars, their shape persistence has yet to be formally analyzed. The potential for conformational freedom in the cyanostar is suggested by the crystal structure that shows accommodation of a shallow, bowl-like shape (Figure 1c). Cyanostar also possesses bowl chirality (Figure 1b)12,19 and, like other bowl-shaped compounds,20 is therefore expected to undergo bowl inversions21 that offer access to multiple conformations.</p><p>Bowl-shaped molecules can be shape-persistent. For example, they have been conformationally preorganized as chiral hosts by imposing extremely high inversion barriers as a means to achieve enantioselective recognition and asymmetric catalysis.5,19-20 In contrast, rapidly-inverting bowls that exhibit high conformational freedom have started to find applications such as in supramolecular polymerization by cofacial association.22 Aida and coworkers designed the first living and stereoselective supramolecular polymerization by using corannulene derivatives.22b In Aida's polymerization scheme, shape persistence of the monomers is critical for propagating the chirality in the growing self-assembled polymers; whereas flexibility in the initiator bowls allows production of either enantiomeric form of the monomer. Unlike corannulenes, however, cyanostar has a cavity in its center, a feature that provides a unique model for testing the shape persistence of cyanostars upon guest binding.</p><p>Herein, we report that conformationally rich flexibility can coexist with shape persistence in the cyanostar macrocycle. This assertion is based on the results from density functional theory (DFT), molecular dynamics (MD),11,23 and NMR experiments. Although as many as 332 conformations are thermally accessible by single cyanostars, the Boltzmann-weighted variation in atomic positions relative to the most stable conformer leads to ~87% retention of the molecule's size,24 indicative of high shape persistence. Unlike the crystal of the 2:1 complex, the single macrocycle has a ruffled rim with two olefins rotated up and three down. The ensemble-averaged bowl shape of cyanostar is further verified by through-space 1H-1H NMR measurements based on nuclear Overhauser effect (NOE), which closely matches with theory to within ±2%. We also test the use of a diglyme guest and cofacial stacking of a second macrocycle as perturbations. We see the shape is retained based on the conformational analysis. By evaluating the conformational landscape, we are able to show that the steric hindrance between the two cofacial macrocycles, rather than the interactions with the weakly bound diglyme, is responsible for the shift in conformer population to enhance shape persistence. The correlations between theory and experiment substantiate the refined definition of shape persistence to enable inclusion of macrocycles that are both conformationally flexible and geometrically well-defined.</p><!><p>To investigate the conformational landscape and examine the shape persistence of a single cyanostar macrocycle, MD simulations and DFT were used (details of the two methods can be found in Supporting Information). To date, cyanostars have only been observed as guest-bound dimers,12, 25 and the strong tendency of cyanostar to form such 2:1 complexes has precluded our ability to examine single cyanostars experimentally.</p><p>The shapes of the cyanostar's conformations were analyzed geometrically. To facilitate data mining of the many MD-generated geometries, dihedral angles between the olefins and their neighbouring phenyl groups were measured to characterize local tilting (ΦCN, Figures 1c and S4) and the overall shape of the bowl. Using these dihedrals two isomerization phenomena were found. First, up-and-down motions of cyano-olefins change the bowl chirality of cyanostar (Figure 2a). Second, in-and-out motions change the orientation of cyano groups relative to the cavity but leave the bowl chirality unchanged (Figure 3a).</p><!><p>The up-and-down motions are closely associated with changes in bowl chirality of the cyanostar. Therefore, use of local chirality to augment the traditional definition of bowl chirality provides a means to describe the conformational diversity of cyanostars. Bowl chirality is usually defined globally20 by looking from the top into the bottom of the bowl (Figure 1b) and then using priority rules to define when symmetry is first broken when moving away from the benzene in either a clockwise (P) or counter-clockwise (M) direction. Here, we identified the P face to be the specific face of a single cyanostar in which a clockwise direction is observed. For simplicity, all top views of cyanostar are shown with P faces. Thus, cyano groups oriented towards the readers will have P local chirality and are oriented "up", and others with local M chirality are oriented "down" (Figure 2a).</p><p>To begin the conformation count, there are four pairs of up-and-down combinations of olefins that are possible (Figure 2b). They can be named using local chiralities: P5, M1P4, o-M2P3, m-M2P3; where o and m refer to the ortho and meta relationship of the two olefins with M chirality. Global chirality can also be interpreted from this nomenclature by the majority rule: monomers that have more P local chirality will be P in the globally.</p><p>The thermal accessibility of the four types of all-out conformations (Figure 2) was confirmed by MD simulations and independently by DFT geometry optimization (Figure 2). Furthermore, the root-mean-square deviation (RMSD) of atomic positions for the DFT-optimized P5 conformer matches with the X-ray solid-state structures12 to within 0.1 Å, reflecting the accuracy of DFT methods.</p><!><p>The rotation energy landscape of a single olefin was computed (Figure 3a) by utilizing the MD-generated distribution of dihedral angles. Four rotamers were identified (see minima in Figure 3a and representative images in Figure 3b) along with two types of barriers (rocking and rolling). The two global minima are out rotamers (ΦCN~ ±30°) and the high-energy minima are in rotamers (ΦCN~ ±120°). These rotamers can either be up (P) or down (M). The free energy difference between the two types of rotamers is sufficiently low (2.8 kcal / mol) that the in rotamers are thermally accessible.</p><p>The same energy profile was computed using DFT and the same four rotamers were found (Figure 3b). Interestingly, DFT shows that a complete 360º rotation is possible. With a barrier of 6.5 kcal / mol (Figures S2 and S3), this motion would be expected on a 10-ns time scale (assuming a typical 1013 pre-exponential factor for unimolecular reactions18). However, this motion was never observed in the MD simulation that covered a 400-ns time period. We attribute this apparent contradiction to differences in the calculation methods: In DFT, all of the internal coordinates are fully relaxed other than the rotating olefin; whereas in the MD simulations – and in reality – all internal modes are thermally excited. Thus, the rotations of nearby olefins in the MD simulation hinder the 360° rotation of any individual olefin.</p><!><p>Based on the up-down and in-out orientations of cyano groups, there will be 1024 mathematically possible conformations for single cyanostars. However, many combinations are not energetically accessible, except for a group of 332 conformations that are predicted to be thermally accessible (Supporting Information). These 332 conformations create a free energy landscape of equal populations of P and M chirality at both local and goal levels (Figure 4a and b). When the degeneracies are factored out, the landscape of 332 states can be simplified into 34 unique geometries, or 17 pairs of enantiomers, that exist as local minima (Figure S1).26 Overall, theory predicts a very rich conformational landscape for the single cyanostar macrocycle.</p><p>At equilibrium, the single macrocycle is not a perfect bowl but instead is ruffled with two out of the five cyano groups on opposite sides. DFT predicts that 82% of the total population will be P2M3 and M2P3 (Figure 4a), while MD predicts 66%. The relative stabilities of the various conformers can be understood by assessing dipole-dipole interactions between cyano-olefins, which carry a strong 5.3 D dipole moment. This value is comparable to the 5.4 D dipole moment of an analogous benzene-triazole-benzene triad that has been widely used in CH-based anion binding.4,14,27 Repulsions between local dipoles favor alternating orientations of the cyano groups. With five olefins, m-M2P3 or m-P2M3 conformation has the best up-down alternation and is thus the global minimum. The perfect bowl conformation present in the crystal structure of the dimers,12 P5 or M5, is the least stable with all dipoles pointing in the same direction. However, this perfect bowl provides a better environment for guest binding with all CH hydrogen bond donors focused towards the center of the dimer.</p><!><p>The 332 thermally accessible conformers undergo rapid isomerization and racemization (Figures 4b, S6, and S8). Although most bowl-shaped compounds have one global inversion,20a the simultaneous inversion of all five cyano groups in the cyanostar was never observed in MD simulations. Rather, local inversions involving movements of a single olefin contribute 94% of all stereoisomerizations, the two-olefin inversion pathways are limited (5%), and all others are rare. Consequently, the energetic profile for rocking single olefins between different all-out conformers was explicitly characterized by DFT (Figure 5).</p><p>The rocking motions are rapid. DFT-derived barriers (Figure 5) range from 0.7 to 1.7 kcal / mol, and are in good agreement with the MD ensemble-averaged rocking barrier of 1.5 kcal / mol (Figure 3). The very low barriers highlight again the flexibility of cyanostars at 298 K. It has been suggested11,28 that at 298 K, a barrier of 24 kcal / mol is a practical limit to distinguish between rigid and flexible conformers. Others consider slow exchange on the NMR time scale (16 kcal / mol) to demarcate rapid versus slow dynamics.29 With either standard, cyanostar rocking motions are much faster.</p><p>Rapid interconversions were confirmed using lifetime correlation functions that were calculated from the MD data (see Section 8.1 of Supporting Information). The expedited loss of coherence between local chiral centers was reflected by a short relaxation time of 35 ps. Beyond 200 ps, the correlation coefficient dropped to zero, suggesting that the olefins have completely lost the memory of their past status of chirality.</p><!><p>Cyanostar macrocycles are flexible: They rapidly convert between multiple conformations. Now we have to determine if they are shape-persistent.</p><!><p>The shape persistence of single cyanostars was determined from the coordinates of all 332 cyanostar conformers. Specifically, the Boltzmann-weighted RMSD relative to the global minimum provides a temperature-dependent determination of the shape deformation. To calibrate the relative significance of the shape changes, we refer the RMSD to the mean molecular radius (assuming the volume of the molecule is distributed as a sphere). Accordingly, a normalized shape-change index, Δσ, can be expressed: Δσ=∑iRMSD(i)•p(i)∕(3VCPK∕4π)1∕3 Where p(i) is the relative population of conformer i, and VCPK is the CPK volume of the molecule. The basic logic of the shape-change index is consistent with the concepts of shape similarity,24 and of conformational heterogeneity.30 Both concepts have been widely used in biology31 and in developing algorithms for conformational searches.32</p><p>Cyanostar has a small shape-change index, Δσ. Using DFT-optimized geometries, the typical RMSD from m-P2M3 to other out conformers of a single cyanostar is 0.4 Å (Table S2). The Boltzmann-weighted RMSD across all conformers was found to be 0.7 Å. Considering the mean molecular radius of 5.5 Å of cyanostar, the normalized shape change index (Δσ) is only 0.13 for single cyanostars in solution at room temperature. In other words, the flexibility of cyanostar produces, on average, only 13% variation in its radius. Thus, there is a corresponding 87% shape persistence that originates in the relatively small RMSD values. The modest variations in shape can also be seen from the MD simulations (see Movies S1 and S2). When the intensity of the stimulus is lowered by reducing the temperature in the thermal bath to 218 and 178 K, the shape persistence increases to 91% and 93%, respectively. These theoretical findings unambiguously show cyanostars are shape-persistent as well as being flexible.</p><p>Interestingly, the noticeable dynamic motions of the single cyanostar macrocycle originate entirely from the olefinic units while the aromatic units remain quite static (see Movie S1). Thus it can be understood that the aromatic component of the backbone is contributing to the shape persistence of cyanostar and the rocking-and-rolling olefins are responsible for the flexibility. This observation may be the reason why cyanostar macrocycles are more flexible than other aromatic-based receptors.13,14</p><!><p>The accuracy of the theoretical calculations were verified experimentally using quantitative NOE experiments.33 NMR spectra obtained at 212 K are consistent with fast exchange between conformers on the NMR time scale (see Section 11 of Supporting Information). The relatively static aromatic components provide a reference point to determine intramolecular proton-proton distances from olefinic proton, Hc, to the aromatic ones, Ha and Hd. These enable calculation of the ensemble-averaged distances and thus the bowl shape of single cyanostars. Specifically, a ratio of two proton-proton distances, n, was measured from a 1H-1H 2D NOESY experiment (Figure 7). This distance ratio (n) was shown from DFT (Figure 7b) to decrease steadily with increasing dihedral angle. Thus, the average depth of the bowl and the shape of single cyanostars can be determined.</p><p>The quantitative NOESY experiment indicates that the conformational ensembles generated by the DFT and MD approaches are quite accurate (Table S22). The range of possible distance ratio (n) can be related to a perfectly planar cyanostar (n = 1.9) and a conformation with the olefin making a ~60 angle (n ~ 1.4). The data analysis on NOE experiments yielded n values of 1.6, clearly indicating that cyanostars on average have the ruffled out conformation in solution. The distance ratio obtained from the NOE experiment is smaller than the crystal structures (P5, n = 1.7) , which is consistent with the existence of multiple conformations, including in rotamers with a distance ratios of n ~ 1.3. To our satisfaction, distance ratios predicted by theory deviate only by 0.03 from experimental values (2% error), with MD slightly overestimating planarity (1.63) and DFT slightly underestimating it (1.57).</p><!><p>With the shape persistence of flexible cyanostars confirmed for the single macrocycle at various temperatures, we made the same assessment as a result of guest binding.</p><!><p>The shallow free energy surface of single cyanostars suggests that even modest external forces are sufficient to perturb the conformer distribution, shape persistence and flexibility of cyanostars. To test this idea, the impact of guest binding was examined using the cyanostar-diglyme model system seen in the 2:1 binding stoichiometry in the crystal.12 Specifically, we focus here on the meso-dimer formed by the rim-sharing arrangement of one bowl with P chirality and the other with M chirality.34 The weak perturbation introduced by diglyme binding is manifested by the modest 2:1 binding constant, β2, of only 30 M−2 (2 kcal / mol; see Section 10 of Supporting Information). MD simulation was the primary modeling tool for the 2:1 complex, as its size of ~300 atoms prevents DFT from efficiently characterizing the entire conformation space.</p><p>MD simulations of the 2:1 cyanostar-diglyme complex revealed substantial motion of hosts and guest within the complex. The bound diglyme was observed to gyrate within the cavity, and occasionally displayed escape and rebinding dynamics (Figure 8a, e, and i). Upon cooling to 218 K, the dissociation is diminished enough to resemble the corresponding crystal structures (Figure 8a). Cyanostars in the dimers showed partial rotations, although restricted by the steric gearing of tert-butyl groups between the pair of stacked macrocycles (Figure 8b, f, and j).</p><p>Consistent with our hypothesis, binding of diglyme to the dimers dramatically alters the conformational landscape of cyanostars. Compared to the flexible free macrocycle (Figure 4), the population of conformers in the 2:1 complex becomes highly concentrated in the newly emerged global minimum of P5 or M5 with a shape of perfect bowls (Figure 8d, h, and l), predominating 85% of the time. The free energy differences between out and in conformers upon diglyme binding are raised from 2.7 to 3.9 kcal / mol; consequently, the overall population of in-rotamers dropped from 30% for the single macrocycle to 4% in the 2:1 complex. Rocking motions were infrequent and resulted in negligible bowl inversions: at 298 K, only 0.1% of all cyanostars in the dimer had an inversion of global chirality from their P (or M) starting point. Only 3% were inverted locally (Figure 8d, h, and l). The rigidification of cyanostar in the 2:1 complex upon diglyme binding was accompanied by suppressed olefin rotations and an extended chirality lifetime beyond 800 ns (Figure 6, vide supra). The steady state correlation coefficient remained high, indicating that >96% of the original chirality configuration was preserved (Figure S34). The number of thermally accessible conformers is reduced upon formation of the 2:1 complex around diglyme from 332 to 20 (P5, M1P4, o-M2P3, and m-M2P3 with their five-fold degeneracy).35</p><p>The shape persistence of the cyanostars in the 2:1 complex increased over the single macrocycle. Using the MD-simulated population and DFT-optimized geometries of single cyanostars with the corresponding chirality, the shape change index (Δσ) associated with the thermal energy present at room temperature was estimated to be 2%. This finding indicates that the shape persistence of cyanostar was greatly enhanced in the 2:1 complex up to 98%.</p><p>The change in conformational landscape for the sandwich complex can be attributed to either one or two structural features (Figure 9). First, steric hindrance between tert-butyl groups on one macrocycle and cyano groups from the other macrocycle could play a role (Figure 9). Rocking any olefins from the top macrocycle towards the other macrocycle generates more steric contacts. Second, hydrogen bonding between cyanostar and diglyme could also be a factor. The perfect bowls maximize guest binding when all of the olefinic CH donors are pointed towards the center between the two macrocycles (Figure 9, "better binding" label). Rotating any P olefins into M olefins would redirect the hydrogen bond donor away from the bound guest.</p><!><p>MD simulations show that steric hindrance from cofacial stacking and not hydrogen bonding from guest binding alters the conformational landscape. This finding emerges from an examination of a putative 1:1 complex, in which the second cyanostar partner was absent. The equilibrium distribution of conformers for the 1:1 complex is found to be almost identical to the distributions for the case of the free cyanostar (Figure 10b). Even the lifetime correlations and relaxation of local inversions are indistinguishable from those in the case of a single macrocycle (see Section 8 of Supporting Information). The only noticeable change is a slightly increased free energy differences between the in and out rotamers by 0.2 kcal / mol, reflected by the reduced number of in rotamers in the 1:1 complex (Figure 10b). The negligible changes between the free macrocycle and the 1:1 cyanostar-diglyme complex indicate that steric hindrance between sandwiched macrocycles play a decisive role36 in reducing the flexibility of olefinic units and thus enhancing the overall shape persistence in the 2:1 complex.</p><!><p>In conclusion, we have shown that flexibility can coexist with shape persistence in cyanostar macrocycles. This coexistence appeared to be a contradiction until we defined shape-persistent molecules as having highly similar shape in response to an external perturbation. When placed in a thermal bath, the conformers were calculated to have similar shapes and resulted in 87% shape persistence. The ensemble averaged geometries matched experimental values determined by NOE experiments and corresponded to an overall puckered bowl for the single cyanostar. The shape persistence and flexibility is unperturbed in response to binding a diglyme guest molecule. Yet, cofacial stacking of a second cyanostar macrocycle shows enhanced shape persistence of 98% and leads to perfect chiral bowls being the preferred ensemble averaged structure. These findings show that macrocycles need not be completely rigid to enjoy the same advantages of traditional shape-persistent macrocycles.</p>

PubMed Author Manuscript

Possible Existence of \xce\xb1-Sheets in the Amyloid Fibrils Formed by a TTR105\xe2\x80\x93115 Mutant

Herein, we combine several methods to characterize the fibrils formed by a TTR105\xe2\x80\x93115 mutant in which Leu111 is replaced by the unnatural amino acid aspartic acid 4-methyl ester. We find that this mutant peptide exhibits significantly different aggregation behavior than the wild-type peptide: (1) it forms fibrils with a much faster rate, (2) its fibrils lack the long-range helical twists observed in TTR105\xe2\x80\x93115 fibrils, (3) its fibrils exhibit a giant far-UV circular dichroism signal, and (4) its fibrils give rise to an unusual amide I\xe2\x80\xb2 band consisting of four distinct and sharp peaks. Based on these results and also several previous computational studies, we hypothesize that the fibrils formed by this TTR mutant peptide contain both \xce\xb2- and \xce\xb1-sheets.

possible_existence_of_\xce\xb1-sheets_in_the_amyloid_fibrils_formed_by_a_ttr105\xe2\x80\x93115_mutan

INTRODUCTION<!>RESULTS AND DISSCUSION<!>CONCLUSIONS<!>Sample Preparation<!>Atomic Force Microscopy Measurements<!>Transmission Electron Microscopy Measurements<!>UV-Vis Absorption and Fluorescence Measurements<!>Circular Dichroism Measurements<!>Fourier Transform Infrared Spectroscopic Measurements<!>Two-dimensional Infrared Measurements

<p>Amyloids formed by the transthyretin (TTR) protein have been linked to various diseases, including Senile Systemic Amyloidosis, Familial Amyloid Polyneuropathy, Familial Amyloid Cardiomyopathy, and Central Nervous System Selective Amyloidosis.1–3 As such, numerous studies have focused on TTR amyloid fibrils, ranging from structure determination to elucidation of the underlying fibrilization mechanism.4–9 In particular, an 11-residue segment of TTR, TTR105–115 (sequence: YTIAALLSPYS where the numbering is consistent with the full-length protein) has been widely used as a model system in this regard, as this segment is known to form 'cross-β' amyloid fibrils similar to those formed by the full-length protein.10,11 Recently, Griffin, Dobson, and co-workers have characterized the atomic structure of TTR105–115 fibrils,11 using a combination of structural techniques, including magic angle spinning nuclear magnetic resonance (NMR), cryoelectron microscopy (cryo-EM), X-ray diffraction, scanning transmission electron microscopy (STEM), and atomic force microscopy (AFM). They found that these fibrils are formed from four, six, or eight two-sheet proto-filaments which are aligned in a head-to-tail manner, with each protofilament consisting of parallel, in-register β-sheets that stack in an antiparallel fashion (Figure 1). In addition, their structure models suggested that structured water exists in the fibril core11 which initially motivated us to introduce a site-specific infrared (IR) probe into the sequence of TTR105–115 and use both linear and nonlinear IR spectroscopic techniques to assess the dynamics of such confined or structured water, a topic that has attracted considerable attention in the past few years.12 Specifically, we mutated Leu111 to aspartic acid 4-methyl ester (DM), which is a well-established protein IR hydration probe.13 The choice of this mutation is based on the fibril structure (Figure 1 and Figure S1 in SI), which shows that 50% Leu111 sidechains are buried in a water-inaccessible region and the rest are exposed to confined water. Much to our surprise, our spectroscopic results suggest that this TTR105–115 mutant (hereafter referred to as TTR-111DM) forms fibrils that are distinctly different from those formed by the wild-type peptide, as the former consist of both β- and α-sheets.</p><p>The α-sheet structure was first proposed by Pauling and Corey as a secondary structural element of proteins.14 As indicated (Figure S2 in SI), the structure of an ideal α-sheet is similar to that of an ideal β-sheet as both have the same meridional repeat distance (4.75 Å) and the same average hydrogen bond distance (2.3 Å); however, they differ in the orientation of their respective backbone NH and CO groups. In the β-sheet structure, the NH and CO groups alternate on either side of the sheet, while all the NH (CO) groups in an α-sheet are aligned on one side of the peptide backbone pleat.14–16 Therefore, each NH/CO group in an α-sheet would form two hydrogen-bonds (H-bonds) with two CO/NH groups on a neighboring strand.15 Comparably, β-sheets are more stable than α-sheets, which lead Pauling and Corey to reject the α-sheet structure as an "important" configuration in proteins.14,17 Indeed, very few α-sheet structures have been found in native proteins,15 although they can be induced to form in short peptides consisting of alternating D- and L-amino acids.18,19 Interestingly, however, α-sheet conformations are frequently observed in molecular dynamics (MD) simulations of amyloid-forming proteins, including TTR, β2-microglobulin, lysozyme, the prion protein, and polyglutamine, particularly in the regions of the protein which are considered the most amyloidogenic experimentally.15,16,20–22 In this regard, the α-sheet has been postulated to be a common intermediate state in the formation of amyloid fibrils and, also, the underlying structure of the toxic amyloid oligomers.15 Nevertheless, to the best of our knowledge, this structure has never been observed experimentally to exist in either transiently populated amyloid oligomers or mature fibrils, making it an elusive target for further studies. Thus, the experimental confirmation of this structure in amyloid fibrils will open up new avenues for providing a better under-standing of the biological role of α-sheets.</p><!><p>As shown (Figure S3), like the wild type TTR105–115 peptide,11 TTR-111DM readily aggregates and forms fibrils at concentrations of a few millimolar. However, the amide I′ (amide I in D2O) bands of these peptides, when aggregated, show significant differences (Figure 2). The amide I band, which arises mainly from the backbone C=O stretching vibrations, is a well-established IR reporter of protein conformations. This is because the amide I band of a polypeptide manifests the underlying vibrational coupling between the individual amide units, an interaction depending on its secondary structure.23 For example, the amide I′ band of amyloid fibrils consisting of either parallel or antiparallel β-sheets is characterized by a strong and narrow peak located at a frequency of less than 1630 cm−1, whereas fibrils composed of antiparallel β-sheets also give rise to a weak amide I′ peak at a frequency of 1680 cm−1 or higher.23–25 Therefore, the FTIR results in Figure 2 provide the first evidence indicating that the Leu111 to DM mutation in TTR105–115 leads to a change in the underlying fibril structures. Consistent with the fibril structures of Fitzpatrick et al.,11 the amide I′ band of aggregated TTR105–115 peptides shows characteristics of parallel β-sheets,26–28 as it is dominated by a pair of narrow peaks located at ~1625 (strong) and ~1665 cm−1 (weak), respectively. Further two-dimensional infrared (2D IR) measurements show that a cross-peak at (ωpump, ωprobe) = (1665 cm−1, 1625 cm−1) is developed at early waiting times (Figure S4 in SI), indicating that these peaks arise from the same fibrillar species and not from heterogeneity in the sample.</p><p>In comparison (Figure 2), the FTIR spectrum of an aggregated TTR-111DM sample in the amide I′ region contains more resolvable spectral features. As expected, the ester carbonyl of the DM sidechain gives rise to an additional IR band outside the amide I′ profile, which can be well fit by a Gaussian function with a peak frequency of 1744.8 cm−1 and a width (i.e., FWHM) of 7.2 cm−1 (Figure S5 in SI). According to the study of Pazos et al.,13 this frequency indicates that the ester carbonyls in the fibrils are situated in a dehydrated environment, an unexpected result. Based on the fibril structures of TTR105–115 (Figure 1 and Figure S1 in SI), 50% of the sidechains at position 111 would face towards the hydrated core of the fibril and, hence, should produce an IR band at a frequency of 1730 cm−1 or lower due to H-bonding interactions with water. To verify that the fibrils produced by both the wild-type and mutant TTR105–115 peptides indeed contain confined water, we measured the FTIR spectra of the respective fibril samples in the form of a dry film. As shown (Figure S6 in SI), the data clearly show that even for fibril samples that have been dried on the surface of a Ge crystal under vacuum for at least 24 hours, the D2O IR band12 at ~2575 cm−1 is still present. This result indicates that fibrils formed by both the wild-type and mutant TTR peptides can host trapped water, as initially suggested.11 Thus, the fact that the DM sidechain in the TTR-111DM fibrils is not located in a hydrated environment suggests that the corresponding Leu111 to DM mutation changes the fibril structure(s). A more convincing evidence in support of this notion comes from the amide I′ band of the mutant sample, which consists of four resolvable, narrow peaks, at ~1616, ~1652, ~1662, and ~1672 cm−1, respectively (Figure 2). The peaks at 1616 and 1652 cm−1 likely correspond to those observed in TTR105–115 fibrils,26 with a modest shift toward lower wavenumbers. This similarity suggests that the fibrils formed by TTR-111DM contain parallel β-sheets. The question is then what produces the other two spectral features.</p><p>One possibility is that the 1662 and 1672 cm−1 peaks arise from non-aggregated peptides and residual trifluoroacetic acid (TFA) in the fibril sample, respectively. This is because the amide I′ band of a disordered peptide is centered around 1650 cm−1 and TFA, which was used in the peptide synthesis and purification, exhibits a strong IR signal at 1672 cm−1. To test this possibility, we followed the aggregation/fibrillization process of the TTR-111DM peptide by measuring its FTIR spectra as a function of time. As shown (Figure 3), the amide I′ band obtained at 'zero time' is dominated by a broad feature centered at ~1650 cm−1, which directly rules out the possibility that the 1672 cm−1 peak originates from TFA. Furthermore, all of the fine spectral features are developed concomitantly, at the expense of the broad 1650 cm−1 feature, which, therefore, argues against the idea that the 1662 cm−1 peak corresponds to non-aggregated peptides. This argument is further corroborated by the ester carbonyl signal of the DM sidechain. If a significant amount of peptide monomers were still present, one would expect the presence of a broad peak at 1725 cm−1, as that observed at 'time zero', in the spectrum collected at 315 minutes, which is not observed (Figure 3 inset). In fact, the fairly narrow bandwidth of the ester carbonyl band of the fully aggregated sample provides strong evidence indicating that the DM sidechains in the fibrils sample a rather homogenous environment. In support of this picture, the 2D IR spectrum of the ester carbonyl band at T = 0 shows little, if any, elongation (Figure S7 in SI). Thus, taken together these results indicate that the four narrow amide I′ peaks of TTR-111DM originate from the underlying fibril structure, rather than from any sample heterogeneity. Moreover, the time dependence of the amide I′ band of TTR-111DM (Figure 3) indicates that its fibrils are formed within ~5 hrs, which is much faster than the fibrilization rate of the wild-type peptide measured under similar experimental conditions.11</p><p>Theoretical calculations have predicted that the amide I′ frequency of the α-sheet is different from its β-sheet counterpart. For example, the density functional theoretical study of Torii29 indicated that the α-sheet conformation of a glycine dipeptide gives rise to a strong amide I′ peak between 1670 – 1690 cm−1 and a less intense one between 1630 – 1650 cm−1. Similarly, the quantum mechanics calculation of Huo and coworkers30 on the amide I band of an alanine dipeptide predicted that the α-sheet conformation would exhibit a strong peak at 1653 cm−1, with two smaller ones at 1623 and 1681 cm−1. Therefore, based on these theoretical predictions, a more plausible interpretation of the atypical amide I′ band shape of the TTR-111DM fibrils is that it contains contributions from both β- and α-sheets. Because of the narrowness of the ester carbonyl vibrational band, it is unlikely that the aggregated TTR-111DM sample is composed of two types of fibrils, with one consisting of α-sheets and the other of β-sheets. Therefore, we hypothesize that the fibrils are assembled from protofibrils consisting of both β- and α-sheets. To further validate this notion, we carried out 2D IR measurements on TTR-111DM fibrils. Should those 4 amide I′ bands indeed share the same structural origin, we expect to observe 2D IR cross-peaks developed between them due to either vibrational coupling or energy transfer. As shown (Figure 4), the 2D IR spectrum obtained with <XXXX> polarization configuration at T = 0 shows cross-peaks between the 1662 and 1672 cm−1 bands. In addition, 2D IR spectra measured with <XXYY> polarization configuration show enhanced cross-peaks between the 1652 cm−1 and two higher-frequency bands and those obtained at longer waiting times indicate the development of cross-peaks between the 1616 cm−1 and other three bands (Figure S8 in SI). Taken together, these 2D IR results pro-vide strong evidence indicating that the unique amide I′ band shape of the TTR-111DM fibrils does not arise from sample heterogeneity, but instead supporting the hypothesis that it manifests the difference in the underlying backbone-backbone H-bond arrangements.</p><p>The protein amide A band mainly arises from the backbone N-D stretching vibrations (in D2O). Therefore, the difference between the backbone-backbone H-bonding patterns of the β- and α-sheets is expected to affect not only their amide I′ bands, as observed (Figure 2), but also their amide A vibrations. Indeed, the aggregated TTR105–115 and TTR-111DM samples show distinct difference in their amide A bands, as seen from ATR-FTIR spectroscopic measurements (Figure S6 in SI). This provides additional evidence supporting the notion that the underlying fibrillar structures of these two peptides are not identical.</p><p>Further supporting evidence comes from the far-UV circular dichroism (CD) spectra of TTR105–115 and TTR-111DM. As indicated (Figure 5), the CD spectrum of an aggregated TTR105–115 sample is similar to that observed previously.31 However, in comparison the CD spectrum of an aggregated TTR-111DM sample is not only different in shape from that of TTR105–115 but also exhibits a giant CD signal at 205 nm. This is an amazing result, as, to the best of our knowledge, such a giant CD signal has never been observed in a peptide or protein system. It is well known that certain amino acids, such as tryptophan, can produce additional CD signals (i.e., a CD couplet) in the far-UV spectral region due to exciton couplings.32,33 Therefore, the giant CD signal observed for the TTR-111DM aggregates is most likely due to additional contributions from the DM sidechains, as methyl acetate, a model compound for the sidechain of DM, exhibits a strong far-UV absorption band, peaked at ~205 nm in water (Figure S9 in SI). Since the magnitude of an exciton CD couplet is sensitively dependent on the relative positions (i.e., distance and orientation) of the two coupled chromophores,34 the greatly enhanced CD signal at 205 nm suggests that in the fibrils, the DM sidechains from two in-register sheets must be in close proximity. This agrees with the linear and 2D IR results showing that the ester group of DM is not only buried in a dehydrated environment but also experiences little, if any, heterogeneity. In support of this picture, the far UV CD spectrum of TTR-111DM monomers only show typical spectral features associated with unstructured peptides (Figure S10 in SI). It is also possible that the giant CD signal is due to exciton couplings involving both electronic transitions arising from the peptide backbone and the DM sidechains. Yet, another possible scenario is that the underlying fibril structure supports a supramolecular chirality, leading to a large in-crease in the CD signal, as has been observed for chiral helical stacks.35–38 Irrespective of the interpretation, these CD spectra once again indicate that TTR-111DM form fibrils with a distinct structure.</p><p>The most direct evidence indicating that the fibril structures of the wild-type and mutant aggregates are dissimilar comes from the AFM images of individual fibrils formed by TTR105–115 and TTR-111DM. It is apparent that the dimension perpendicular to the long fibril axis (hereafter referred to as d) is larger for the TTR-111DM fibrils than for the TTR105–115 fibrils (Figure 6). A more quantitative assessment further substantiates this notion as it indicates that the height (h) of the TTR105–115 fibrils is about 10–12 nm, which is on the same order as measured previously,11 whereas the TTR-111DM fibrils have a h of about 4–6 nm and a d about 25–45 nm (Figure 6). This aspect ratio seems to suggest that the TTR-111DM fibrils adopt a ribbon-like structure; however, the fact that these fibrils contain confined water, even under dry conditions (Figure S6 in SI), argues against this picture. Moreover, a two-dimensional fast Fourier transform (2D-FFT) analysis of individual fibrils reveals, as shown (Figure S11 in SI), that the wild-type fibrils have an intrinsic twist along the long fibril axis, with a periodicity of ~83 nm, a value similar to that reported in other studies for TTR105–115 (95 ± 10 nm).11 On the contrary, the corresponding 2D-FFT analysis of the TTR-111DM fibrils indicates that they have no detectable twists or pitches (Figure S11 in SI). In this regard, the morphology of the TTR-111DM fibrils resembles that of tape-like fibrils observed in various amyloid aggregates.39 For example, the AFM image of a tape-like fibril formed by apo-α-lactalbumin shows that it has a h of ~5 nm and a d of ~26 nm and exhibits no detectable twists along the fibril axis.40</p><p>Hayward and Milner-White have predicted through molecular dynamics simulations that parallel α-sheets tend to assemble into nanotube-like structures.41 Therefore, to provide further information on the morphology of TTR-111DM fibrils, we examined them using transmission electron microscopy (TEM). As indicated (Figure 7 and Figure S12 in SI), the TEM images also shows that this mutant peptide assembles into fibrils that lack the long-range helical twists observed in TTR105–115 fibrils,11 which have a cross-over distance of ~95 nm. Thus, these results suggest that the TTR-111DM peptide forms either non-twisting fibrils or fibrils with a twist that is not detectable by the current method, hence consistent with the AFM result. A closer examination of the TEM images (Figure S12 in SI) reveal, however, that the fibril width (9 ± 2 nm) is ~3–5 times smaller than that determined via AFM measurements. One likely interpretation of this discrepancy is that the experimental conditions used in TEM measurements (e.g., staining and vacuum) lead to disassembly of thicker fibrils and convert them into thinner ones. Another possibility is that the procedures used to prepare AFM samples promote thicker fibril formation.42 Irrespective of the underlying cause of this discrepancy, the fact that both types of images show that the TTR-111DM fibrils do not exhibit the commonly observed, long-range helical twists suggests that conversion of a thinner fibril to a thicker one is through lateral association of protofilaments without braiding, a mechanism that has been earlier proposed to explain the formation of flat, tape-like fibrils.40</p><p>To ensure that the non-twisting fibril morphology observed in both AFM and TEM measurements is not due to their special experimental conditions, we used the induced circular dichroism (ICD) method43 to further characterize the twist of TTR-111DM fibrils in solution. The ICD method uses the induced CD signal of an amyloid-bound dye, for example thioflavin T (ThT), to characterize the supramolecular chirality of amyloid fibrils. It has been demonstrated that fibrils with distinct helical twists can display a strong ICD signal in the absorption spectrum region of the dye.39,43 As shown (Figure S13 in SI), in the presence of TTR-111DM peptide, the ThT fluorescence intensity is significantly increased, indicating of ThT binding to fibrils; however, the same solution does not show any appreciable ICD signals (Figure S13 in SI). In agreement with Loksztejn et al.43 fibrils with periodic twists such as an Aβ16–22 derivative, Aβ16–22F19K*,44 was also found to have an observable ICD signal (Figure S14 in SI). Thus, this result corroborates the notion that TTR-111DM forms fibrils without long-range twists.</p><p>All of the experimental results support the notion that the Leu111 to DM mutation in TTR105–115 significantly changes the fibrilization energy landscape of this short peptide, resulting in fibrils of different structure and morphology. However, the spectroscopic methods used in this study do not allow us to assess the detailed molecular arrangements of the peptide molecules in the fibrils. Nevertheless, based on the IR data, we speculate that the protofibrils formed by TTR-111DM could have a structure as depicted in Figure 8, where each sheet is a mix of α- and β-sheet characters with the DM sidechains packed between two sheets and hence in a dehydrated environment. While further studies using high-resolution structural techniques are required to validate this model, it is nevertheless consistent with the study of Armen et al.,20 which showed, via MD simulations, that at low pH, an α-sheet intermediate is formed in the 105–115 region of the full-length TTR monomeric protein. Furthermore, the authors postulated that the α-sheet may be an important pathological conformation in neurodegenerative amyloidosis. Incidentally, the DM mutation used in the current study is similar to that (i.e., Leu111 to Met) found in the full-length TTR aggregates associated with a Danish variant of familial amyloid cardiomyopathy.45 Therefore, it would be interesting to explore, in future studies, whether TTR-111DM aggregates exhibit a stronger toxicity toward human cells.</p><!><p>In summary, we have employed a combination of biophysical techniques to characterize the structural characteristics of the fibrils formed by TTR105–115 and a Leu111 to DM mutant (TTR-111DM). We found that the TTR-111DM fibrils exhibit quite unique spectroscopic and morphological properties: (1) their DM sidechains give rise to a single and narrow IR band at ~1745 cm−1, signifying a rather homogenous fibril population; (2) their amide I′ band consists of a new pair of strongly coupled peaks; (3) they produce a giant far-UV CD signal; and (4) they have no detectable pitches, or twists, along the long fibril axis. Taken together, these results not only indicate that the fibrils formed by TTR-111DM are structurally different from those formed by the wild-type peptide, but are also, according to previous simulations studies, best explained by a view that the TTR-111DM fibrils consist of both β- and α-sheets. However, it is worth noting that IR spectroscopy is unable to provide a full atomic view about the fibril structure in question and, hence, we cannot rule out other interpretations of our spectroscopic results. Nevertheless, because of the elusiveness of α-sheet and its potential role in amyloid diseases, we hope that our work will inspire further structural studies on the TTR-111DM fibrils, using computational and high-resolution structural methods.</p><!><p>The details about peptide synthesis and purification are given in the Supporting Information. All peptide samples were prepared initially by dissolving the corresponding lyophilized peptide in solvent composed of 10% acetonitrile (ACN) and 90% D2O (pD 2) with a final monomer concentration of ~4 mM. Procedures for fibril formation are described in the Supporting Information.</p><!><p>AFM samples were prepared by placing ~5 μL of the aggregated peptide sample in question directly on a clean mica sheet/strip, which was then rinsed with 200 μL of Millipore water and was subsequently dried using N2 gas. AFM images were obtained on a Bruker Dimension Icon Atomic Force Microscope and were processed using the free Gwyddion software.</p><!><p>TEM samples were prepared by placing ~3 μL of an aggregated peptide solution on a thin carbon film (deposited on a copper grid) which was initially made hydrophilic through glow discharging. After 1 minute, the solution was gently blotted with filter paper and then ~3 μL of a 1% uranyl acetate solution (pH 4.5) was added to stain the sample. The resultant solution was then blotted, which was followed by another round of staining using the same procedure. TEM image was obtained with a JEOL 1010 transmission electron microscope operated at 80 KV, equipped with a Hamamatsu digital camera (250,000 magnification) and the images were processed using the free AMT Advantage Image Capture software.</p><!><p>UV-Vis absorption spectra were collected on a Jasco V-650 UV-Vis spectrophotometer using a 1.0 cm quartz cuvette at room temperature. Fluorescence spectra were taken on Jobin Yvon Horiba Fluorolog 3.10 fluorometer.</p><!><p>Circular dichroism (CD) spectra were collected on an Aviv CD 62A DS spectrometer with an averaging time of 20 s. For fibril samples, the solution was held between two CaF2 windows separated by a 6 μm spacer; whereas for monomer samples, a regular quartz CD cuvette with a pathlength of 1 mm was used. For all presented CD spectra, a background spectrum measured with only the corresponding solvent but otherwise under the same conditions has been subtracted.</p><!><p>Fourier Transform Infrared (FTIR) spectra were collected on a Nicolet 6700 FTIR spectrometer with a spectral resolution of 1 cm−1 and an average of 256 scans, using a sample holder made with two CaF2 windows and a 50 μm Teflon spacer. Attenuated total reflectance Fourier transform IR (ATR-FTIR) spectra were collect using a Horizon ATR accessory (Harrick Scientific Products, Inc.) for the Nicolet 6700 spectrometer. The aggregated peptide sample in question was first placed onto to the germanium (Ge) crystal and was then dried under vacuum for at least 24 hours before measurement.</p><!><p>2D IR spectra were collected on a homebuilt 2D IR setup that has been described in detail previously.46 Briefly, three femtosecond IR pulses (k1, k2, and k3, respectively) were focused onto the sample, which was sandwiched between two CaF2 windows separated by a 25 μm Teflon spacer, in a box-CARS geometry to generate the desired third-order response. This response was then heterodyned with a local oscillator (LO) pulse, and the combined signal was further dispersed by a monochromator and detected with a 64-pixel liquid nitrogen-cooled IR array detector (Infrared Associates, FL). By scanning the time delay (τ) at 2 fs steps between the k1 and k2 pulses from −3.5 to 4.0 ps, the rephrasing and nonrephasing data were measured and added up to obtain the absorptive 2D spectrum for a fixed delay time (T) between k1/k2 and k3 pulses.</p>

PubMed Author Manuscript

HCM and DCM cardiomyopathy-linked \xce\xb1-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human \xce\xb1-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations.

hcm_and_dcm_cardiomyopathy-linked_\xce\xb1-tropomyosin_mutations_influence_off-state_stability_and_c

Introduction<!>Molecular Dynamics<!>DNA Construction and Protein Purification<!>Tropomyosin Binding and Reconstitution of Thin Filaments<!>Calcium-Regulated Motility<!>Myosin-dependent activation<!>Statistics<!>Molecular Dynamics of Mutant and Wild-Type Tropomyosin<!>Mutant tropomyosin flexibility<!>Altered local affinity of mutant tropomyosins interfere with myosin-driven thin filament motion<!>Velocity/pCa Relationship<!>Myosin-dependent activation<!>Discussion<!>

<p>Tropomyosin is a coiled-coil protein that binds along the long-pitch helix of actin. Tropomyosin, in concert with the troponin complex, inhibits myosin-binding, which leads to inhibition of striated muscle contraction that causes muscle relaxation. To explain the kinetics of this process, McKillop and Geeves [1,2] introduced a three-state kinetic model for thin filament activity and its regulation, describing blocked, closed and open states, where only the open-state is fully activated, the blocked-state is fully inhibited and the closed-state represents an intermediate off-state between the two end states. Subsequent support for the kinetic scheme came with structural evidence [3], showing that tropomyosin alternates between occupying one or another of three defined positions on the actin filament, viz. B-, C- and M-states. Here, the three structural states display different levels of obstruction and exposure of the myosin-binding site on actin, depending on calcium binding to troponin and myosin-binding on actin. In each of these schemes, tropomyosin acts as an inhibitor in two of these states (blocked/B and closed/C), absent myosin interactions, with the blocked/B-state being stabilized by the calcium-free troponin complex [4–7]. The probability of tropomyosin occupying either of the two off-states depends on a number of factors in addition to cytosolic calcium concentration altering troponin conformation. For example, ionic strength, pH, isoform [8], stretch [9] as well as a variety of mutations, may alter tropomyosin affinity for, or position on, actin [10].</p><p>Thin filaments examined structurally at low intracellular levels of calcium reveal that tropomyosin, in fact, is constrained toward the outer domain of successive actin molecules blocking myosin-binding sites along thin filaments (the B-state position). Calcium-binding to the TnC-subunit of troponin induces a conformational change in the entire troponin complex that promotes tropomyosin movement toward the inner domain of actin (the C-state position). This transition, while still in an off-state, partially frees the "closed" myosin-binding site and facilitates weak non-force producing myosin-interaction, which then likely auto-induces even further tropomyosin movement to an open M-state position and leads to full muscle activation [3,11]. The correspondence between steps in the structural and kinetic descriptions suggest that they are manifestations of the same process.</p><p>Molecular dynamics simulations guided by reconstructions of muscle thin filament electron micrographs reveal that each tropomyosin molecule interacts with seven consecutive actin monomers by means of a locally repeating pattern of weak electrostatic interactions [12,13]. Energy landscape calculations [14,15] additionally show that tropomyosin position on actin is determined by a shallow interaction-potential well close to its corresponding location in the low-calcium blocked/B-state [14–16]. Absent myosin, the electrostatic interaction energy between actin and tropomyosin is significantly weaker in the open/M-state [15,16]; however, binding is typically robust in the open/M-state because, in addition to interacting with actin, tropomyosin is normally trapped topologically on actin in a binding channel formed by the inner edges of actin and the bound myosin-head [17]. Thus, disease-linked mutations that either weaken or strengthen interactions between actin and tropomyosin in the blocked/B-state or those flanked by myosin in the open/M-state will inevitably affect actomyosin interactions and hence, in distinct ways, contractility.</p><p>Our objective is to determine the effects of the mutations on actin-tropomyosin interactions and their impact on strong-binding of myosin cross-bridge formation during activation of contractility. We approach this objective by studying the performance of reconstituted thin filaments containing wild-type and a variety of disease-causing mutant tropomyosins. In the current work, we investigated two mutations, where based on energy landscape determination one has been predicted to effectively stabilize (E40K tropomyosin) and the other to strongly destabilize (E62Q tropomyosin) blocked/B-state thin filaments [14]. Indeed, we find that these mutations disrupt the regulatory switching mechanism in markedly distinct ways, in one case leading to decreased and in the other increased calcium-sensitivity of thin filament activation. The E40K point mutation occurs at an "e-heptad position" [18], normally participating in "e-g" pairing. e-g pairs in coiled-coils such as tropomyosin form an electrostatic ridge linking component chains of the dimer together while shielding hydrophobic, a and d, core residues from solvent ( Fig. 1A) [19]. In contrast, the E62Q mutation occurs at an f-heptad position [20], which would be solvent exposed in the canonical heptad repeat and in a position frequently engaged in tropomyosin-actin interaction. Thus, the effect of this mutation is likely to differ markedly from that of E40K ( Fig. 1B, C). Indeed, in the current study, myosin-dependent in vitro motility of actin filaments (in the presence and absence of troponin) showed reduced velocity when E40K tropomyosin was included in assays and conversely increased velocity when E62Q was added. These results are suggestive of an E40K mutation-induced stabilization of the tropomyosin in the blocked/B-state, and alternatively a destabilization by E62Q, as implied by prior energetics [14] and now corroborated by molecular dynamics measurements in the current work. Our computational analysis indicates that local and long-range alterations in tropomyosin conformation and in tropomyosin-actin interactions appear to underlie the altered regulatory switching. Our new data highlight likely residue-level specific perturbations, which translate into distinct disease-causing mechanisms, namely they suggest how two tropomyosin mutations influence blocked/B-state binding stability in different ways. In addition, the functional effects noted here cannot simply be based on changes in tropomyosin coiled-coil flexibility, frequently invoked to describe the action of tropomyosin mutations without convincing direct support.</p><!><p>Molecular dynamics was performed on isolated tropomyosin as detailed by Li et al. [21] and on the actin-tropomyosin complex as described previously [13]. In the latter case, a model of two tropomyosin coiled-coil dimers extending from either side of an overlapping domain on F-actin was represented by tropomyosin residues 134-1 on the N-terminal side of the overlap nexus linked to residues 284-234 on the C-terminal side, while the tropomyosin model associated with five actin subunits [13]. The mutations at positions 40 and 62 incorporated into this model are located in the middle of the structure used. Amino acid substitutions to characterize mutant tropomyosins were made in VMD [22] and 30 ns of MD of the acto-tropomyosin model in explicit solvent was carried out at 300 K as previously using the program NAMD [23]. The average structure throughout MD was calculated in CHARMM [24]. The frame of MD closest to average structures determined by its low Root Mean Square Deviation (RMSD) to backbone atoms was used for display in Figure 2.</p><p>The persistence length and local flexibility (δ) of isolated tropomyosin (i.e. free of actin) was assessed over the last 10 ns of a 30 ns MD calculation as in Li et al. [21,25]. Here, CHARMM was used for MD [24] performed in implicit solvent. The apparent persistence length (PLa) was calculated by the tangent correlation method as previously described [21,25], using the relationship ‹cos(θ[s(t)])›s(t)=exp[−s/PLa], where s is a segment of increasing arc length (of up to 300 Å) over which θ[s(t)] was measured at times t of the MD simulation. PLa is a measure of deviations of tropomyosin from a notional straight rod.</p><p>The local bending flexibility of tropomyosin (δ) was measured over the last 10 ns of simulation by taking each tropomyosin snapshot from MD trajectory files and dividing the individual tropomyosin conformers into overlapping segments consisting of nine successive residues acquired along the entire length of the molecule (i.e. residues i − i+9, i+1 − i+10, i+2 – i+11, i+3 – i+12, … i + 275 − i+284). Each separate segment was centered on and fitted to corresponding residues in the time-averaged tropomyosin structure and angular deviation between that segment and the average structure was calculated (i.e. the tangent of the segment to the super-helical average was quantified). The mean deviation determined at any position along tropomyosin during the 10 ns period defined the local δ [21,25].</p><p>The dynamic persistence length (PLd), a test of the overall flexibility of a curved rod-like tropomyosin, was calculated from an average of all local delta values using the equation cos‹δ›=exp[−s/Pd], where s, the nine residue segment, for each local delta value was 14.4 Å. These methods and corresponding algorithms are detailed previously [21,25].</p><!><p>Mutagenesis of wild-type human striated α-tropomyosin (Tpm1.1), containing the N-terminal extension Met-Ala-Ser, was detailed previously [14]. E40K mutant tropomyosin was generated by similar methods but with the primer pair: 5′ AGATGAGCTGGTTAGCCTGCAG 3′ (FORWARD PRIMER) 5′ TTCAGCTGCTTGCTACGATCCTC 3′ (REVERSE PRIMER). Mutant tropomyosin was prepared as described in [26].</p><!><p>To determine the apparent affinities of our tropomyosin variants for actin we performed co-sedimentation experiments with F-actin. In these experiments 7 μM F-actin was mixed with various amounts of human recombinant tropomyosin containing met-ala-ser and either the E40K mutation, E62Q mutation, or Wild-Type sequences. F-actin and tropomyosin were combined and incubated for 30 minutes. Tropomyosin bound to F-actin was isolated by centrifugation at 100,000 g, in an Airfuge (Beckman-Coutler) for 30 minutes. The resulting supernatant was then removed and the pellet was washed with a low salt solution to remove unbound proteins. The pellet was solubilized in an equal volume of gel running buffer containing 6M Urea and 10 mM β-mercaptoethanol and tropomyosin bound to F-actin was then determined via SDS-PAGE. Gels were stained with Coomassie Blue and bands corresponding to tropomyosin and actin were quantified via densitometry and ImageJ analysis. The amount of tropomyosin bound to actin was determined by first normalizing the actin in each lane to the total amount of actin used in the experiment. Then the amounts of tropomyosin were scaled to these new values and plotted versus the amount of free tropomyosin added to the solution. The resulting data were fit to a Hill-type cooperative binding model Y=Bmax*[Tm]h/(Kdh + [Tm]h) using GraphPad Prism 7.04 Software to determine apparent affinity and binding cooperativity.</p><p>Actin filaments were reconstituted for in vitro motility assays by incubating TRITC labeled actin (1 μM) with either wild-type, E40K, or E62Q tropomyosin (0.6 μM) overnight at 4°C. For experiments involving fully regulated thin filaments, human recombinant cardiac troponin complex (0.6 μM) was incubated with actin-tropomyosin for at least one hour. Reconstituted thin filaments were stored at 4 °C and used within five days.</p><!><p>In vitro motility assays were performed with porcine skeletal muscle myosin and porcine cardiac actin at 30 °C as described previously [14] with solutions prepared as described [14]. Prior to addition to the flow cell assay chamber, the reconstituted thin filaments were diluted 1:200 in actin buffer containing HDTA, instead of EGTA, to remove any potential for residual EGTA in the flow cell prior to addition of motility buffer at the desired pCa to the flow cell [27]. The reconstituted thin filaments were incubated with surface-bound myosin for three minutes followed by another three-minute incubation of 300 nM troponin/tropomyosin to insure binding of regulatory proteins. Motility was induced by the addition of motility buffer containing 1 mM ATP at various pCa levels from pCa 10 to 4.</p><!><p>To examine how tropomyosin mutations impact myosin-activated thin filament motility, we measured actin sliding velocity as a function of increasing myosin concentration. In this assay, actin filaments, either fully-regulated with troponin and tropomyosin or complexed with just tropomyosin, translocate at maximum velocity if, at all times during filament movement, at least one myosin head is interacting with the actin filament. The probability of strong cross-bridge formation is related to (1) the number of available myosin heads (loading concentration, ρ) and target sites, which is in part controlled by the activity of the tropomyosin, and (2) the fraction of time that the myosin molecule remains bound r (duty cycle). Thus, the probability that at least one head is interacting with actin at all times is given by the statement 1−(1−r)ρ. Therefore, velocity will be altered as a function of r and ρ according to the equation: Vobs= Vmax [1−(1−r)ρ] where Vobs is the observed velocity of the actin filament and Vmax is the maximal sliding velocity at saturating myosin concentrations [28,29].</p><!><p>Standard tests of significance and of significant differences of points in plots measuring filament motility were done by comparing standard errors, where minimum p-values less than 0.05 were considered significant. Hill curves were plotted and Hill coefficients (nH) calculated by fitting velocity data (V) versus calcium concentration according to the equation: V=Vmax[Ca2+]nHpCa50+[Ca2+]nHTests of significance of Hill coefficients were performed using the "compare function" in the program Prism (GraphPad 7825, La Jolla, CA 92037 USA). Here, significance and significant differences between plotted curves was determined by an extra sum-of squares F-test where differences between wild-type and mutant curves with p-value less than 0.05 were considered significant.</p><!><p>In order to examine the impact of the E40K and E62Q mutations on the structure of, and interactions between, tropomyosin and actin, we performed molecular dynamics simulations of wild-type and mutant tropomyosin on actin, as described in the Materials and Methods and previously [13]. Figure 2 shows that the introduction of either the DCM mutant, E40K ( Fig. 2C) or HCM mutant, E62Q, ( Fig. 2D) alters the interactions of tropomyosin side chains and actin. In the case of the E40K mutant, a number of negatively charged residues on actin could potentially interact favorably with the mutant lysine side chain; however, most of these are too distant from the mutant side chain to have a significant effect. The closest of these on actin, E93 and E334, have a minimum distance of 8.2 Å and 7.3 Å to the mutant K40 tropomyosin side chain during the MD simulations, suggesting a small impact to tropomyosin-actin association. In contrast however, side chain interactions within the tropomyosin molecule itself are affected by the mutation. While side chains at e-position E40 and g-position R35 form stereotypical (i,i+5) inter-chain e-g pair ( Fig. 2A), salt bridges in the crystal structure [19] and in MD models of wild-type tropomyosin, mutant K40 and R35 do not ( Fig. 2C). Thus, introducing the DCM-associated mutation, E40K, makes this interaction unfavorable by replacing the negatively charged glutamic acid with a positively charged lysine side chain. During the simulation, the loss of the salt bridge between E40 and R35 is compensated by a new inter-chain salt bridge formed between the side chains of tropomyosin R35 and e-position E33 ( Fig. 2C). This rearrangement (see Fig. 1D) is noted at the beginning of the MD production runs of both actin-tropomyosin in explicit solvent and isolated tropomyosin in implicit solvent. In both cases, the alteration is constant throughout simulation (see Supplementary Item 1). Moreover, the effect can be propagated locally resulting in changed inter-chain pairing in N- and C-terminal directions accompanied by new K40-E42 side chain orientation and altered E-33-K29-D28,E26 bonding. This side chain redistribution in the mutant is likely to affect the structure-function relationships of the tropomyosin coiled-coil ( Fig. 2E), creating further alterations in the actin-binding residues C-terminal to the mutation site. In particular, tropomyosin residue S45 is shifted in relation to the actin filament, forming a novel hydrogen bond interaction between E40K tropomyosin and the side chain of R28 of actin. Consistent with previous energy landscape determinations [14], these new interactions are expected to increase the affinity of the E40K mutant tropomyosin for actin. Therefore, evidently more energy will be required to move the mutant from the B-state position during regulatory transitions on the thin filament.</p><p>Based on previous work [14], HCM mutant E62Q, unlike E40K, is predicted to have a lower affinity for the B-state position on actin. In the wild-type model, the E62 side chain is positioned to make salt bridge interactions with two positively charged side chains on actin, R147 and K328 ( Fig. 2B). Although mutation of E62 to glutamine is relatively conservative, the amide side chain of the glutamine cannot form an ionic bridge with the two positively charged residues on actin. In the simulations, the loss of the negative charge on tropomyosin results in loss of interaction between Q62 of tropomyosin with actin residues R147 and K328 ( Fig. 2D). This divergence from the wild-type model also occurs at the beginning of the MD production run and is stable throughout simulation (Supplementary Item 2). Moreover, additional reduction in affinity is expected from the movement of the tropomyosin coiled-coil away from the actin surface in the simulation ( Fig. 2F), also affecting interaction strength in the vicinity of the mutation, even though there was no apparent concomitant mutation-induced change in coiled-coil flexibility or overall equilibrium binding of E62Q mutant tropomyosin binding with actin (see below; Fig. 3 and 4). Thus, the E62Q mutation is expected to have a lower local affinity for actin than that for the wild-type, and thus would require less energy to move from the B-state to the C- and M-states.</p><!><p>The semi-rigidity of tropomyosin contributes to tropomyosin's cooperative motions on actin filaments and hence its regulatory position [21]. Typically stiffness or flexibility of an object is defined by its flexural and torsional mobility as well as its linear elasticity. Here, flexural variance of the tropomyosin mutants was measured during MD, namely we quantified the extent of their bending fluctuations and compared the data to that of the wild-type tropomyosin. To determine overall tropomyosin flexibility, we assessed persistence length, a measure of thermal bending variance ( Table 1). Persistence length measurements of rod-like structures like tropomyosin determine the aggregate bending fluctuations about that structure and thus reflects the material properties of the molecule. In order to best assess material properties of tropomyosin in a simple and meaningful fashion, isolated tropomyosin molecules were examined, unconstrained by extra-molecular interaction. In contrast, once bound to actin, tropomyosin's inherent bending fluctuations will be curbed by head-to-tail polymerization, limited by the topological organization of tropomyosin on the F-actin helix, and dampened further by electrostatic interactions with the actin substrate, where its bending may be dominated by motions of the actin filament and not by tropomyosin's intrinsic molecular flexibility.</p><p>The structure of actin-free tropomyosin is characteristically curved. Indeed, to a varying extent tropomyosin is pre-shaped to match to the helical pitch of F-actin [21,30]. This innate curvature of tropomyosin spuriously contributes to standard calculation of persistence length, usually measured against uniformly straight reference structures. Tropomyosin's bending deviations from such an idealized straight rod yields a so-called "apparent" persistence length (PLa) (approximately 90 to 130 nm), which over-estimates flexibility. We therefore determined a "dynamic" persistence length, PLd, the true measurement of flexural stiffness that accounts for the average curvature of the molecule (see Materials and Methods section). We accomplished this by measuring flexural deviations from tropomyosin's average curved shape [21,25]. Our results show that the E62Q mutation does not affect tropomyosin flexibility. However, E40K is more flexible and, on average, straighter than the wild-type ( Table 1).</p><p>As mentioned, persistence length measures the aggregate flexural rigidity of rod-like structures and is most useful for assessing isomorphous materials. However, the amino acid sequences of each tropomyosin actin-binding pseudo-repeat differ (see Fig. 1) and hence tropomyosin flexibility is not uniform along its length [21,25]. Thus, local alteration in curvature and bending dynamics along the tropomyosin coiled-coil may also be mutation specific. We therefore also assessed the effects of the disease-linked mutations on local tropomyosin flexibility. For these measurements, we determined the bending variance (local δ) one residue at a time over all tropomyosin residues during the entire course of simulation relative to residues in the average structure. The local delta angle provides a measure of tropomyosin super-helical flexibility, where a larger angle reflects greater deviation from the average for that residue during the simulation and therefore indicates greater local flexibility. As expected, the E40K mutation alters tropomyosin local flexibility near to the site of the mutation ( Fig. 3) where the changed e-g pairs have realigned, and this change in flexibility is stable over time (Supplementary Item 3). The local effect apparently is propagated toward the N-terminus of the mutant structure ( Fig. 3). In addition to the localized effects observed, the simulations also show that the E40K mutation has prominent delocalized effects toward the C-terminus, where a and d heptad positions are less frequently occupied by canonical Leu, Ile, or Val residues and e-g pairing is irregular. The alterations in tropomyosin flexibility and shape parallel the changed pattern of tropomyosin-actin interactions observed in MD of E40K on actin ( Fig. 2). In contrast, measurements of E62Q tropomyosin persistence length and local delta show that, unlike E40K tropomyosin, both the tropomyosin E62Q local and aggregate flexibility is similar to wild-type ( Fig. 3, Supplementary Item 3). This is expected since residue 62 occupies an f-heptad position, viz. it protrudes orthogonally from the tropomyosin coiled-coil and apparently is not required for coiled-coil rod-stability, but instead is involved in actin interaction [12].</p><!><p>Mutant tropomyosin molecules were expressed in E.coli and purified as described previously [14]. Mutant tropomyosin interaction with F-actin was assessed via SDS-PAGE and cosedimentation as described previously [31]. Despite the altered actin-tropomyosin interactions propagated by the tropomyosin mutations and observed via MD simulations, no obvious differences in apparent affinity values were found for F-actin and mutant tropomyosin versus those for F-actin and wild-type tropomyosin ( Fig. 4). Thus, equilibrium binding and the formation of the tropomyosin polymer on actin appear to be unaffected by these tropomyosin mutations.</p><p>To determine other possible functional effects of the altered tropomyosin-tropomyosin and tropomyosin-actin interactions revealed by the MD simulations, we evaluated the cooperative activation of thin filaments containing mutant and wild-type tropomyosins in the absence of troponin ( Fig. 5). Using an in vitro motility assay, we measured the impact of the tropomyosin mutations analyzed above on actin sliding velocity and on the fraction of regulated filaments that were motile. At the low concentrations employed in the motility assay, the presence of wild-type tropomyosin decreased maximal velocity when compared to that of tropomyosin-free actin controls, a phenomenon well described by others [32]; nonetheless, the presence of E62Q tropomyosin was not inhibitory, consistent with destabilization of the B-state positioning observed structurally.</p><p>Compared to the motility of control tropomyosin-free F-actin, more myosin was required to maximally stimulate actin-tropomyosin-based motility judging from corresponding plots of the fraction of filaments which were motile (fraction "%" motile) (regardless of the presence or type of mutant or wild-type tropomyosin). In contrast, thin filaments containing E40K tropomyosin required a similar amount of myosin to reach maximal activation, while filaments containing wild-type E62Q-mutant tropomyosin reached maximal velocity at lower concentrations ( Figure 5B), again consistent with destabilization of the B-state by the E62Q tropomyosin mutation ( Fig. 1).</p><!><p>To test if weakening or enhancing the interaction between tropomyosin and actin alters calcium-regulated actomyosin activity, we again measured the impact of the two tropomyosin mutations on actin sliding velocity and the fraction of regulated filaments motile for filaments now containing both tropomyosin and troponin. As shown in Figure 6, wild-type tropomyosin/troponin fully inhibited thin filament motility at low calcium concentrations and, as calcium concentration was increased, actin filament movement increased cooperatively over a narrow pCa range. Compared to wild-type, troponin-tropomyosin regulated actin filaments containing the HCM mutant E62Q activated at lower calcium (i.e. they were sensitized) when assessed either by sliding velocity or by fraction motile. Conversely, actin filaments containing the DCM mutant E40K required higher calcium to maximally activate sliding velocity and fraction motile (i.e. they were desensitized) ( Fig. 6, Table 2).</p><p>Interestingly in the case of the E62Q mutant, the Hill-Coefficient for the actin sliding velocity relationship was lower than that for wild-type; however, fraction motile values were in fact not very different from each other. In contrast, the Hill-Coefficient for the E40K mutant was lower for both the measured fraction motile and sliding velocity. A slightly reduced maximal filament sliding speed was also observed for E40K.</p><!><p>Strong binding of myosin to actin during the closed- to open-state thin filament transition is known to contribute in part to the activation of muscle thin filaments [1]. In order to ascertain if any of the differences observed in the regulated motility assay can be attributed to differences in the ability of myosin to activate thin filaments containing mutant tropomyosins, we studied the effect of myosin loading concentration on the in vitro motility of calcium-regulated thin filaments at submaximal calcium activation levels. While the maximal sliding velocity and fraction motile are similar to that seen at pCa 7 in the pCa curves ( Fig. 7 inset), the rate of velocity increase as a function of myosin loading ( Fig. 7A) shows no significant differences. This suggests that at the level of detection neither the inhibition nor the enhancement of strong cross-bridge binding to regulated thin filaments is an obvious cause of the mutation-dependent differences in thin filament activation measured ( Fig. 6).</p><!><p>In this study, we examined two mutations in the human cardiac tropomyosin molecule linked to HCM and DCM. First we assessed their structural impact on tropomyosin coiled-coil behavior and then their corresponding functional influence on thin filament motility and thus on myosin interactions with actin.</p><p>Tropomyosin is a stereotypical coiled-coil protein with unique repeating heptad (a-g) motifs that determine its super-helical structure [33]. The HCM mutation, E62Q, occurs at an f position [20] replacing a charged glutamate residue normally in a favorable position to interact with basic binding partners on actin [12,13]. The E40K mutation, on the other hand, resides at an e position in a heptad [18] and is part of the e-g paired inter-chain ionic interaction normally facilitating dimeric coiled-coil structural stability and stiffness [19]. Using a combination of computational modeling and in vitro motility, we measured structural effects of altered mutant tropomyosin coiled-coils on actin-tropomyosin interaction and then correlated these with functional studies and phenotypical impact.</p><p>Consistent with molecular dynamics simulations performed here and previous energy landscape data [14], in vitro motility data on E62Q tropomyosin suggests that the mutation disrupts interactions between tropomyosin and actin that normally stabilize the B- thin filament state. Alternatively, E62Q may also destabilize the intermediate closed/C off-state, which we have not directly assessed by our approaches, but which would be consistent with energy landscape measurements [see Fig. 3a in reference 14]. The effects, in turn, will increase the number of motile filaments at low calcium levels and causes less inhibition of motility at low myosin concentrations than elicited by wild-type tropomyosin ( Fig. 5 and 6).</p><p>The E40K mutation decreases cooperativity of activation ( Fig. 6; Table 2), consistent with a reduction in tropomyosin flexural rigidity noted during MD simulations, and evident from the decrease in persistence length ( Table 1) as well as the increased local structural flexibility ( Fig. 3). Here, the increased flexibility of the mutant parallels subtle molecular rearrangements in the coiled-coil. The disruption of the e-g pair between E40 and R35 by the E40K mutation observed during MD, and simultaneous rearrangement of neighboring pairs, is consistent with this notion. In addition, MD simulation of E40K tropomyosin once bound to actin shows that such rearrangements appear to deform the overall tropomyosin coiled-coil causing global alterations in contacts with actin ( Fig. 2), consistent with the stabilization of the B-state and reduced calcium-sensitivity noted in Figure 6.</p><p>Our motility data and those of others [34] on the E40K mutant could be explained by a weakening of the open/M-state itself and responsiveness of actin-tropomyosin to strong myosin binding. In turn, this mechanism [34] might indirectly favor an already strengthened blocked-state. Our data provides no obvious direct support for this possibility. In fact, our previous results [14,15] show that the open/M-state binding of wild-type tropomyosin to myosin-free actin already is exceedingly weak. Moreover, residue 40 of tropomyosin does not appear to come into contact with myosin during strong crossbridge binding on F-actin [17], and thus it is not apparent how the mutation could directly impact strong binding without concomitant major conformational changes taking place.</p><p>Increased tropomyosin flexibility is frequently considered a hallmark of increased calcium-sensitivity, biasing movement away from the blocked- (or closed-) states. Here, we have found that this "rule of thumb" is likely to be an over-simplification (e.g. see Table 3). Indeed, the E62Q variant, featuring a mutation that leads to enhanced calcium-sensitivity is no more flexible than the control. And in fact, the E40K mutant, which leads to decreased thin filament stiffness, decreases calcium-sensitivity. Evidently the two mutations examined in the current study influence regulatory function by impacting actin-tropomyosin-myosin binding and not by tropomyosin flexibility.</p><p>Our results, examining tropomyosin function and the effect of mutations at the single molecule level are consistent with several studies that examined the effects of the E62Q and E40K tropomyosin mutations in skinned fibers [35], myofibrillar samples [36], reconstituted thin filaments [14,34,37], and isolated proteins [36]. The results of the present work extend those previously evinced and demonstrate that disruption of specific contacts within tropomyosin molecules or at the acto-tropomyosin interface can be subtle and involve both local and long-range effects. In turn, these may affect the position of tropomyosin on actin and ultimately alter the inhibitory or activating states of the thin filament. Mutations that either increase or decrease interactions between B-state tropomyosin and actin are generally associated with corresponding decreased or increased calcium-sensitivity (e.g., [38]) and often times mutations that increase calcium-sensitivity parallel a loss of tropomyosin inhibitory function. However, the effects of disease-linked point mutations on contractile protein structure and protein-protein interactions are not always easily predicted directly from sequence considerations. Thus, relationships between mutation type and disease categories are not necessarily obvious. The multi-faceted combination of computational and experimental biochemistry used here allow alterations in thin filament regulation to be understood at a fundamental molecular level. This information can potentially help guide targeted treatment modalities well in advance of compensatory cascades affecting disease presentation.</p><!><p>Supplemental figure 1: Salt bridge switching in E40K tropomyosin (orange) compared to wild-type (green). Interatomic distances are shown between the side chains of E/K40 and R35 (A) or R35 and E33 (B) during the MD simulation as determined in VMD. The atoms used in the distance determination were the delta carbon of E33 or E40, the zeta carbon of R35, and the zeta nitrogen of K40.</p><p>Supplemental figure 2: The E62Q mutant loses interactions with actin residues K238 and R147 during the MD simulation. Shown is the distance between the delta carbon of tropomyosin residue Q62 and the zeta carbon of R147 (A) or the zeta nitrogen of K328 (B) of actin as measured during the MD simulation.</p><p>Supplemental figure 3: Local delta angles for residues 32 (A-C), 40 (D-F), or 62 (G-I) measured over the course of the MD simulation. Delta angles for individual residues were measured compared to the average structure over the same time period at intervals of 20 ps (see Materials and Methods). Values were graphed over simulation time for wild-type tropomyosin (A, D, G), E40K mutant tropomyosin (B, E, H), and E62Q mutant tropomyosin (C, F, I). The average value over the entire simulation time shown is indicated by the dotted line and the average and standard deviation values are shown in the corner of each graph.</p>

PubMed Author Manuscript

Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

Abstract5‐hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non‐enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein–DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single‐base resolution. We employ chemical oxidation to transform 5hmU to 5‐formyluracil (5fU), followed by the polymerase extension to induce T‐to‐C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T‐to‐C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU‐rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.

sequencing_5‐hydroxymethyluracil_at_single‐base_resolution

<!>Experimental Section<!>Conflict of interest<!>

<p>F. Kawasaki, S. Martínez Cuesta, D. Beraldi, A. Mahtey, R. E. Hardisty, M. Carrington, S. Balasubramanian, Angew. Chem. Int. Ed. 2018, 57, 9694.</p><p>DNA‐base modifications can profoundly influence biology and a number of modified bases have been identified in the genomes of a variety of organisms.1 5‐hydroxymethyluracil (5hmU) is produced through oxidation of thymine both enzymatically and non‐enzymatically2 and can influence the binding of proteins to DNA.2b, 3 It has also been suggested that 5hmU might lead to genomic instability as it can be removed by DNA repair enzymes to create potentially mutagenic lesions4 and affect the stability of DNA duplexes.5 When incorporated at some promoter sites, 5hmU has been shown to affect transcription by bacterial RNAP, therefore it may have a significant effect on microbial biology.6 In mammals, reported levels of 5hmU vary by cell‐ and tissue types.2b, 7 Increased levels of 5hmU autoantibodies have been reported in cancer cases,8 and blood 5hmU mononucleoside levels have been studied as a marker of cancer risks and invasiveness.9 We previously reported a method to map 5hmU at moderate resolution by chemical enrichment of 5hmU‐containing DNA fragments followed by sequencing.10 A method for single‐base sequencing of 5hmU would enable the identification of individual modification sites in genomes. A single‐molecule real‐time (SMRT) sequencing approach could in principle be applied to map 5hmU at single‐base resolution, however the intrinsic signature signal for 5hmU is rather weak unless the base is further modified.11 Mapping 5hmU at single‐base resolution is a worthy challenge that could transform genome‐wide analysis of 5hmU. Herein, we describe a chemical approach for single base‐resolution sequencing of 5hmU and demonstrate its utility in various sequence contexts.</p><p>The conceptual basis for sequencing 5hmU involves chemical oxidation of 5hmU to 5fU, which ionizes under mild alkaline pH owing to the electron‐withdrawing exocyclic aldehyde (pK a at N3=8.1 for 5fU vs. 9.3 for 5hmU).12 The ionized form of 5fU can base‐pair with G (Figure 1), causing a T‐to‐C base change that marks the original 5hmU sites. The oxidation of 5hmU to 5fU was carried out using KRuO4.13 The T‐to‐C change is then established during a polymerase‐dependent single extension, which is subsequently amplified by PCR. To rule out T‐to‐C changes that arise for reasons other than 5hmU (for example, pre‐existing mutations, non‐5hmU DNA damage), sequencing is compared to a "no‐oxidation" control in which there has been no conversion of 5hmU to 5fU.</p><p>Structure of thymine‐base modifications and experimental designs. A) Canonical T:A, 5hmU:A, and 5fU:A base pairs and the 5fU:G base pair. B) The workflow to sequence 5hmU. C) Sequences of the oligonucleotide models (ODN).</p><p>As proof of concept, we first employed a synthetic oligonucleotide with two 5hmUs at defined positions (ODN1), and the base readout profiles at each 5hmU site as well as proximal non‐modified thymine sites were quantified (Table 1 and the Supporting Information, Tables S1 and S2). Sequencing was performed on an Illumina next generation sequencing (NGS) platform (a schematic summary of the sequencing data analysis is shown in the Supporting Information, Figure S1). Under optimised conditions, the proportion of total sequencing reads generating a "C" signal, at the 5hmU‐modified sites was high ("%C"=39 % and 30 %, Table 1 and Table S1) compared to unmodified T sites (1.4 %, Table 1; Wilcoxon rank‐sum test, p‐value=0.003 for both 5hmU sites). In the control experiment in which no oxidation of the DNA was carried out (that is, 5hmU is not converted to 5fU) the proportion of sequencing reads exhibiting a "C" signal at 5hmU‐modified sites were low (2.2 and 2.7 %, Table 1) and comparable to the levels of unmodified T (that is, 1.2 %, Table 1) (Wilcoxon rank‐sum test, p‐value >0.01, for both 5hmU sites). Thus, individual 5hmU sites could be resolved from unmodified T and detected by sequencing. We found that the T‐to‐C percentage change depends on the concentration of dATP during single‐extension PCR. A 500‐fold decrease in concentration of dATP compared to other nucleotide triphosphates was optimal (Table 1 and Table S1).</p><p>Sequencing readout for 5hmU‐modified ODN1.</p><p>[a] Steps as shown in Figure 1. Step 3 was carried out at 37 °C with 10 mm MgSO4 and dNTP mix (final concentrations: 250 μm for dCTP, dGTP, and TTP and 500 nm for dATP) using Bst DNA Polymerase, Large Fragment. See Supporting Information for details. [b] The proportion of reads giving T, C, or other signal (that is, A, G, insertion, and deletion) at the 5hmU‐modified sites over all reads. Mean±SD values of technical triplicates (at least two data out of three were obtained in coverage depth of greater than 1000×) are shown. [c] Mean values for seven proximal Ts.</p><p>The strength of the T‐to‐C signal change for 5fU depended on the choice of polymerase (Table S2), and we chose Bst DNA Polymerase, Large Fragment (obtained from New England Biolabs) for further study owing to its capability to induce T‐to‐C signal change at 5hmU sites without introducing noise at unmodified sites. When using a separate ODN model modified with varying levels of 5hmU at two defined positions (0–26 %, ODN2), the strength of the "C" signal at 5hmU (percent C counts over the sum of C and T counts, %C/(C+T)) increased linearly with the level of 5hmU (Figure S2 a). At a sequencing coverage depth of 100× in ODN2, 5hmU was detectable down to an incorporation level of 15 % (fold‐change of "C" signal compared to the no‐oxidation control, Figure S2b, data available at https://github.com/sblab-bioinformatics/5hmUseq).</p><p>To investigate potential sequence context bias in our approach, we prepared the oligonucleotide model ODN3, with a randomised base flanking each side of a single 5hmU site, therefore representing the 16 possible trinucleotide sequence contexts (N1‐5hmU‐N2, N1 and N2=A or T or G or C). While we observed some context‐dependent variability in the %C/(C+T) signal at the 5hmU‐modified sites, the calling of 5hmU relative to no‐oxidation control was clear and unambiguous in all cases (Figure 2), indicating that the method is suitable for detecting 5hmU in all trinucleotide sequence contexts.</p><p>C signal in synthetic ODN3. The proportion of reads giving "C" signal over the total sum of C+T reads in N1‐5hmU‐N2 trinucleotide contexts in ODN3.</p><p>We then applied the method to map 5hmU in the genome of the eukaryotic pathogen Trypanosoma brucei (Figure 3).14 We mapped 5hmU on chromosome 2, and observed 161 Ts with significant 5hmU signal (0.02 % of all Ts on the chromosome), as defined using a FDR threshold (against "no‐oxidation" control) <0.1.15, 16 As determined using a simulated random distribution, these sites showed significant (p‐value=0.0019) overlap with 5hmU regions obtained using our previously reported chemical enrichment strategy (for details see Supporting Information).10, 13, 16</p><p>5hmU signal in chromosome 2 of Trypanosoma brucei. Ts with significant (FDR<0.1) single‐base resolution 5hmU sites and also regions of 5hmU peaks obtained using the previously reported chemical enrichment‐based method (top panel). Magnified view of three loci with 5hmU signal after normalization with the no‐oxidation controls (bottom panel). Arrows indicate the significant sites (FDR<0.1) [15] and bottom bars are 5hmU regions from enrichment mapping.</p><p>In conclusion, we have demonstrated a chemical method to detect and sequence 5hmU at single‐base resolution. We further envisage the method could be extended to detect 5fU by removing the oxidation step and normalising T‐to‐C conversion relative to conditions insensitive to 5fU such as libraries prepared without the single‐extension step.</p><!><p>Sequencing experiments were carried out on a Mi‐Seq instrument using Miseq reagent kit v3 (Illumina). Quantification of 5hmU by LC‐MS2 analysis was carried out on a Q Exactive™ Hybrid Quadrupole‐Orbitrap Mass Spectrometer (Thermo Scientific) equipped with a nanospray ionization source, coupled to an Ultimate RSLCnano LC system (Dionex). Detailed experimental protocols and commercial sources of reagents are included in the Supporting Information (PDF). All sequencing data have been deposited in the ArrayExpress database at EMBL‐EBI (https://www.ebi.ac.uk/arrayexpress/) under accession number E‐MTAB‐6456. All the code developed for the data analysis has been released in the manuscript's GitHub page (https://github.com/sblab-bioinformatics/5hmUseq).</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Highly Emissive 9‐Borafluorene Derivatives: Synthesis, Photophysical Properties and Device Fabrication

AbstractA series of 9‐borafluorene derivatives, functionalised with electron‐donating groups, have been prepared. Some of these 9‐borafluorene compounds exhibit strong yellowish emission in solution and in the solid state with relatively high quantum yields (up to 73.6 % for FMesB‐Cz as a neat film). The results suggest that the highly twisted donor groups suppress charge transfer, but the intrinsic photophysical properties of the 9‐borafluorene systems remain. The new compounds showed enhanced stability towards the atmosphere, and exhibited excellent thermal stability, revealing their potential for application in materials science. Organic light‐emitting diode (OLED) devices were fabricated with two of the highly emissive compounds, and they exhibited strong yellow‐greenish electroluminescence, with a maximum luminance intensity of >22 000 cd m−2. These are the first two examples of 9‐borafluorene derivatives being used as light‐emitting materials in OLED devices, and they have enabled us to achieve a balance between maintaining their intrinsic properties while improving their stability.

highly_emissive_9‐borafluorene_derivatives:_synthesis,_photophysical_properties_and_device_fabricati

<!>Introduction<!><!>Introduction<!>Synthesis of donor‐functionalised borafluorenes<!><!>Molecular structures of the donor‐functionalised 9‐borafluorenes<!><!>Molecular structures of the donor‐functionalised 9‐borafluorenes<!>Photophysical and electrochemical properties<!><!>Photophysical and electrochemical properties<!><!>Photophysical and electrochemical properties<!><!>Theoretical studies<!><!>Theoretical studies<!>Fabrication of OLEDs based on the donor‐functionalised borafluorene compounds<!><!>Conclusion<!>Experimental Section<!>Conflict of interest<!>

<p>X. Chen, G. Meng, G. Liao, F. Rauch, J. He, A. Friedrich, T. B. Marder, N. Wang, P. Chen, S. Wang, X. Yin, Chem. Eur. J. 2021, 27, 6274.</p><!><p>Three‐coordinate boron‐containing materials have attracted considerable attention over the last several decades due to the conjugation between the vacant p orbital on boron and the π electrons of conjugated systems. This conjugation leads to desirable optical and electronic properties that, in turn, enable applications in optoelectronics and sensing materials.[ 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 ] For example, several groups have shown that three‐coordinate boron, as an electron‐acceptor, combined with electron‐donor groups, giving D–A systems, can exhibit thermally activated delayed fluorescence (TADF), which is favourable for organic light‐emitting diode (OLED) devices.[ 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ] Among three‐coordinate boranes, boroles are unique because of their 4π‐electron five‐membered‐ring structure, which presents antiaromatic character, according to Hückel's rule. Due to their antiaromaticity, boroles exhibit high Lewis acidity, and thus instability towards ambient conditions (i.e., air and water), [30] unless the vacant orbital on boron is sterically protected. The first borole, pentaphenylborole, was reported half a century ago by Eisch et al.; [31] however, its crystal structure was determined only a little over 10 years ago.[ 32 , 33 ] Recently, Marder and co‐workers used a very bulky and highly electron‐deficient 1,3,5‐tris(trifluoromethyl)benzene (FMes) group to stabilise boroles. Interestingly, the stability of these FMes‐protected boroles was improved significantly and, at the same time, their low reduction potentials and pronounced antiaromaticity were maintained. [34] Dibenzoboroles, also widely known as 9‐borafluorenes, exhibit significantly enhanced stability because of their reduced antiaromatic character, due to the delocalisation of π electrons over the fused biphenylene backbone.[ 35 , 36 ] Benefiting from their easy accessibility, 9‐borafluorenes have been explored as a platform for chemical sensors, [37] novel ring‐extension reactions[ 38 , 39 , 40 , 41 , 42 ] and small‐molecule activation. [43] However, their enhanced stability typically comes at the expense of their acceptor properties, as the LUMO is typically much higher in energy. Thus, the challenge is to retain the low‐lying LUMO of a simple borole, while greatly enhancing the stability of the system.</p><p>To explore the potential applications of 9‐borafluorenes in functional materials, multiple D–A systems with 9‐borafluorene as the acceptor unit and an amine as the donor have been studied (Figure 1). Yamaguchi and co‐workers observed strong solvatochromic behaviour of both the absorption and emission of I and II (Figure 1), which suggests a high degree of charge‐transfer character and strong Lewis acidity.[ 44 , 45 ] In contrast, Zhao and co‐workers observed only limited solvatochromism in both the absorption and emission of III and IV, which indicates localised excitation character (Figure 1). [46] This could be due to highly efficient electron delocalisation in the ground state, as a result of the rigid ladder framework. More recently, Marder and co‐workers reported a strategy to enhance the π‐accepting ability (i.e., low‐lying LUMO) of borafluorenes while maintaining their stability, that is, to overcome the stability–property trade‐off that is normally encountered in 9‐borafluorenes. [47] They synthesised a trifluoromethylated borafluorene with a dimethylamino group on the exo‐aryl moiety at the position para to boron, namely p‐NMe2‐FXylFBf (V; Figure 1), which exhibits a reduction potential of around −1.28 V (vs. Fc/Fc+), and the twisted D–A structure results in TADF with a delayed fluorescence lifetime of around 1.6 μs. However, its low quantum yield (ca. 0.03) limits its application in optical materials. The above reports show that D–A systems with borafluorene acceptors often exhibit interesting photophysical and electrochemical properties, which greatly widens the potential applications of borafluorene derivatives in chemistry and materials.</p><!><p>Previously reported borafluorenes and those discussed in the current work.</p><!><p>In this study, we expanded upon this strategy for enhancing the chemical and thermal stability of 9‐borafluorenes by introducing nitrogen‐based electron donors of varying donor strength at the para position of the exo‐aryl moiety of 9‐borafluorenes. The subtle changes in the electronic structure provide insight into the fundamental properties of borafluorene D–A systems. As o‐trifluoromethyl groups have proven to be excellent protecting groups for borafluorenes in previous work, bis(trifluoromethyl)phenylene was selected as the spacer.[ 48 , 49 , 50 ] Unsubstituted 9‐borafluorene was chosen as acceptor, considering the ease of synthetic access and comparability with other 9‐borafluorene derivatives.</p><!><p>The borafluorene precursor 9‐ClBF was prepared according to the literature (Figure 2), [49] and the donor‐functionalised bis(trifluoromethyl)benzene compounds were synthesised by Buchwald–Hartwig coupling reactions[ 51 , 52 , 53 ] (see the Supporting Information). The donor‐functionalised bis(trifluoromethyl)benzene was treated with nBuLi in Et2O at −78 °C for 30 min, then warmed to room temperature and stirred for another 3 hours to give the corresponding lithiated donor‐functionalised bis(trifluoromethyl)benzene. The lithiated species were subsequently mixed with 9‐ClBF in dry toluene at −78 °C, and then warmed to room temperature to give FMesB‐Cz, FMesB‐Ac and FMesB‐Ptz in overall yields of 23, 29 and 29 %, respectively (Figure 2). These compounds are light‐yellow powders and are stable in air. They were fully characterised by NMR spectroscopy and HRMS (see the Supporting Information).</p><!><p>Synthetic route to FMesB‐Ac, FMesB‐Ptz and FMesB‐Cz.</p><!><p>Single crystals of the three compounds were obtained by recrystallisation from hexane, and the structures are shown in Figure 3. The bond lengths between the boron and the neighbouring carbon atoms are similar to those of the FMes‐capped 9‐borafluorene (F MesBf). [49] The bis(trifluoromethyl)phenyl groups adopt orientations almost orthogonal to the planes of the borafluorene moiety in all of the compounds, with torsion angles of 75.5(2) and 77.0(2)° for FMesB‐Cz, 83.5(1)° for FMesB‐Ac and 82.8(1)° for FMesB‐Ptz. All of the dihedral angles are slightly smaller than those in the compounds reported by Marder and co‐workers, which could be due to less steric hindrance in our compounds. [47] It is noteworthy that the donor groups also exhibit a highly twisted configuration with respect to the bis(trifluoromethyl)phenyl group, with torsion angles of 75.3(2) and 76.6(2)° for FMesB‐Cz, 80.9(1)° for FMesB‐Ac and 83.2(1)° for FMesB‐Ptz. The above structural features reveal that two twisting nodes exist in these compounds, which can largely block the electron communication between the donor and acceptor, thus suppressing the charge‐transfer behaviour. In contrast, p‐NMe2‐FXylFBf, with one twisting node between the trifluoromethylated borafluorene and the bis(trifluoromethyl)phenyl group, exhibits strong charge‐transfer character in its emission spectra. [47] The shortest B⋅⋅⋅F distances are 2.518(9) and 2.575(9) Å for FMesB‐Cz, 2.512(4) and 2.611(4) Å for FMesB‐Ac, and 2.524(2) and 2.592(2) Å for FMesB‐Ptz, which are much shorter than the sum of the van der Waals radii of boron and fluorine (3.39 Å). [54] This has previously been observed and discussed for boranes and boroles with o‐CF3‐aryl moieties[ 34 , 48 , 50 , 55 , 56 ] and can be explained by the lone‐pair electrons of the fluorine atoms interacting with the empty p orbital of the boron centre.</p><!><p>(a) Molecular structures determined by single‐crystal XRD analysis at 298 K for FMesB‐Cz and at 180 K for FMesB‐Ac and FMesB‐Ptz. Thermal ellipsoids are drawn at the 50 % probability level. Hydrogen atoms have been omitted for clarity. Selected bond lengths [Å] for FMesB‐Cz: B1−C1 1.562(3), B1−C12 1.567(3), B1−C13 1.580(3); for FMesB‐Ac: B1−C1 1.553(3), B1−C12 1.560(3), B1−C13 1.584(3); for FMesB‐Ptz: B1−C1 1.562(2), B1−C12 1.561(2), B1−C13 1.581(2). The pictures show the crystals under a UV lamp. (b) Optimised geometries and AIM analysis of FMesB‐Cz, FMesB‐Ac and FMesB‐Ptz, showing the B⋅⋅⋅F bond paths (purple lines) and BCPs (red points).</p><!><p>To gain a deeper understanding of these weak interactions, DFT calculations were conducted. We performed full geometry optimisations at the B3LYP/6‐31G** level of theory, starting from the crystal structure coordinates. In these optimised structures, the B⋅⋅⋅F distances of FMesB‐Cz and FMesB‐Ac are 2.620 and 2.618 Å, respectively. In the case of FMesB‐Ptz, the B⋅⋅⋅F distances are 2.615 Å for B1⋅⋅⋅F1 and 2.621 Å for B1⋅⋅⋅F4, because of the bending of the phenothiazine moiety. An atoms‐in‐molecules (AIM) analysis, performed with the Multiwfn software package, [57] revealed that the electron densities (ρ) and its Laplacians (∇2 ρ) at the bond critical points (BCPs) of these molecules are ρ=0.0135 ea 0 −3 and ∇2 ρ=0.0514 ea 0 −5 (a 0 is the Bohr radius) for FMesB‐Cz, ρ=0.0135 ea 0 −3 and ∇2 ρ=0.0506 ea 0 −5 for FMesB‐Ac, and ρ=0.0135 ea 0 −3 and ∇2 ρ=0.0506 ea 0 −5 (BCP1) and ρ=0.0134 ea 0 −3 and ∇2 ρ=0.0503 ea 0 −5 (BCP2) for FMesB‐Ptz. All of these values are small and comparable to those of FMes‐capped dithienylborane compounds, which indicates similar weak B⋅⋅⋅F interactions. [50] In addition, typical π⋅⋅⋅π interactions with a head‐to‐tail packing mode are observed in the crystal structures of FMesB‐Cz, FMesB‐Ac and FMesB‐Ptz, with the distances between the borafluorene plane and the centroid of the respective benzene ring of the amine donor group being 3.524(6) and 3.805(6) Å as well as 3.583(7) and 3.815(7) Å for the two symmetrically independent molecules of FMesB‐Cz, respectively, and 3.570(3) and 3.732(3) Å for FMesB‐Ac. Due to the bending of the phenothiazine moiety, the molecular packing of FMesB‐Ptz shows a non‐parallel relationship between neighbouring molecules, which results in two packing distances between the phenothiazine and borafluorene moieties of around 3.482(1) and 4.091(1) Å, respectively (see Figure S1 in the Supporting Information).</p><!><p>The UV/Vis spectra of the compounds were recorded in CH2Cl2 solution (1×10−5  m); they exhibit an intense absorption at around 260 nm, and a weaker absorption at around 280–300 nm (Figure 4). All the compounds exhibit weak absorption in the visible range, with the onset value of the absorption tailing to beyond 400 nm.</p><!><p>UV/Vis spectra (black line) of donor‐functionalised borafluorene compounds (a) FMesB‐Cz, (b) FMesB‐Ac and (c) FMesB‐Ptz in CH2Cl2, and photoluminescence spectra in different solvents (c=1×10−5  m) and in the solid state (colour lines, as indicated in the figures). (d) Cyclic voltammograms of the donor‐functionalised 9‐borafluorene compounds recorded in CH2Cl2 using nBu4NPF6 (0.1 m) as the electrolyte and at a scan rate of 100 mV s−1.</p><!><p>The photoluminescent (PL) spectra show that these compounds exhibit bright‐yellow emission in hexane, with maxima in the range 545–550 nm, and relatively high quantum yields (PLQYs) (ca. 32 % for FMesB‐Cz and over 45 % for the other two compounds, Figure 4 and Table 1). Relatively long fluorescent lifetimes (134–140 ns in hexane) were observed for all of the compounds, which seems to be an intrinsic behaviour of borafluorenes and was reported by Rupar [58] and Marder[ 47 , 59 ] and their co‐workers. Almost no solvatochromism was observed in the emission spectra of FMesB‐Cz, but partial quenching was observed in more polar solvents (see Figure S2 in the Supporting Information). For compound FMesB‐Ac, solvatochromic behaviour was observed as the fluorescence maximum redshifted from 545 nm in hexane to around 650 nm in CH2Cl2 solution, with a significantly quenched intensity, similarly to that observed for the 9‐borafluorene compounds reported by Yamaguchi and co‐workers. [44] In the case of FMesB‐Ptz, which has a stronger donating group, the fluorescence quantum yield dropped dramatically in polar solvents (e.g., ca. 1 % in CH2Cl2). Interestingly, all the compounds exhibit significantly quenched and blueshifted fluorescence in DMF solution, with the maximum emission wavelength shifted to around 400 nm for FMesB‐Cz, and around 450 nm for FMesB‐Ac and FMesB‐Ptz (see Figure S4). This phenomenon may be attributed to the coordination of the donor solvent DMF to the boron centre of the borafluorenes. A similar blueshift upon coordination to DMF, with increased luminescence, was observed by Yamaguchi et al. [45] Other intramolecular [56] and intermolecular [47] adducts have been reported to exhibit excited‐state dissociation, retaining the emission wavelength of the "free" borafluorene, which indicates that the adducts of DMF with our compounds persist in their excited states. To confirm this hypothesis, we recorded the 11B NMR spectra of the borafluorene compounds in DMF (see Figure S4). In comparison with the 11B NMR chemical shifts in chloroform (ca. 65 ppm), the resonances of these compounds in DMF are shifted significantly upfield to around 11 ppm, revealing the formation of four‐coordinate boron centres.</p><!><p>Photophysical properties of the donor‐functionalised borafluorenes.</p><p>Compd</p><p></p><p>298 K</p><p>78 K</p><p></p><p>λ abs [nm][a]</p><p>λ PL [nm] sol[b]/film[c]</p><p>PLQY [%] sol[b]/film[c]</p><p>τ [ns] sol[b]/film[c]</p><p>λ PL [nm]</p><p>τ [ns]</p><p>FMesB‐Cz</p><p>355</p><p>550/551</p><p>31.8/73.6</p><p>139.4/0.6, 107.9</p><p>534[d]/533[e]</p><p>3.7, 125.5[d]/60.8, 160.4[e]</p><p>FMesB‐Ac</p><p>355</p><p>545/551</p><p>45.4/57.3</p><p>140.3/0.6, 114.8</p><p>536[d]/534[e]</p><p>3.6, 121.8[d]/7.6, 155.7[e]</p><p>FMesB‐Ptz</p><p>390</p><p>548/606</p><p>46.3/22.0</p><p>134.1/0.3, 50.9</p><p>534[d]/525[e]</p><p>9.3, 124.0[d]/0.8, 167.6[e]</p><p>[a] Onset wavelength values of UV/Vis absorption of donor‐functionalised 9‐borafluorene compounds in hexane (1×10−5  m). [b] Data obtained in hexane. [c] Data obtained as neat films. [d] Data obtained at 78 K in a CH2Cl2 frozen glass (1×10−5  m). [e] Data obtained at 78 K in a 3‐methylpentane frozen glass (1×10−5  m).</p><!><p>The low‐temperature photophysical properties of the three compounds were also recorded in frozen 3‐methylpentane and CH2Cl2 at around 78 K (see Figure S6 in the Supporting Information and Table 1). The fluorescence maxima occur at 534–536 nm in CH2Cl2 and at 525–534 nm in 3‐methylpentane,and exhibit a well‐resolved band structure showing different vibrational modes. All of the compounds exhibit two lifetimes, as shown in Figure S6; one is shorter than the other typically by more than 100 ns, and the longer lifetimes are dominant. The long lifetimes of around 121–126 ns in frozen CH2Cl2 are slightly shorter than those in 3‐methylpentane (ca. 155–168 ns). The PL spectra of the compounds are almost identical, which indicates that the emissions can mainly be attributed to local excitation (LE) transitions localised on the 9‐borafluorene moiety.</p><p>In the solid state, both FMesB‐Cz and FMesB‐Ac exhibit bright‐yellow fluorescence in both the crystalline form and in neat films, with identical emission maxima at 551 nm, relatively high quantum yields of up to 73.6 % for FMesB‐Cz (Figure 4 and Table 1) and a lifetime of around 108 ns in neat films (see Figure S5 in the Supporting Information). In contrast, FMesB‐Ptz exhibits pale redshifted orange fluorescence both in the crystal and in the neat film, and the lifetime is shortened to 50.9 ns in the neat film. To interpret this phenomenon, 5 wt% doped poly(methyl methacrylate) (PMMA) films of all three compounds were prepared. The PL spectra of these film samples are almost identical to their PL spectra in hexane, and the lifetimes are also comparable with the data in hexane (see Figure S5), which indicates that the redshifted PL spectrum of FMesB‐Ptz in the neat film could be due to intermolecular interactions (e.g., the formation of excimers) in the excited state. The thermal stabilities of these compounds were determined by thermogravimetric analysis (TGA). The donor‐functionalised compounds exhibit much higher decomposition temperatures (>250 °C) than the compound without a donor (F MesBf, T d=148 °C; see Figure S3). This excellent thermal stability indicates that the donor‐functionalised 9‐borafluorene compounds are promising candidates for applications in materials science.</p><p>The electrochemical properties of these 9‐borafluorenene derivatives were examined by cyclic voltammetry (CV) in CH2Cl2 (Figure 4 d). All of the compounds exhibit reversible reduction potentials, which can be assigned to the reduction of boron. The half‐wave reductive potentials (E 1/2) of these compounds range from −1.90 to −1.95 V versus Fc+/0, which are slightly more negative than that of F MesBF (E 1/2=−1.87 V vs. Fc+/0, see Figure S8 in the Supporting Information), [49] which indicates that the electron‐donating groups weaken the electron‐accepting ability of boron, although the influence is very weak given the highly twisted geometries. In the case of oxidative processes, the phenothiazine‐terminated compound FMesB‐Ptz exhibits a reversible oxidation with E 1/2=+0.46 V versus Fc+/0 and an irreversible oxidation with E onset=+1.01 V versus Fc+/0, whereas the other two compounds exhibit irreversible oxidations with E onset values for FMesB‐Ac and FMesB‐Cz of +0.61 and +0.93 V versus Fc+/0, respectively. The HOMO and LUMO energies of these compounds were estimated from the oxidation and reduction potentials by using the equation E LUMO/HOMO=−5.16−E red/ox (see Table 2). [60]</p><!><p>Electrochemical data and calculated FMO energy levels for the donor‐functionalised borafluorenes.</p><p>Entry</p><p>E red [V]</p><p>E ox [V]</p><p>E LUMO [eV]</p><p>E HOMO [eV]</p><p></p><p></p><p></p><p>Exptl[a]</p><p>Calcd[b]</p><p>Exptl[a]</p><p>Calcd[b]</p><p>FMesB‐Cz</p><p>−1.93</p><p>0.93[c]</p><p>−3.23</p><p>−2.66</p><p>−6.09</p><p>−6.06</p><p>FMesB‐Ptz</p><p>−1.90</p><p>0.46</p><p>−3.26</p><p>−2.73</p><p>−5.62</p><p>−5.58</p><p>FMesB‐Ac</p><p>−1.95</p><p>0.61[c]</p><p>−3.21</p><p>−2.69</p><p>−5.77</p><p>−5.52</p><p>[a] E LUMO/HOMO=−5.16−E red/ox. [b] Calculated by DFT using Gaussian 09 [61] at the B3PW91/6‐311+G* level of theory. [c] Onset of the oxidation potential.</p><!><p>DFT calculations were carried out on all of the compounds, with geometry optimisations conducted at the B3LYP/6‐31G** level of theory. Frequency calculations showed no imaginary frequencies, which indicates that energy minima had been reached. The energies of the frontier orbitals were generated by single‐point energy calculations based on the optimised structures, conducted at the B3PW91/6‐311+G* level of theory. As expected, the LUMOs of the compounds are located on the borafluorene moieties (Figure 5), and the HOMOs of the compounds in this work are mainly located on the donor groups without any contribution from the bis(trifluoromethyl)phenyl group, which could be due to the twisted configuration. In contrast, the HOMO of p‐NMe2‐FXylFBf is distributed over both the dimethylamino and bis(trifluoromethyl)phenyl groups, because the coplanarity of the two groups is favourable for p–π conjugation. [47] Generally, there is good agreement between the calculated and experimental HOMO energies. Although the experimentally approximated LUMO energies are almost identical for the three compounds, varying from −3.21 to −3.26 eV, the calculated energies show larger differences, ranging from −2.66 to −2.73 eV (Table 2). TD‐DFT calculations were conducted on all of the compounds at the PBE0/6‐311+G** level of theory (see Figure S11 and Table S6 in the Supporting Information).</p><!><p>(a) Plots of the frontier orbitals and energy levels of the donor‐functionalised borafluorene derivatives (B3PW91/6‐311+G*//B3LYP/6‐31G**). (b) Optimised geometries and illustration of the transitions of the S1 states of the three compounds (PBE0/6‐31G**, using CH2Cl2 as solvent model).</p><!><p>For FMesB‐Cz, the weakly allowed S0→S1 (oscillator strength f=0.001) and S0→S2 (f = 0.03) transitions can be assigned to the HOMO−1→LUMO and HOMO→LUMO transitions, respectively. For FMesB‐Ac and FMesB‐Ptz, the S0→S1 and S0→S2 transitions can be assigned to HOMO→LUMO and HOMO−1→LUMO, respectively. The weak transition from the donor groups to the acceptor groups in these compounds could be due to the twisted spacer, bis(trifluoromethyl)phenyl, which reduces the overlap between the orbitals and thus reduces the oscillator strength of the transition. Optimisations of the S1 states were conducted for the three compounds at the PBE0/6‐31G** level of theory with additional solvent correction (CH2Cl2). As shown in Figure 5, the S1 state of FMesB‐Ac exhibits charge‐transfer character (mainly arising from a HOMO←LUMO transition), whereas FMesB‐Cz and FMesB‐Ptz reveal localised character with the transition to the S1 state being located on the borafluorene moiety (mainly HOMO−1←LUMO). These results match well the solvatochromic photoluminescent behaviour mentioned above.</p><p>Another important factor that can be obtained from DFT calculations is the antiaromaticity index of the 9‐borafluorene compounds. We calculated the nucleus independent chemical shifts (NICSs) as an index of aromaticity by means of DFT at the B3LYP/6‐31G** level of theory based on the optimised structures.[ 62 , 63 ] NICS(1)zz values, which represent the NICS value 1 Å above and below the centre of the ring, are listed in Figure S10 in the Supporting Information. The results indicate that the NICS(1)zz values of the borole rings are around +26, which is almost identical to that of F MesBf (NICS(1)zz=+26.05, calculated by using the same method as us), and indicates moderate antiaromaticity. This value is similar to the NICS(1)zz values previously reported for other borafluorene derivatives. [35] These results suggest that the electron‐donating groups have almost no influence on the aromaticity of the borole ring.</p><!><p>To explore the application of these functionalised borafluorenes in organic electronics, vacuum‐deposited OLEDs based on FMesB‐Cz and FMesB‐Ac as emitting dopants were fabricated to investigate their electroluminescence (EL) performance. In this work, 4,4′‐cyclohexylidenebis[N,N‐bis(4‐methylphenyl)aniline] (TAPC) was used as the hole transport material, 1,4,5,8,9,11‐hexaazatriphenylenehexacarbonitrile (HATCN) was used as the hole injection layer material, 1,3,5‐tris[3‐(3‐pyridyl)phenyl]benzene (TmPyPB) was used as the electron‐transport material and 9,10‐di(2‐naphthyl)anthracene (ADN) was used as the host material. Devices with the configuration ITO/HATCN (2.1 nm)/TAPC (34 nm)/5 wt% emitting dopant:ADN (15 nm)/TmPyPB (21 nm)/LiF (1 nm)/Al (100 nm) were fabricated and characterised. As shown in Figure 6 and Table S2 in the Supporting Information, the devices based on FMesB‐Cz and FMesB‐Ac achieved turn‐on voltages of between 3.6 and 3.8 V, and produced EL spectra exhibiting a similar yellow‐green colour with identical emission peaks at 552 nm, resembling their PL spectra in the solid state. The devices based on FMesB‐Cz and FMesB‐Ac achieved a maximum luminance of 22 710 cd m−2 at 11 V and 22 410 cd m−2 at 10 V, respectively. The maximum external quantum efficiency (2.4 %) and current efficiency (7.5 cd A−1) of the FMesB‐Ac‐based device were higher than those of the FMesB‐Cz‐based device. These results suggest that the better EL properties of FMesB‐Ac may be attributed to its higher solid‐state efficiency. Furthermore, both devices exhibited excellent performances, with external quantum efficiencies of up to 2.1 % at 1000 cd m−2, which are comparable to those reported for other boron‐containing OLED devices.[ 64 , 65 , 66 , 67 , 68 ]</p><!><p>EL characteristics of OLED devices based on FMesB‐Cz and FMesB‐Ac. (a) Configuration and energy diagram of the devices, and photographs showing their emission colours. (b) Structures of the molecules used in the devices. (c) EL spectra of the devices at a luminance of around 1000 cd m−2. (d) Luminance (L)–voltage (V)–current density (J) characteristics for the two devices. (e) Power efficiency (η p), current efficiency (η c) and external quantum efficiency (η ext) versus luminance (L) curves for the two devices.</p><!><p>A series of 9‐borafluorene derivatives functionalised with carbazole, acridine and phenothiazine as donor groups with a bis(trifluoromethyl)phenyl spacer have been prepared in moderate yields, and all of the compounds exhibited good stability. The crystal structures of these compounds indicate that the bis(trifluoromethyl)phenyl group inhibits the donor–acceptor interaction to some extent. Furthermore, typical π‐stacking interactions are observed between the borafluorene and donor groups of neighbouring molecules in the solid state. Electrochemical studies and DFT calculations indicate that the introduction of donor groups influences the energy levels of the borafluorene group only slightly. Although the compounds are almost colourless in dilute solution, which is attributed to the weakly allowed S0→S1 transition, some of the compounds exhibit strong yellowish emission in solution and the solid state with quantum yields of up to 73.6 % for FMesB‐Cz in neat film. FMesB‐Ptz shows a relatively weak and redshifted emission in the neat film and crystal, which could be attributed to intermolecular interactions. All of the compounds exhibit excellent thermal stability with much higher decomposition temperatures than the non‐donor analogue, which demonstrates their potential for application in materials. OLED devices were fabricated with two of the highly emissive compounds as light‐emitting materials. Both devices exhibited strong electroluminescence at 550 nm, and a maximum luminance intensity greater than 22 000 cd m−2. This is the first example of borafluorene derivatives being used as light‐emitting materials in OLED devices. Finally, this study has demonstrated that the strategy of balancing the stability and intrinsic properties of 9‐borafluorene greatly expands its prospects of application in the field of materials.</p><!><p>Crystallographic data: Deposition numbers 2000574 (FMesB‐Cz), 2000564 (FMesB‐Ac), and 2000565 (FMesB‐Ptz) contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Observations of tetrel bonding between sp³-carbon and THF

We report the direct observation of tetrel bonding interactions between sp 3 -carbons of the supramolecular synthon 3,3-dimethyl-tetracyanocyclopropane (1) and tetrahydrofuran in the gas and crystalline phase. The intermolecular contact is established via s-holes and is driven mainly by electrostatic forces. The complex manifests distinct binding geometries when captured in the crystalline phase and in the gas phase. We elucidate these binding trends using complementary gas phase quantum chemical calculations and find a total binding energy of À11.2 kcal mol À1 for the adduct. Our observations pave the way for novel strategies to engineer sp 3 -C centred non-covalent bonding schemes for supramolecular chemistry.

observations_of_tetrel_bonding_between_sp³-carbon_and_thf

Introduction<!>Results and discussion<!>details). Shown in Table<!>Summary and concluding remarks<!>Conflicts of interest

<p>Non-covalent interactions are key forces that drive phenomena such as host-guest chemistry, molecular aggregation, crystallization and protein folding. 1,2 In recent years, important intermolecular interactions like hydrogen and halogen bonding 1,3-7 have been contextualized as s-hole interactions. [8][9][10] A s-hole can be seen as a Lewis acidic site along the vector of a covalent bond, the location of which coincides with the s* orbital of that bond. The extreme outcome of a s-hole interaction can be the breaking and/or making of a s bond, such as in the formation of I 3 À from molecular I 2 and I À . 11,12 A similar rationale can be applied to so-called 'p-hole interactions' involving electron-decient aromatic rings, 13,14 or polarized double bonds with related covalent bond-forming chemistry such as in aldol-type reactions. In principle, sand p-hole interactions should be available with all the non-metallic elements of the periodic table. This includes carbon; [15][16][17] an element of central importance to life and ubiquitous presence in synthetic chemistry. One might thus wonder to what extend carbon can be exploited as locus of Lewis acidity to establish 'tetrel-bonding interactions' (in analogy to halogen-and chalcogen-bonds). 18 Such interactions are well-known for sp 2 -hybridized C-atoms in carbonyls [19][20][21][22][23][24][25][26] and have recently been reported for the sphybridized C-atoms of (coordinated) acetonitrile, 27 carbon monoxide 28 and carbon dioxide. [29][30][31][32] Non-covalent interactions with sp 3 -hybridized carbon atoms are implicated in the advent of canonical S N 2 nucleophilic displacement reactions 12,[33][34][35] and can persist with methyl groups in crystal structures. [36][37][38] However, a supramolecular synthon to predictably generate directional tetrel-bonding interactions centred on sp 3 -C has not yet been experimentally disclosed. We envisaged that 1,1,2,2tetracyanocyclopropane (TCCP) derivatives could full this role. 39,40 These rings are synthetically viable and contain a sterically accessible electrophilic site located roughly on the two sp 3 C-atoms in the (NC) 2 C-C(CN) 2 fragment. This is exem-plied by the molecular electrostatic potential (MEP) map of 3,3-dimethyl-TCCP (1) shown in Fig. 1. The calculated s-hole potential of +44 kcal mol À1 lies in-between the s-holes of water (+55 kcal mol À1 ) and ammonia (+35 kcal mol À1 ), which are prototypical s-hole (i.e. hydrogen bond) donors. The Lewis acidic site of 1 should thus be able to form a tetrel bonding interaction with an electron-rich partner such as the lone pair electron cloud on tetrahydrofuran (THF, estimated at À40 kcal mol À1 ). [39][40][41] Here we report on the verication of this hypothesis by synthesizing 1 and showing thatas anticipated -1 binds to THF via intermolecular sp 3 -C/O interactions, both in the crystalline state and in the gas phase.</p><!><p>Cyclopropane 1 was readily prepared in a one-pot cascade reaction from acetone, malononitrile and molecular bromine (Scheme 1). Presumably, cyclization to 1 proceeds from an intermediate formed by the nucleophilic attack of in situ generated [BrC(CN) 2 ] À on the Knoevenagel condensation product of acetone and malononitrile. 42 The yield of our procedure (83%) is higher than obtained by previously reported methods [42][43][44][45][46][47][48] (max. 72%). 47 All literature procedures with a yield in excess of 50% 42,43,[45][46][47][48] (maximum 72%) 47 use a two-step approach starting from an activated malononitrile derivative 42,43,[45][46][47] and/or use electrochemical synthesis. 47,48 Single crystals suitable for X-ray diffraction measurements (see ESI † for details) were obtained by slow evaporation of a solution of 1 in THF. The molecular model of [1/THF] resulting from the diffraction study is shown in Fig. 2a. All the intramolecular distances and angles within this structure can be considered as normal (not shown). 49 The plane running through the O-and C-atoms of the THF molecule is roughly coplanar with the cyclopropane ring plane in 1 (: plane-plane ¼ 8.2 ). Interestingly, the oxygen atom of the THF molecule is directed towards C1/C3/C4 of the cyclopropane ring in 1, with very short intermolecular distances, in particular sp 3 -C1/O1 of 3.007 Å (C3/C4 /O1 ¼ 3.1 Å, not shown). This is 0.213 Å within the van der Waals radii of O (1.52 Å) and C (1.70 Å) and thus consistent with a bonding interaction. 12,27,39,40,50 Further stacking of [1/THF] in the crystal is aided by weak N1/N2/ C1/C3/C4 interactions (max. 0.067 Å van der Waals overlap, see Fig. S3 †). ‡</p><p>A DFT optimization at the B3LYP 51,52 -D3(BJ) 53 /def2-TZVP 54,55 level of theory of the atomic coordinates found in the crystal structure converged at a nearly identical structure (see Fig. S5 †). The interaction energy (DE) was computed to be À10.1 kcal mol À1 . This is much larger than interactions of dimethyl ether halogen bonded to I-C 6 F 5 (À5.6 kcal mol À1 ) or hydrogen bonded to water (À6.7 kcal mol À1 ) at this same level of theory. 37,56 Interestingly, the [1/THF] structure shown in Fig. 2b was found to be 1.1 kcal mol À1 more stable, representing the true energetic minimum with DE ¼ À11.2 kcal mol À1 (see also Fig. S6 †). The structure is similar to the crystal structure but with the THF oriented almost perpendicular to the cyclopropane plane, with : plane-plane ¼ 83.8 . The distances between the THF-O and the two sp 3 (NC) 2 C-C(CN) 2 atoms display up to 0.297 Å van der Waals overlap, which is 0.084 Å more than observed in the crystal structure. This difference likely originates from the lack of any other interactions in the idealized gas phase computation versus various other potential weak interactions within the crystal of [1/THF].</p><p>Rotational spectroscopy is the technique to experimentally discriminate between the two relative orientations of [1/THF] (Fig. 2) that are so close in energy in the gas phase calculations (1.1 kcal mol À1 ). Thus, we conducted chirped pulse Fourier transform microwave (CP-FTMW) spectroscopy 57,58 to assign the geometry of [1/THF] in the gas phase (see ESI † for 1 are spectroscopic parameters extracted from this experiment together with predicted values based on DFT calculations of [1/THF] with 'coplanar' or 'perpendicular' THF orientations. The experimental rotational constants (in particular B and C) provide a conclusive assignment of the [1/THF] complex in the 'perpendicular' orientation, which is also the DFT-energetic minimum (righthand side of Fig. 2).</p><!><p>To date we were unable to quantify tetrel bonding interactions with 1 in solution, but we did observe a very large and unusual solvent dependency for the 1 H and 13 C NMR resonances of 1 (detailed in Fig. S7 and Table S4 †). For example, the methyl protons of 1, which are g to CN, span a range of 1.39 ppm passing from benzene through toluene, acetonitrile, methanol and chloroform, to acetone. In comparison, the ethoxy methyl protons in ethyl acetate and diethyl ether vary by just 0.34 and 0.12 ppm, despite being closer to functional groups. 64 These results seem to suggest strong and geometrically specic interactions between 1 and most solvent molecules. Based on these preliminary observations, we anticipate that future studies will demonstrate that tetrel bonding interactions with tetracyanocyclopropane derivatives also persist in solution.</p><p>To gain more insight into the physical origins of the [1/ THF] adduct, the 'perpendicular' structure was subjected to a Morokuma-Ziegler inspired energy decomposition, 37,[59][60][61] an 'atoms-in-molecules', 62 and a non-covalent interaction analysis. 63 The energy decomposition analysis revealed that the interaction is mainly electrostatic in origin (52.7%) followed by dispersion (30.7%) and orbital interactions (16.8%). Interestingly, orbital mixing occurred between the HOMO of THF and the LUMO of 1 (À3.86 kcal mol À1 stabilization) and between the HOMOÀ1 of THF and the HOMO of 1 (À4.80 kcal mol À1 stabilization, see Fig. S8 † for details). The 'atoms-in-molecules' analysis of [1/THF] shown in Fig. 3a reveals several bond critical points (bcp's) between the N-atoms of 1 and several CH hydrogens of THF, indicating very weak hydrogen bonding interactions (r z 0.005 a.u.). The densest bcp of r ¼ 0.0115 a.u. is present between the THF O-atom and one of the sp 3 (NC) 2 C-C(CN) 2 atoms (highlighted in yellow). § In line with these results, the NCI plot shown in Fig. 3b clearly reveals that there are two sp 3 -C/O interactions that are mainly electrostatic in origin (blue), and that the C-H/N interactions are mainly dispersive (yellow/green).</p><p>For comparison purposes, a cyclopentane adduct was calculated aer in silico O / CH 2 mutation and geometry optimization of structure [1/THF]. This resulted in the structurally similar [1$$$cyclopentane] adduct shown in Fig. 3c (see ). The adduct is also much less stable with DE ¼ À4.0 kcal mol À1 , which is mainly driven by dispersion (59.4%) followed by electrostatic interactions (22.2%). The NCI analysis of this adduct depicted in Fig. 3d clearly shows that this adduct is only held by dispersive C-H/N interactions (green).</p><!><p>In summary, it was shown that 1 can form [1/THF] complexes in the crystalline state and in the gas phase with a calculated interaction energy of up to À11.2 kcal mol À1 . These complexes are held together by strong polar interactions between the de facto Lewis acidic site in between the sp 3 -hybridized C-atoms of 1 and the Lewis basic THF-O. These results demonstrate that tetrel-bonding interactions with sp 3 -carbon centres can indeed be used to engineer supramolecular complexes, thus paving the way for their exploration in other molecular disciplines, e.g. supramolecular chemistry, crystal engineering and medicine.</p><!><p>The authors declare no conict of interest.</p>

Royal Society of Chemistry (RSC)

Modeling the Influence of Correlated Molecular Disorder on the Dynamics of Excitons in Organic Molecular Semiconductors

In this Letter, we investigate the role of correlated molecular disorder on the dynamics of excitons in oligothiophene-based organic semiconductors. We simulate exciton dynamics using the Frenkel exciton model and we derive parameters for this model so that they reflect the specific characteristics of all-atom molecular systems. By systematically modifying the parameters of the Frenkel exciton model we isolate the influence of spatial and temporal molecular correlations on the dynamics of excitons in these systems. We find that the molecular fluctuations inherent to these systems exhibit long-lived memory effects, but that these effects do not significantly influence the dynamic properties of excitons. We also find that excitons can be sensitive to the molecular-scale spatial correlations, and that this sensitivity grows with the amount of energetic disorder within the material. We conclude that control over spatial correlations can mitigate the negative influence of disorder on exciton transport.

modeling_the_influence_of_correlated_molecular_disorder_on_the_dynamics_of_excitons_in_organic_molec

<!>Parametrization of Frenkel Hamiltonian<!>Exciton Dynamics Without Excited State Forces

<p>The optoelectronic properties of organic molecular semiconductors are known to depend sensitively on how molecules are arranged within the material. [1][2][3][4][5][6][7][8] The inability to exploit this dependence for improving material performance is a problem that hinders the development of electronic applications that incorporate these materials, such as organic photovoltaic (OPV) and light-emitting (OLED) devices. [9][10][11][12][13][14][15] This problem originates, in part, from a lack of theoretical methods that can reliably predict the excited-state electronic properties of materials with disordered or irregular microscopic structure. Many efforts to address this problem have thus been focused on developing ways to accurately include the effects of disorder in traditional theoretical models. 8,[16][17][18][19][20] In this letter, we extend these efforts with a theoretical approach designed to reveal the specific effects of correlated molecular disorder on electronic energy transport in organic molecular semiconductors. Our approach combines classical molecular dynamics (MD) simulations, semiempirical electronic structure calculations, and the Frenkel exciton model, in order to identify the characteristics of molecular correlations in these materials and isolate their influence on the microscopic dynamics of electronic excitations. Using this approach, we find that energy transport in these materials can be very sensitive to the presence of spatial correlations, and that this sensitivity depends on the width of the distribution of excitation energies. We illustrate that fluctuations in these energies play a fundamental role in driving the dynamics of electronic excitations, but we also highlight that these dynamics are insensitive to the temporal correlations that arise due to regular nuclear vibrations.</p><p>These findings suggest that it may be necessary to consider the interplay between correlated and uncorrelated disorder when applying molecular design principles to the development of organic molecular semiconductors.</p><p>Energy transport in organic conjugated systems is determined primarily by the microscopic dynamics of Coulombically bound excited electron-hole pairs, known as excitons. In condensed phase systems, intermolecular electronic coupling can drive excitons to delocalize across many individual molecules. The properties of these delocalized excitons depend on the strengths of electronic couplings and on how they are distributed in space and time. The characteristics of a material's molecular structure that are most relevant to the dynamics of excitons are thus encoded in these electronic coupling distributions. Unfortunately, these distributions are difficult to compute because they require evaluation of the exited state electronic structure and because they tend to vary significantly with small changes in nuclear configuration and therefore must be computed separately for each pair of molecules in the system.</p><p>The computation of electronic properties is not the only challenge associated with modeling exciton dynamics in disordered systems. Accurate modeling also requires the ability to address system sizes large enough to fully accommodate delocalized excitons, and a sampling scheme capable of capturing the effects that arise due to local variations in molecular structure. If excitons delocalize over more than a few molecules, then standard electronic structure methods, such as those based on density functional theory, are generally too computationally expensive to fully meet these requirements. As an alternative, site-based phenomenological models provide a simple and efficient platform for computing the static and dynamic properties of delocalized excitons in extended heterogeneous systems.</p><p>Here, we simulate the properties of delocalized excitons using the Frenkel exciton model 21,22 in which the excited electronic properties of a N-molecule system are expressed in terms of the Frenkel Hamiltonian,</p><p>where |i represents a state with an exciton localized on molecule i, i denotes the energy of that state, and V ij denotes the intermolecular electronic coupling between states |i and |j .</p><p>Because this simple model lacks explicit chemical detail, all aspects of a system's molecular structure must be described implicitly, in terms of model parameters. For this reason, the effects of molecular disorder are often incorporated into this model by dressing static model parameters with additional random components. The characteristics of these random components are often assumed to be Gaussian distributed, spatially uncorrelated, and described by simple dynamics that are either temporally uncorrelated (e.g., white noise) or arising from weak coupling to a bath of harmonic oscillators. 18, [23][24][25][26] Although these assumptions introduce disorder in a well defined and easily controllable manner, it is not obvious to what extent the molecular structure that they imply is physically realistic.</p><p>In our approach, the parameters of the Frenkel Hamiltonian are assigned non-randomly based on the microscopic structure of configurations generated with all-atom MD simulation (see supplementary information for more details). We consider the effect of thermal fluctuations on the statistics of these model parameters, and we quantify the spatial and temporal correlations that are associated with these statistics. By determining the separate influences of these correlations on the dynamics of excitons we can critically assess the assumptions that are commonly applied when parameterizing the Frenkel exciton model for organic molecular semiconductors. We present results for room temperature (T = 300K) condensed phase systems that are made up entirely of assembled sexithiophene (T6) molecules. We choose T6-based materials specifically because they have been well studied both experimentally and theoretically. [27][28][29][30] We consider systems with two different characteristic morphologies: a monolayer film of 150 longitudinally aligned molecules and an amorphous bulk of 343 T6 In Fig. 1 we illustrate how differences in molecular structure influence the characteristics of site energetic disorder. Fig. 1(b) shows the spatial distribution of site energies (i.e., i in Eq. 1) as derived by applying our parameterization method to a single MD configuration of the monolayer film. This plot illustrates that the monolayer film includes significant spatial disorder, because the molecules are not arranged on a regular lattice, and significant energetic disorder, because molecules exhibit a wide range of excitation energies.</p><p>Fig. 1(c) contains a plot of P ( ), the probability for a molecule in a given system to have a site energy . We observe that P ( ) is broad and asymmetric for both the monolayer and the amorphous system, which have a standard deviations of 0.10eV and 0.18eV, respectively. The distribution of the monolayer system is both red-shifted and narrowed relative to that of the amorphous system. These differences reflect the influence of favorable π−stacking interactions between neighboring molecules in the film, which simultaneously limit configurational variations and promotes conjugation through molecular planarization.</p><p>Molecular packing effects in condensed phase systems can lead to the emergence of spatial correlations in molecular and electronic structure. These correlations are often neglected when assigning parameters in the Frenkel exciton model. Our approach to assigning these parameters preserves these correlations, which enables them to be quantified. We define the spatial correlation function for site energies as,</p><p>where • • • denotes an average over all available configurations of a given system, ¯ is the average site energy for molecules in the system, r ij is the center-of-mass separation between the molecules associated with sites i and j, and δ(x) is the Dirac delta function. As this plot illustrates, closely spaced molecules tend to have correlated excitation energies.</p><p>We observe that these correlations die off rapidly with distance, not extending much beyond distances of about 0.5nm (i.e., roughly the nearest neighbor distance), but are slightly longer ranged in the monolayer system. Despite the lack of long-range spatial correlations, it has been found that excitons readily delocalize in both of these systems. 20 The time dependence of the model parameters in Eq. 1 are generated to reflect the evolution of a given system from MD simulation. A representative trace of (t) for a single site of the model monolayer film, as plotted in the inset of Fig. 2(a), reveals that the excitation energy of individual molecules can exhibit significant fluctuations. The temporal correlations in these model parameters can be characterized by computing the time correlation function for site energies, and for intermolecular electronic couplings,</p><p>where the averages implied by • • • include data generated for a system with a given molecular structure and the overbars indicate time average. As illustrated in Fig. 2, both C (t) and C V (t) exhibit non-trivial forms, including distinct short and long time decay profiles along with a remarkably long lived oscillatory feature. For both correlation functions we attribute the initial fast decay (i.e., τ ∼ 100fs) to the dephasing effect from nuclear ballistic motion, and the slower decay (i.e., τ ∼ 500fs) to ring-ring torsional dynamics. We attribute the long lived oscillations, with periods of approximately 20fs and 100fs, to the C-H and C-C bond stretching vibrations, respectively. These correlations are significantly more complicated than what is usually assumed in applications of the Frenkel model to organic molecular semiconductors. 18,23,24,26 Some previous efforts to develop more realistic descriptions of the phonon spectral density have combined MD simulation with excited state electronic structure calculation, [31][32][33][34] however, due to computational cost these efforts have been limited to relatively small system sizes and short time scales.</p><p>To investigate how the molecular correlations in these materials influence the dynamics of excitons we carry out simulations using the time-dependent Hamiltonian in Eq. 1. Specifically, we solve the time-dependent Schrodinger equation to obtain the exciton wavefunction,</p><p>where T is the time-ordering operator and c i (t) is the wavefunction coefficient in the molecular site basis. We then isolate the influence of specific correlations by modifying the Hamiltonian, replacing correlated parameters with uncorrelated random noise. By using the time dependent Frenkel Hamiltonian derived from MD simulations we are able to captures the effect of nuclear motion on the properties of the exciton, however, this approach to dynamics omits the feedback of the exciton on the dynamics of the nuclei. Omitting this feedback yields a significant gain in computational efficiency, however, the resulting electronic dynamics are thus prevented from properly thermalizing. Since the transient effects associated with exciton thermalization are most pronounced on timescales that are longer than we simulate here, 35,36 we expect the errors associated with the omission of this feedback to be small and have no influence on the nature of our conclusions. This issue is discussed more thoroughly in the Supporting Information.</p><p>We quantify the dynamic properties of excitons in terms of their mean-squared displacements (MSD), which we compute from the solution to Eq. 5 using the formula,</p><p>where r i denotes the center-of-mass position of the molecule associated with site i. We compute the MSD by averaging over a nonequilibrium ensemble of trajectories that are each initialized with the exciton localized on a single site. Similarly, the delocalization of excitons can be quantified in terms of the inverse participation ratio, as presented in the Supporting Information. amorphous bulk under different model conditions. In both panels the fully mapped reference condition is represented with a solid blue line, the static condition is represented with a solid red line, the condition with no spatial correlations is represented by a solid yellow line, and the condition with no temporal correlations is represented by a dashed magenta line. The black dashed lines denote MSD between a localized and fully delocalized exciton.</p><p>We compare the exciton dynamics generated under four different sets of model conditions.</p><p>The first condition is the reference condition, in which the parameters of H(t) are mapped directly from the results of MD simulations following the method in Ref. 20. The second condition is a static condition, designed to evaluate the effect of molecular fluctuations on the dynamics of excitons. For the static condition all model parameters are time independent, mapped from a single randomly drawn configuration from the MD simulations. The third and forth set of model conditions modify those of the reference condition by eliminating either spatial or temporal correlations in the model parameters, respectively.</p><p>Under the third set of model conditions, spatial correlations are eliminated by assigning i (0) for each site randomly from the distributions in Fig. 1(c), but the time-dependent component δ i (t) = i (t) − i (0) for each site remains identical to that of the fully mapped reference Hamiltonian. With this set of conditions V ij (t) is left unmodified from the reference Hamiltonian. Under the forth set of model conditions, temporal correlations are eliminated from the time-dependent components δ i (t) and δV ij (t) = V ij (t) − V ij (0) by assigning both as Gaussian white noise with the same variance as the reference Hamiltonian.</p><p>The MSD computed for each of the four conditions are plotted in Fig. 3. We find that under the reference condition, excitons in both the monolayer and the amorphous systems exhibit similar dynamics, with ∼ 100fs of rapid diffusion followed by a plateauing as excitons reach the system boundaries. We observe that for the static conditions the initial exciton dynamics are similar but they taper off early (i.e., t ≥ 50fs) due to the onset of Andersen localization. 37 These results thus indicate that while molecular fluctuations are central to sustaining the excitons dynamics in these systems, their local mobility is primarily determined by the distribution of electronic couplings. Fig. 3 also illustrates that exciton dynamics in these materials are not sensitive to the non-Markovian time correlations exhibited by C (t) and C V (t) in Fig. 2. Specifically, the model conditions without time correlations reveal that the MSD for excitons is essentially unaffected when the dynamics of the fully mapped reference Hamiltonian are replaced with Gaussian white noise. Notably, these results are consistent with a similar finding that non-Markovian effect in biological light harvesting systems do not play a significant role in the exciton dynamics at room temperature. 38 To expand upon this point, we compare the statistics of exciton displacements across many individual trajectories. In Fig. 4 we plot the probability distribution, P (D 2 ), where</p><p>is the squared displacement of an exciton computed from a single trajectory. This figure illustrates that the statistics of exciton displacements are similar for both systems under model conditions with or without time correlations.</p><p>The MSD for excitons generated under model conditions without spatial correlations, as plotted in Fig. 3, reveals that the influence of spatial correlations on exciton dynamics is system dependent. Removing spatial variations from the reference model has minimal effect in the monolayer film, however, in the amorphous system the absence of spatial correlations results in a reduction of exciton diffusivity. Because the spatial correlations for each system are very similar (see Fig. 1(d)), the origin of this system dependence must involve a different aspect of molecular structure.</p><p>Based on the difference in P ( ) between the two systems (e.g., Fig. 1)c, we hypothesize that the sensitivity of exciton dynamics to spatial correlations is mediated by the amplitude of energetic disorder in the system. We test this hypothesis by artificially amplifying the disorder in the i 's in the model amorphous film with and without the inclusion of spatial correlations. As illustrated in Fig. 5, if the amplitude of disorder in this system is systematically increased, then the overall exciton diffusivity decreases (as expected) and the difference between the MSDs with and without spatial correlations grows. In other words, the effect of spatial correlations on exciton dynamics grows more pronounced as the microscopic structure of a system is made more disordered.</p><p>As Fig. 5 summarizes, the importance of spatial correlations on exciton dynamics depends on the amount of energetic disorder in the system. This observation implies that current rule-of-thumb design criteria may be insufficient for guiding ongoing material development efforts. In addition, this implies that control over spatial correlations can possibly mitigate the negative influence of disorder on exciton transport properties. We have also found that exciton dynamics in these systems are insensitive to the details of temporal correlations, however, this insensitivity may not necessarily persist in all organic molecular semiconductors. Simple phenomenological models, such as we have utilized here, provide a convenient and efficient framework for exploring the interplay between molecular structure and opto- electronic material properties. The continued development and application of these modeling approaches is thus important for advancing our understanding of these systems. A useful measure to characterize the extent of delocalization of exciton wavefunction is the inverse participation ratio (IPR) [1] to the finite-size effect. The final value of IPR for the amorphous bulk is higher because of its larger system size compared to the monolayer film.</p><p>We also explore IPRs generated under three other different conditions in Fig. 1: calculations with static Hamiltonian (solid red lines), calculations with no spatial correlation in the static disorder (solid orange lines), and calculations with no time correlation in the timedependent fluctuations (dashed magenta lines). In general, the qualitative behaviors under each condition are very similar to those found in the analysis of MSDs in the main text.</p><p>IPRs generated from the same Hamiltonian but without the time-dependent components (i.e. δV ij (t) = 0 and δ i (t) = 0) results in a significantly reduced IPR due to the Anderson localization effect. Fig. 1 also shows that temporal correlation does not play an important role in exciton transport as the IPR generated from the Hamiltonian without temporal correlation (dashed magenta lines) is nearly identical to the IPR generated from the molecular simulations (solid blue lines). The distributions of IPRs at t = 100f s sampled from 100 initial configurations are plotted in FIG. 2, which again shows that the IPR distributions generated from molecular simulations and from Hamiltonian with white noise to be similar, confirming the minimal effect of temporal correlation. Similar to the findings in the main text, the effect of spatial correlation on IPRs is system dependent: it has little effect on the monolayer film, but reduces the IPR in the disordered film (dashed lines in FIG. 1). FIG. 3 shows the IPR in disordered film with its static disorder artificially increased, and our results demonstrate that the effect of spatial correlation on IPR increases with the magnitude of static disorder.</p><!><p>We follow a recently developed method for mapping the structure of a N -molecule configuration onto the parameters of a corresponding N × N Frenkel Hamiltonian matrix. In this method, molecular configurations are generated using classical molecular dynamics (MD) simulations and each individual configuration is translated into Frenkel model parameters based on the analysis of N single-molecule excited state electronic structure calculations.</p><p>Specifically, for a given configuration we perform a single electronic structure calculation on each individual molecule, treating all other molecules as an effective dielectric medium.</p><p>Electronic structure is computed using a semiempirical Pariser-Parr-Pople (PPP) Hamiltonian with excited state properties computed at the level of configuration interactions singles.</p><p>Intermolecular couplings are evaluated by computing the diabatic coupling between the locally excited molecule pairs through transition densities. A complete description of this method, including information about classical force fields, electronic structure methods, and benchmarking against higher level theories can be found in Ref. [2].</p><!><p>We have chosen to simulate exciton dynamics with a method that omits the effects of excited state nuclear forces. In our method, the dynamics of the classical subsystem evolve on the potential energy surface of the electronic ground state, and are thus unaffected by the state of the exciton. In the absence of feedback between the electronic and nuclear degrees of freedom,the composite system cannot properly thermalize. This simply means that excitons will not relax into an equilibrium energic state, but rather will exhibit behavior associated with the high-temperature limit. Including the effects of excited state forces in our model</p><p>is straightforward yet very computationally expensive so we omit them.</p><p>We justify this omission by recognizing that this approximate method for treating dynamics is accurate in the short time limit. The timesacle for energetic relaxation of excitons is expected to be governed by two timescale. First, is the timescale associated with the molecule reogranization time, i.e., the time for excited molecules to relax on the excited state potential energy surface. This timescale is on the order of 100fs, but for delocalized excitons involves relatively small changes in excitation energy. Second, is the timescale associated with exciton migration on a disordered energetic landsacape. This timescale is determined by the mobility of excitons and the characteristics of the hetereogeneous energetic landscape. In these materials we expect the timescale to be on the order of picoseconds.</p><p>[1] Ishizaki, A.; Fleming, G. R. New J. Phys. 2010, 12, 055004.</p><p>[2] Shi, L.; Willard, A. P. J. Chem. Phys. 2018, 149, 094110.</p>

ChemRxiv

Carboxylato-Pillar[6]arene-Based Fluorescent Indicator Displacement Assays for Caffeine Sensing

In the present work, we have developed a new indicator displacement system based on pillararene for anionic water-soluble carboxylato pillar [6] arene (WP6) and aromatic fluorescent dye safranine T (ST). A large fluorescence enhancement and colour change of ST were observed after complexation with electron-rich cavity in WP6 because of host-guest twisted intramolecular charge-transfer interactions. The constructed pillararene-indicator displacement system can be applied for caffeine selective detection in water.

carboxylato-pillar[6]arene-based_fluorescent_indicator_displacement_assays_for_caffeine_sensing

Introduction<!><!>Materials and Methods<!>Complexation of ST With WP6<!><!>Complexation of ST With WP6<!><!>Complexation of ST With WP6<!><!>Complexation of ST With WP6<!>Complexation of Caffeine, Theophylline, and Theobromine With WP6<!>Fluorescent Indicator Displacement<!><!>Conclusion<!>Data Availability Statement<!>Author Contributions<!>Funding<!>Conflict of Interest<!>Publisher’s Note<!>Supplementary Material<!>

<p>Fluorescent indicator displacement assays (F-IDAs) are typically used to convert synthetic receptors into optical sensors in supramolecular chemistry. In F-IDAs, the competitive binding principle is used: after binding a fluorescent indicator to the receptor, when a competing analyte is introduced into the indicator–receptor pair, the indicator is discharged from the receptor to induce a fluorescence change (Wiskur, et al., 2001;Nguyen and Anslyn, 2006). Macrocyclic hosts typically provide ideal receptors for use because of their particular composition and excellent functions. The macrocyclic hosts, such as cyclodextrins (Crini, 2014; Pal, et al., 2015), calixarenes (Koh, et al., 1996; Hennig, et al., 2007; Guo and Liu, 2014; Zheng, et al., 2018), cucurbiturils (Florea and Nau, 2011; Praetorius, et al., 2008; Barrow, et al., 2015; Sonzini, et al., 2017) and pillararenes (Wang P, et al., 2014; Bojtár, et al., 2015; Bojtár, et al., 2016; Hua, et al., 2016; Bojtár, et al., 2017; Hua, et al., 2018; Xiao, et al., 2018, Xiao, et al., 2019a; Xiao, et al., 2019b), combined with various dyes have been applied as receptors in F-IDAs for specific and selective sensing in drugs, biomolecules, or other organic compounds.</p><p>This study established an FID assay with a water-soluble pillararene for caffeine detection. Caffeine is the most widely consumed psychostimulant drug worldwide. Appropriate caffeine intake may enhance alertness, attention, and nerve cell activity and decrease the possibility of type 2 diabetes. However, excessive intake of caffeine may possibly cause a headache, high blood pressure, irregular small muscle movement, and allergy, especially in teenagers and pregnant women (Nehlig, et al., 1992; Rapuri, et al., 2001; Smith, 2002; Lovallo et al., 2005). Caffeine detection can be realised with costly and complex methods, such as HPLC-MS and immunoassay (Wu, et al., 2000; Oberleitner, et al., 2014). Therefore, caffeine detection remains inconvenient for public usage. Thus, we realised novel host–guest recognition between water-soluble pillararene (WP6) and safranine T (ST) and revealed the operation of this host–guest recognition motif as an FID assay in caffeine detection (Scheme 1). The assay seems selective for theophylline and theobromine.</p><!><p>Chemical structures and cartoon presentations of WP6, ST and caffeine and illustration of the turn-off fluorescence detection of caffeine through indicator displacement process.</p><!><p>The reagents used were marketable and applied directly without further purification. WP6 (Yu et al., 2012) was synthesized by following the known procedures. Nuclear magnetic resonance (NMR) spectra were obtained using the Bruker Avance III HD 400 spectrometer with the deuterated solvent as the lock and the residual solvent as the internal reference. Fluorescence spectra were obtained by using the Agilent Cary Eclipse fluorescence spectrophotometer. To prevent the dilution effect during titration, WP6 stock solutions were produced using the same ST solution. The measurement was repeated three times for each experiment. Displacement assay for theophylline and theobromine was performed at pH 7.2 with WP6 at varying concentrations of theophylline and theobromine, respectively. All the experiments were conducted at room temperature (298 K).</p><!><p>To study the host–guest complexation between WP6 and ST, 1H NMR spectroscopy was first performed. Given that the complex solubility of neat D2O did not occur at the mM scale, DMSO-d 6 cosolvent was supplemented. According to Figure 1, ST aromatic protons in the complex shifted upfield to varying degrees. This result revealed that ST was encapsulated by WP6 cavity and protons on ST were shielded by the electron-rich cyclic structure when the inclusion complex formed (Wang Y. et al., 2014). The characteristic signal broadening of the protons on ST was observed because of the shielding effects of the aromatic host (Li et al., 2010). Furthermore, protons on WP6 revealed minor chemical shifts resulting from host–guest interactions between WP6 and ST.</p><!><p>Partial 1H NMR spectra (400 MHz, D2O:DMSO-d 6 = 1:1, 298 K) for (A) 3 mM WP6, (B) 3 mM WP6 and 10 mM ST, (C) 10 mM ST.</p><!><p>The formation of host–guest complex between WP6 and ST was further confirmed through UV-vis absorption spectroscopy. (Figure 2). A broad absorption band above 555 nm, corresponding to the charge-transfer interaction between electron-rich WP6 and electron-deficient ST, was observed. Furthermore, after adding WP6 to ST, a red shift appeared, which indicated that a representative charge-transfer complex was formed (Wang Y et al., 2014). The fluorescence titration of ST with WP6 was performed under ambient temperature in water. According to. Figure 3A, an enhancement in fluorescence and a red shift in the emission spectra were observed with the progressive supplement of WP6, which indicated that a strong supramolecular complex was formed. These changes may arise from the formation of twisted intramolecular charge transfer (TICT) state when ST occupied the WP6 cavity in the aqueous buffer. Under the TICT state, the phenyl or phenazinyl group is assumed to rotate around bonds that connect them to the central single bond. The twisting movement is subjected to restriction of the encapsulated ST guest, leading to enhanced fluorescence (Grabowski et al., 2003; Bojtár, et al., 2015).</p><!><p>UV-vis spectra for (A) WP6, (B) ST, and (C) ST in the presence of 50 equiv. of WP6 (1 × 10−3 M) in PBS (pH = 7.2). The inserted photo displays the colour changes related to ST with the addition of WP6.</p><p>(A) Changes of the fluorescence intensity in ST (0.02 mM) upon the titration of WP6 (0–25 equiv.) in PBS (λex = 523 nm, λem = 584 nm, pH = 7.2). The inserted photo exhibits an enhancement in fluorescence in water under excitation at 365 nm via the UV lamp at 298 K. (B) Fluorescence titration for the competitive displacement of ST (0.02 mM) from WP6 (0.3 mM) using caffeine (0–150 equiv.) in PBS at pH 7.2 (λex = 523 nm, λem = 584 nm). The inserted photo exhibits the corresponding fluorescence quenching in water under excitation at 365 nm via the UV lamp at 298 K.</p><!><p>The association constant (K a) and the optical spectroscopic data of ST and corresponding WP6 complex are listed in Table 1, which presents a comparison with the data of ST complexes with β-cyclodextrin, ST⊂β-CD (Zhang et al., 2005), disulphide bridged β–cyclodextrin, ST⊂SS-β-CD (Yang et al., 2017), and γ-cyclodextrin, ST⊂γ-CD (Wang et al., 2012). An association constant of K a = (1.50 ± 0.06) × 104 M−1 was obtained using a nonlinear fitting to the fluorescence spectra, measured by titration experiments. The stoichiometry of 1:1 for the complexes was tested with the molar ratio approach, based on the fluorescence data related to WP6-ST mixtures.</p><!><p>Association constants (K a) and optical spectroscopic data for the complexes of ST with WP6 and other macrocycles.</p><!><p>The optimal association constant revealed system applicability to FID. The spectroscopic data for complex ST⊂WP6 were similar to the spectroscopic data for the complex of the dye containing SS-β-CD, ST⊂β-CD (Yang et al., 2017), which proved the similarity of polarity of these two macrocycles. However, because of the weak interactions, the association constant with the uncharged cyclodextrin was lowered by an order of magnitude.</p><!><p>Next, for evaluating analyte complexation, 1H NMR spectra were obtained for caffeine, theophylline, and theobromine. Supplementary Figure S1 reveals that all the proton signals of caffeine shifted upfield at various extents, which indicated that caffeine was threaded into the host cavity. Furthermore, according to the 2D NOESY spectrum (Supplementary Figure S4), NOE correlation signals were obtained between protons Ha-d of caffeine and proton H1 on WP6, verifying WP6's assignment for the caffeine threaded structure. Signals from the NMR spectra for theophylline and theobromine exhibited similar changes after adding WP6 (Supplementary Figures S2, S3).</p><p>Next, fluorescence titrations were performed at 298 K in PBS at pH 7.2 for estimating the binding behaviours of WP6 with caffeine, theophylline, and theobromine in a quantitative manner. Job plots (Supplementary Figure S8) drawn using fluorescence titration data suggest WP6 and the three guests in a 1:1 host–guest complex of the aqueous solution, respectively. Based on the nonlinear curve-fitting approach (Supplementary Figures S10, S11), the measured association constants (K a) were (2.51 ± 0.24) × 104 M−1, (9.30 ± 0.04) × 103 M−1, and (9.14 ± 0.08) × 103 M−1 for caffeine, theophylline, and theobromine, respectively. The K a value for the binding of caffeine is approximately an order of magnitude greater than the binding of theophylline and theobromine.</p><!><p>Next, the indicator displacement process was used to measure the nonfluorescent host–indicator complex to detect caffeine. Figure 3B displays the typical process of displacement titration. When caffeine was gradually added into a mixed PBS solution with ST and WP6, fluorescence intensity quenching was apparent. The result proved that the added caffeine was able to rival with ST to push the indicator from the WP6 cavity. The following optical changes could be attributed to the formation of the caffeine⊂WP6 complex, which exhibited higher stability compared with ST⊂WP6. Furthermore, the 'turn-off' fluorescence changes resulting from caffeine addition could be observed by naked eyes by using a simple UV lamp (Figure 3B ). The results indicate that the applicability of the ST⊂WP6 complex to an F-IDA for sensing anticancer drug caffeine.</p><p>The ST⊂WP6 system as a caffeine sensor exhibited higher selectivity than theophylline and theobromine. Theophylline and theobromine at the same concentration (2 mM) were added to the solution of the ST⊂WP6 complex, respectively. Figure 4 displays changes of the fluorescence ratio I/I0 in the ST⊂WP6 complex on the addition of theophylline and theobromine, respectively. Negligible fluorescence changes were observed following the addition of theophylline and theobromine. Under the used conditions, theophylline and theobromine induced limited interference in the caffeine selective responses, suggesting the prominent selectivity of the method for caffeine. This selectivity can be attributed to the difference in binding constants between the host WP6 and the guests.</p><!><p>Fluorescence spectra for ST (0.02 mM) + WP6 (0.35 mM) with caffeine (2 mM), theophylline (2 mM), and theobromine (2 mM), respectively.</p><!><p>In conclusion, a new host–indicator composed of an electron-deficient dye ST and anionic water-soluble pillar[6] arene WP6 was developed. After the ST⊂WP6 complex was formed, the twisted intramolecular charge-transfer-induced fluorescence enhancement and solution colour changes were apparent. Furthermore, this supramolecular system was successfully applied as a fluorescent indicator displacement assay to detect caffeine. Large signal modulation and a selective response towards caffeine against theophylline and theobromine were observed.</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>QD designed the work. YX and KG made contributions to the experiments and collective data. The paper was written by QD. All authors extensively discussed the results, reviewed the manuscript, and approved the final version of the manuscript to be submitted.</p><!><p>This work was supported by the Research and Cultivation Foundation of Henan University of Engineering (PYXM202009).</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.2021.816069/full#supplementary-material</p><!><p>Click here for additional data file.</p>

PubMed Open Access

Recent Progress in Natural-Product-Inspired Programs Aimed To Address Antibiotic Resistance and Tolerance

Bacteria utilize multiple mechanisms that enable them to gain or acquire resistance to antibiotic therapies during the treatment of infections. In addition, bacteria form biofilms which are surface-attached communities of enriched populations containing persister cells encased within a protective extracellular matrix of biomolecules, leading to chronic and recurring antibiotic-tolerant infections. Antibiotic resistance and tolerance are major global problems that require innovative therapeutic strategies to address the challenges associated with pathogenic bacteria. Historically, natural products have played a critical role in bringing new therapies to the clinic to treat life-threatening bacterial infections. This Perspective provides an overview of antibiotic resistance and tolerance and highlights recent advances (chemistry, biology, drug discovery, and development) from various research programs involved in the discovery of new antibacterial agents inspired by a diverse series of natural product antibiotics.

recent_progress_in_natural-product-inspired_programs_aimed_to_address_antibiotic_resistance_and_tole

INTRODUCTION<!>TEIXOBACTIN, A PROMISING NEW ANTIBIOTIC: iChip DISCOVERY, TOTAL SYNTHESIS, AND NOVEL ANALOGUES<!>DEFINING RULES TO TARGET GRAM-NEGATIVE BACTERIA AND CONVERTING A DEOXYNYBOMYCIN ANALOGUE TO A BROAD-SPECTRUM ANTIBIOTIC<!>DEVELOPING TOTAL SYNTHESIS PLATFORMS FOR NEW, FULLY SYNTHETIC ANTIBIOTICS<!>PROMYSALIN: A SPECIES-SPECIFIC ANTIBIOTIC THAT TARGETS SUCCINATE DEHYDROGENASE IN Pseudomonas aeruginosa<!>RECENT ADVANCES OF SIDEROPHORE CONJUGATES FOR \xe2\x80\x9cTROJAN HORSE\xe2\x80\x9d TARGETED DELIVERY OF ANTIBIOTICS TO PATHOGENIC BACTERIA<!>ClpP PROTEASE-ACTIVATING AGENT ADEP4 FOR PERSISTER CELL AND BIOFILM ERADICATION<!>HALOGENATED PHENAZINE BIOFILM-ERADICATING AGENTS<!>CONCLUSIONS

<p>The discovery and routine administration of antibiotics to treat bacterial infections led to their recognition as "wonder drugs" in the mid-20th century.1–9 To date, antibiotics are still considered one of the most significant interventions in human medicine. The efficacy and selectivity antibiotics exert made many believe that bacterial infections would be a thing of the past. However, instead of the routine eradication of bacterial pathogens in the clinic, we have witnessed an alarming rise in the failure to treat bacterial infections due to the generation of antibiotic-resistant "superbugs"3,5 and antibiotic-tolerant persister cells and biofilms.10–12</p><p>Clinically approved antibiotics operate via the selective inhibition of critical targets that impede replication and growth or eradicate bacterial cells. It is interesting to note that relatively few bacterial targets have been exploited for antibiotic treatments in the clinic. The mechanisms of action for our arsenal of antibiotics include the inhibition or disruption of (1) cell wall synthesis (e.g., β-lactams,13 vancomycin14), (2) protein synthesis (e.g., tetracyclines,15 macrolides,16 aminoglycosides,17 linezolid18), (3) RNA poly-merase (e.g., rifamycins19–21), (4) nucleic acid synthesis (e.g., quinolones22–25), (5) folate biosynthesis (e.g., sulfonamides, trimethoprim),26 and (6) cell membrane (e.g., polymyxins;26 Figure 1).4,7</p><p>Upon exposure to antibiotics, bacteria can expediently evade their action using one or more well-defined resistance mechanisms.3–6,8,27,28 Bacterial resistance has led to the catastrophic rise of roughly 2 million annual infections, resulting in 23 000 deaths in the United States.29 In Europe, more than 33 000 deaths were attributed to antibiotic-resistant bacterial infections in 2015.30 Universal acquired resistance mechanisms can prevent antibiotics access to the aforementioned intracellular targets. The cell wall of Gram-negative bacteria consists of inner and outer membranes, which upon mutation can act as a permeability barrier to antibiotics leading to reduced penetration into the cells.31,32 Alternatively, bacteria can actively eliminate, or "pump out", antibiotics before they bind their corresponding intracellular target via the action of efflux pumps (e.g., efflux of macrolides).31,32 Mutations and modifications of the target's binding site are, collectively, another resistance mechanism bacteria utilize to acquire resistance as the resulting changes negatively impact drug–target interactions of antibiotic therapies. Specific examples of mutation and modification to impede antibiotic binding sites include (1) mutation and alteration of penicillin binding proteins to negate the effects of penicillin, (2) structural alterations in DNA gyrase to prevent fluoroquinolone action, and (3) methylation of critical residues within the rRNA of bacterial ribosomes to eliminate the binding of aminoglycosides.33–35 Additionally, bacteria can enzymatically modify antibiotics to generate inactive agents, and examples include (1) hydrolysis of β-lactam antibiotics by β-lactamase enzymes to cleave and destroy the critical β-lactam warhead (pharmacophore) required for antibacterial activities and (2) modification, or functionalization (e.g., acylation), of hydroxyl and/or amine groups critical for aminoglycoside antibiotics to hydrogen bond (bind) to bacterial ribosomes by aminoglycoside-inactivating enzymes.29 In addition, bacteria can overproduce targets to bypass the effects of antibiotics (e.g., trimethoprim; Figure 1).29,35</p><p>Innate antibiotic tolerance is a distinct microbiological phenomena from acquired resistance and is now recognized as the underlying cause of chronic and recurring bacterial infections.36 Using a communication process known as quorum sensing, bacteria secrete and sense small organic signaling molecules to monitor their population density and coordinate virulent behaviors.37–41 Bacteria use quorum sensing to coordinate surface attachment and subsequent biofilm formation, where dense communities of bacterial cells are encased within matrixes of polysaccharides and extracellular DNA.42–46 Once within a biofilm, bacterial cells divide at a much slower rate compared to their planktonic (free-floating) counterparts or they do not replicate at all. The subpopulation of biofilm cells that enter into a dormant state are known as "persister cells" and are characterized as metabolically inactive.12,47–51 Extracellular polymeric substances (EPSs), or biofilm matrix, provide an additional layer of protection to surface-attached communities, enabling these bacteria to thrive in harsh conditions (e.g., host immune response).52,53</p><p>It is important to note that all classes of FDA-approved conventional antibiotics were initially discovered as bacterial growth inhibitors against actively dividing cultures. Therefore, it should be no surprise that clinically used antibiotics are essentially ineffective against nonreplicating biofilm infections. With the 17 million new cases of biofilm infections in addition to >500 000 deaths each year in the United States that result from these infections, there is a critical need to identify compounds that effectively eradicate biofilms through growth-independent mechanisms (Figure 2).10,41</p><p>The current need for new and effective antibiotic therapies has motivated several research groups to initiate drug discovery programs inspired by various antibiotic natural products, which may be known or recently discovered. The Lewis group has created a new technology to facilitate isolation of novel antibiotics from previously inaccessible sources, while the Myers lab has developed novel total synthesis platforms for the discovery of new antibiotics. The Hergenrother group elegantly utilized a collection of diverse small molecules to establish guidelines that will allow the targeting of clinically imposing Gram-negative pathogens. The Miller group has pioneered a Trojan horse strategy and continues to make advances in this area by taking advantage of siderophore systems for bacterial import in unique and elegant ways to impart activity against Gram-negative organisms. Recent work by the Wuest lab has focused on a natural product with narrow-spectrum activity against Pseudomonas aeruginosa. Both broad- and narrow-spectrum-based strategies have been employed, but scientists agree that care must be taken to minimize resistance development against these novel agents. With biofilms being inherently tolerant to current antibiotics, the Lewis group and our lab have identified agents capable of eradicating persister cells and established biofilms. Our group has identified a series of synthetically tunable halogenated phenazine small molecules derived from a marine antibiotic that potently eradicates bacterial biofilms through a rapid iron starvation mechanism. Fortunately, recent advances in chemistry, molecular biology, and chemical biology have yielded a bevy of powerful techniques that can be employed to better understand problems associated with pathogenic bacteria and enable the discovery of novel natural-product-inspired therapies. This Perspective will detail each of these innovative and exciting antibiotic discovery strategies (Figure 3).</p><!><p>Bacteria compete for finite resources in their environment and often produce antibiotics to compete with each other. Many antimicrobials used in the clinic today have been derived from natural products produced by bacteria. Large numbers of organisms cannot be grown successfully through traditional culturing methods and are thus named "unculturable".1 It is believed that approximately 99% of all species in external environments are uncultured.54 However, recent development of iChip technology by the Lewis group has enabled the growth of previously uncultured bacteria (Figure 4).54,55 This new technology has enhanced the potential for many more types of bacteria to be investigated and screened for the identification of new antibiotics.</p><p>By use of the iChip, extracts from 10 000 bacterial isolates were grown and screened, which led to one isolate (β-proteobacteria, named Eleftheria terrae) that demonstrated antibacterial activity against S. aureus. From the Eleftheria terrae extract, teixobactin 1 was isolated and structurally elucidated using mass spectrometry, NMR, and Marfey's analysis.55 Teixobactin 1 proved to be a unique antibiotic containing a cyclic depsipeptide moiety composed of four amino acid residues, including an enduracididine residue, that are appended to a seven amino acid linear chain bearing a methylphenylalanine. It is also interesting to note that 1 has four d-amino acid residues incorporated into its impressive structure. The biosynthetic gene cluster consists of two nonribosomal peptide synthetase (NRPS) genes named txo1 and txo2 where 11 modules are encoded. The depsipeptide 1 showed excellent antibacterial activities in minimum inhibitory concentration (MIC) assays against significant, Gram-positive pathogens, including S. aureus (MIC = 0.25 μg/mL against methicillin-sensitive and methicillin-resistant strains; 1 also demonstrated antibacterial activities against intermediate vancomycin-resistant S. aureus, VISA), enterococci (MIC = 0.5 μg/mL, vancomycin-resistant enterococci strains, VRE), M. tuberculosis (MIC = 0.125 μg/mL), Clostridium dif f icile (MIC = 0.005 μg/mL), and Streptococcus pneumoniae (MIC ≤ 0.03 μg/mL, penicillin-resistant strain) as reported by Lewis and co-workers.54</p><p>A common technique for determining the mechanism of action for antibiotics is to generate resistant mutants. Lewis and colleagues were not able to develop resistant mutants of S. aureus or M. tuberculosis, indicating that teixobactin's target may not be a protein. This hypothesis was supported by the inhibition of peptidoglycan synthesis by 1, while no effect was observed on biosynthesis of DNA, RNA, or proteins. Because true vancomycin resistance in S. aureus is rare and vancomycin is known to bind lipid II, Lewis and co-workers hypothesized that teixobactin could also exert its activity through interaction with this target. When whole cells (S. aureus) were treated with 1, a significant amount of the peptidoglycan precursor undecaprenyl-N-acetylmuramic acid-pentapeptide (UDP-Mur-NAc-pentapeptide) accumulated in a similar fashion to cells treated with vancomycin. Competition enzyme binding assays indicated that 1 does not inhibit MurG, FemX, or PBP2 enzymes directly, while the addition of lipid II prevented 1 from inhibiting growth against S. aureus. These combined results demonstrate that 1 specifically interacts with the peptidoglycan precursor rather than interfering with the activity of the enzymes. Teixobactin retained activity against VRE which has a modified lipid II, suggesting that it binds at a different site than vancomycin.54</p><p>The therapeutic potential of 1 was also reported by Lewis and co-workers.54 Teixobactin retained its antibacterial potency in the presence of serum and demonstrated good chemical and microsomal stability and low toxicity. The pharmacokinetic parameters showed that after a single 20 mg per kg dose (iv injection) of 1 in mice, serum concentrations were maintained above MIC values for 4 h. Animal efficacy studies were then performed with 1 in a mouse septicemia model against MRSA at a dose that leads to 90% death; however, treatment with 1 (iv at 20 mg/kg, 1 h after infection) resulted in the survival of all mice, and a later experiment determined the PD50 (protective dose at which half of the animals survive) of 1 to be 0.2 mg/kg. 1 was also shown to be efficacious in a thigh infection of S. aureus and a lung infection model of S. pneumoniae, causing a 6 log10 reduction of colony forming units (CFU) per mL.54</p><p>Following the initial report of teixobactin's discovery, two total syntheses of 1 have been published utilizing solid-phase technology in separate reports by Payne and co-workers56 and Li and co-workers.57 In both total syntheses of 1, macro-lactamization at the less hindered Thr8-Ala9 connection has proved to be the fruitful cyclization approach (see conversion of 5 to 6, Figure 5A, by Li and co-workers) as opposed to a macrolactonization strategy. Following the macrolactamization step, Li and co-workers coupled linear peptide 3 to cyclic peptide 6 using a key ligation step in pyridine/acetic acid before final HPLC conversion to 1. Yuan and co-workers developed a stereoselective and scalable synthetic route to the rare l-allo-enduracididine (L-allo-End)58 moiety of 1, which played a critical role in the total synthesis reported by Li and co-workers.</p><p>In addition to total synthesis campaigns, solid-phase chemistry has enabled initial medicinal chemistry efforts of 1 by multiple groups, including Albericio,59 Singh,60–63 Su and Fang,64 Rao,65 Li,66 and Chan.67 Several of these groups have probed the structural requirements of the l-allo-enduracididine moiety at position 10 of 1. If this unique residue is not required for antibacterial activities, then more simplified analogues of 1 can be prepared more readily. Albericio and co-workers were the first to report the replacement of the unusual l-allo-enduracididine residue of 1 by substituting with L-arginine; however, this new analogue lost significant antibacterial activities against S. aureus (MIC = 1.6 μg/mL) and B. subtilis (MIC = 0.40 μg/mL) based on MIC value comparison (1 reported MIC values with ≥8-fold more potent activities when compared to this analogue).59 Fang and Su and co-workers synthesized nine teixobactin analogues, which establish that (1) a guanidine or amine group at position 10, (2) the hydroxyl group of serine (position 7), (3) the N–H proton of the terminal phenylalanine (position 1) were all critical for antibacterial activity. During these investigations, Fang and Su also reported that position 4 can tolerate various side chain size and functional group content.64</p><p>The initial reports of teixobactin analogues gave insightful information into the SAR of 1; however, a "first wave" of synthetic analogues reported by Albericio, Su, Fang, and co-workers59,64 lost a substantial amount of antibacterial activity and appeared to be less than exciting lead molecules. Rao and co-workers65 recently reported the synthesis of 21 new teixobactin analogues, including simplified and equipotent analogue 7 (Figure 5B). This work demonstrated that the N-Me-d-Phe residue at position 1 of teixobactin (1) tolerates additional hydrophobic groups on the phenyl ring and (2) does not require the methyl group on the amine, as the primary amine was well tolerated. These structural modifications combined with a l-lysine substitution of the l-allo-enduracididine residue at position 10 resulted in equipotent analogue 7, which also demonstrated outstanding in vivo efficacy in a S. pneumonia mouse model of infection (100% survival rate of mice infected with bacterial loads that lead to 90% death in controls).</p><!><p>Gram-negative pathogens are a major clinical problem due to their lack of sensitivity to many commonly used anti-biotics.68,69 Quinolones were the last new class of antibacterial agents active against Gram-negative bacteria introduced into the clinic in 1968.1 Despite extensive high-throughput screening efforts against Escherichia coli with large compound libraries of synthetic compounds, no lead compounds were translated into clinical agents.70 With two cellular membranes and a lipopolysaccharide-coated outer membrane, Gram-negative bacteria pose significant challenges for compound entry.68,71–73 Compounds capable of penetrating the outer membrane do so through porins, which are narrow trans-membrane proteins lined with charged amino acids. Once inside Gram-negative bacterial cells, small molecules are susceptible to removal through the function of efflux pumps; therefore, in order for a compound to accumulate within Gram-negative bacteria and engage its intracellular target, compounds are required to enter through porins faster than being removed via efflux.68</p><p>Hergenrother and co-workers noted that despite initial efforts by others, we have a limited understanding of the physicochemical properties enabling compound accumulation in Gram-negative bacteria.68,72,73 A structurally diverse set of 100 compounds with unique scaffolds rapidly accessed from available natural products were used to systematically analyze small molecule accumulation in E. coli (whole cells) using a liquid chromatography with tandem mass spectroscopy (LC–MS/MS) method. This approach using whole cells allowed multiple variables affecting compound accumulation to be accounted for, such as multiple porins, efflux pumps, and lipopolysaccharide composition. Conventional antibiotics with known high (i.e., tetracycline) and low (i.e., novobiocin) accumulation properties were evaluated as controls and reported to validate the method.68</p><p>Natural-product-derived compounds were selected for this study as most clinically used antibacterial drugs are either natural products or derivatives. The compound collection utilized by the Hergenrother lab was synthesized by this team, so these compounds are synthetically accessible with tunable properties to enable structure–activity relationships geared toward accumulation in Gram-negative bacteria. The library of 100 compounds utilized for this work, which was generated utilizing the "complexity-to-diversity" approach,74–78 included positively charged, negatively charged, and neutral compounds.68 The chemical structure, molecular weight, ClogD7.4, and charge of all compounds were tracked with accumulation results in E. coli.</p><p>From the initial accumulation studies, the primary factor that governed accumulation in E. coli was charge, with 12 of 41 positively charged compounds being the most likely to accumulate. Interestingly, 8 of the 12 positively charged compounds were primary amines.68 In follow-up SAR investigations, replacement of the primary amine with different functional groups (e.g., carboxylic acid, amide, alcohol) or amines bearing more substitutions significantly reduced accumulation in E. coli. Although the presence of a primary amine was important for accumulation, it alone was insufficient; therefore, chemoinformatics was utilized to understand which factors contribute to amine accumulation. This work revealed that the flexibility, as measured by the number of rotatable bonds, and shape of a compound are important factors that govern accumulation. Compound shape was described by "globularity" to inform the three-dimensionality. Shape was scored on a scale from 0 globularity for a "flat" compound (e.g., benzene) to 1 globularity for a "spherical" compound (e.g., adamantane).</p><p>On the basis of the extensive data set generated by Hergenrother and co-workers, the following guiding principles for accumulation in E. coli were developed: (1) compounds are most likely to accumulate if they contain a nonsterically encumbered amine, (2) contain some nonpolar functionality (typically not a problem for organic compounds), (3) contain a rigid structure and (4) have low globularity.68 These guidelines are referred to as the "eNTRy rules" (N, ionizable nitrogen, primary amine is best; T, low three-dimensionality, globularity score of ≤0.25; R, relatively rigid, ≤5 rotatable bonds).73 These guidelines were validated retrospectively with antibacterial agents presented by O'Shea and Moser.79 In addition, the role of porins was examined by testing the high-accumulation antibiotic controls and test compounds in a strain bearing a diminished number of porins.68 As predicted, a decrease in accumulation was observed for all compounds tested in the strain with fewer porins. Molecular modeling was performed with select test compounds and antibacterial agents that transverse the major porin of E. coli, OmpF, revealing a key interaction between the necessary primary, or non-hindered, amine and acidic residues (most often Asp113) that allow high accumulating compounds.</p><p>These well-defined guidelines were instructive in converting an analogue of the Gram-positive antibiotic deoxynybomycin (DNM, 9) into the broad-spectrum agent, 6DNM-NH3 (11, Figure 6).68 DNM is an inhibitor of DNA gyrase,80 however, only active against Gram-positive pathogens, presumably due to minimal accumulation in Gram-negative bacteria.68 DNM possesses shape and rigidity features that align with the eNTRy rules; however, no amine group is present. Analogue 6DNM 10 possesses an aliphatic bridge expanded by one methylene unit to give a six-membered ring, compared to DNM's (9) five-membered ring.</p><p>As one would expect based on the small structural differences between analogues, DNM 9 and 6DNM 10 possess the same activity profile (S. aureus MIC = 0.06–1 μg/mL; E. coli MIC > 32 μg/mL). However, 6DNM-NH3 11 has incorporated a primary amine appended to the aliphatic region of the six-membered ring, and as predicted based on the eNTRy rules, 11 demonstrates significantly higher levels of accumulation compared to 6DNM 10 (11, accumulation = 1114 nmol per 1012 CFU; 10, accumulation = 298 nmol per 1012 CFU). In line with eNTRy rule design and observed increases regarding E. coli accumulation, 6DNM-NH3 demonstrates broad-spectrum antibacterial activities (11, S. aureus MIC = 0.03–0.5 μg/mL; E. coli MIC = 0.5–16 μg/mL), including activities against several Gram-negative pathogens (11, A. baumannii MIC = 2–16 μg/mL; K. pneumoniae MIC = 1–8 μg/mL; E. cloacae MIC = 0.5–4 μg/mL; P. aeruginosa MIC = 16 μg/mL).68 Steered molecular dynamics simulations indicated that 6DNM-NH3 11 was able to pass through the porin OmpF with the assistance of the key interaction between the primary amine of 11 and Asp113 within the porin channel; however, 6DNM 10 was incapable of this interaction and required distortion in Asp113 and neighboring residues to allow passage. This ground-breaking work will undoubtedly pave the way for the discovery and development of antibiotics against Gram-negative pathogens.72</p><!><p>One of the primary methods to discover and develop new antibacterial agents over the past 60 years has been semisynthesis, which is chemical synthesis using natural products (e.g., antibiotics) as starting points.81 Early examples of semisynthesis in the antibacterial arena include the conversion of (1) streptomycin to dihydrostreptomycin (via a chemoselective reduction of the aldehyde in streptomycin to a primary alcohol to yield a new, more stable antibacterial agent) and (2) chlorotetracycline to tetracycline (via hydro-genation; tetracycline was later isolated). With these early success stories, medicinal chemists realized the value of semisynthesis for antibacterial discovery and utilized this approach to advance other antibiotic classes (e.g., macrolides, synthetic transformation of erythromycin to azithromycin in four steps); however, this approach has significant limitations as deep-seated modifications are not always accessible via semisynthesis. Myers and co-workers are working to address these limitations by developing modular total synthesis platforms, and they have made significant progress generating novel tetracycline81–88 and macrolide antibiotics89–93 bearing structural modifications not possible through current semisynthetic approaches.</p><p>In the mid-1990s, the Myers lab began work on fully synthetic tetracycline antibiotics.82 This program led to the implementation of a high-yielding, diastereoselective Michael–Claisen annulation between benzoate esters related to 13 and chiral "AB enone" 14, resulting in the constructive C-ring "stitching" of new tetracycline antibiotics.81–86 The "diversity element" of this route can be clearly seen in structure 15 (Figure 7), which bears four positions poised for structural modification. On the basis of structure–activity relationship knowledge, the diversity element tracks to positions that do not negatively impact bacterial ribosome inhibition by tetracyclines. This incredible chemistry platform has enabled >3000 fully synthetic tetracyclines to date,81,82,86 several of which have proven to be promising clinical candidates (e.g., 24, 25) including ervacycline 26 (XERAVA), which recently received FDA approval for the treatment of complicated intra-abdominal infections (cIAI).94 As presented in Figure 7D, ervacycline 26 has very potent, broad-spectrum activity against several pathogenic bacteria, including methicillin-resistant Staphylococcus aureus.</p><p>The Myers lab has also recently established a total synthesis platform for the discovery of new macrolide antibiotics.89–93 Myers and co-workers reported the generation of >300 fully synthetic macrolides (FSMs) that incorporate structural design features that combine structure–activity relationship knowledge (macrolactone, C9 ketone/amine replacement, 14- or 15-membered macrocycle, incorporation of desosamine sugar at C5) with resistance liabilities (replacement of C3-clasinose sugar with ketone to give ketolides; Figure 8A) to inform the synthesis of new macrolides.92 Eight simple building blocks (30–33, 38–40, 47; Figure 8B) were utilized to assemble key left-handed fragment 44 (amine) and right-handed fragment 43 (ketone), which were then joined, or converged, through a stereoselective reductive amination reaction followed by sequential macrolactonization and click reactions to yield 48 (a 14-membered azaketolide). The Myers team noted that many past macrolide synthesis efforts have failed at the critical macrolactonization step; therefore, special attention was made to rigidify the substrate via the cyclic carbamate group, which led to an impressive 78% yield for the macrolactonization step (on gram scale). Several clinically effective and promising macrolides have cyclic carbamate moieties fused to the C12/ C13 position of the macrolide, including telithromycin, cethromycin, and solithromycin 50. An additional benefit of the cyclic carbamate moiety is that the aniline residue of solithromycin 50 is known to bind a unique pocket in the bacterial ribosome,92 making such structures better inhibitors of this target and more effective antibacterial agents.</p><p>A series of 15-membered azaketolides, including FSM 49, was synthesized using a similar route to the 14-membered azaketolides (Figure 8B).92 This new route required modification to only three of the building blocks (34, 35, and 41, the original glycosyl donor reported by Woodward et al.95) used to synthesize the 14-membered azaketolides. The key left-handed fragment 44 was also utilized in this route and underwent an analogous (1) reductive amination with right-hand aldehyde 45, (2) macrolactonization (94% yield, 1.9 g scale), (3) click reaction sequence to afford 15-membered azaketolide 49. Myers and co-workers also utilized the key right-hand aldehyde 45 to access a series of 14-membered ketolides, including the clinical candidate solithromycin 50 and approved drug telithromycin (not shown). Intermediate 42, which was accessed through a stereoselective addition of ketone 36 with alkynyl lithium 37, was transformed into a Grignard reagent via the hydromagnesiation of the acetylenic moiety and subsequently reacted with aldehyde 45 to join the key left- and right-hand fragments of the ketolide series. Following a subsequent oxidation/deprotection sequence, the key macrolactionization step was successfully carried out in 66% yield (1.7 g scale) to produce the desired 14-membered macrolactone scaffold. A final reaction sequence involving C2 fluorination and installation of the cyclic carbamate (bearing the important aniline residue) led to the synthesis of solithromycin 50 in 14 steps and 16% yield from 34 and 35.</p><p>This total synthesis platform was utilized to generate >300 FSM antibiotic candidates by varying building blocks in addition to modifying readily diversifiable structural features at multiple positions around the macrolactone architecture.92 This proved to be a powerful synthetic medicinal chemistry tactic as deep-seated variations at select positions within FSM scaffolds were accessed, which are not possible using semisynthetic approaches. Following chemical synthesis, 305 FSMs were evaluated for antibacterial activities against a diverse panel of Gram-positive and Gram-negative pathogens. Potent antibacterial agents were identified among each of the FSM subclasses (14-membered azaketolides, 15-membered azaketolides, 14-membered ketolides).</p><p>Following the initial screen, the most promising FSMs were evaluated against an expanded panel of bacteria with genetically characterized resistance mechanisms to erythromycin (ermB, ribosome-modifying methyltransferase; mefA, efflux; Figure 9) and other antibiotics (MRSA and VRE strains).92 Several analogues (e.g., 49, 51, 52; Figure 9) demonstrated significant improvements in activity against this macrolide-resistant panel of pathogenic bacteria. FSM 51 (FSM-100573) is a 14-membered ketolide that demonstrated remarkable activities during these investigations. In fact, 51 outperformed every current clinically used macrolide antibiotic against extremely challenging strains, such as S. pneumoniae with both ermB and mefA genes. FSM 51 also demonstrated significant improvements against Gram-negative pathogens, such as A. baumannii and P. aeruginosa.</p><p>This incredible synthesis platform has created unprecedented opportunities to explore and develop novel macrolide antibiotics. The synthesis approach to FSMs is highly convergent from multiple building blocks; however, key advanced left-handed and right-handed intermediates were strategically utilized in divergent pathways to access multiple FSM subclasses (e.g., left-handed fragment 44 was used to synthesize 14- and 15-membered azaketolide scaffolds; right-handed fragment 45 was used to synthesize 15-membered azaketolide and 14-membered ketolide scaffolds). Similar to Myers' tetracycline synthesis platform, one can envision accessing thousands of FSM from the chemistry described here. Myers and co-workers have truly inspired scientists and clinicians with this ground-breaking work, which has great potential to lead to new clinical agents that overcome bacterial resistance in human patients.</p><!><p>Broad-spectrum antibiotics target essential pathways in bacteria, including cell wall and protein synthesis, and are capable of treating a wide range of infections.96 However, misuse and prolonged exposure to broad-spectrum antibiotics can result in undesirable side effects (e.g., inflammatory diseases, obesity) as commensal population dynamics can be dramatically altered by these agents.96–98 "Narrow-spectrum" antibiotics (e.g., amoxicillin) target large subsets of bacteria, such as Gram-positive versus Gram-negative bacteria, or anaerobes versus aerobes; however, species-specific agents against significant human pathogens would be extremely useful and could avoid the undesired side effects that result from the misuse of our current antibiotic arsenal.96</p><p>Over the past several years, Wuest and co-workers have developed an exciting medicinal chemistry and chemical biology program around the species-selective agent promysalin 53 (Figure 10A), which targets the major Gram-negative pathogen Pseudomonas aeruginosa.96,99–103 The multispecies community found in the root systems of various plants is known as the rhizosphere,104,105 and in this environment, bacteria are known to utilize chemical warfare strategies for colonization and to defend themselves. Pseudomonads are highly prevalent in the rhizosphere, and these bacteria produce an array of secondary metabolites with biological activities that promote survival, including antibiotics, virulence factors, biosurfactants, siderophores (to obtain iron from their environment).99 Promysalin is a secondary metabolite isolated from Pseudomonas putida found in rice plants.106 This natural product induces swarming and biofilm formation in P. putida; however, it demonstrates unique species-specific antibacterial activity against P. aeruginosa (growth inhibition, IC50 = 0.83 μg/mL; note, MIC values have not been reported for the promysalin work described here).</p><p>Wuest and co-workers reported a convergent and concise total synthesis of (−)-promysalin 53 (Figure 10).99 In the original isolation paper by De Mot and co-workers,106 the three stereogenic centers of promysalin were not defined. Therefore, Wuest's total synthesis of promysalin involved a unique combination of bioinformatics (reannotation of the biosynthetic gene cluster via AntiSMASH computation) and stereoselective approaches (reagent control, substrate selection; Figure 10B) to yield four diastereomers of promysalin for structural elucidation and biological assessment.</p><p>All four diastereomers of the myristic acid fragment 60 (diastereomers 60a–d) of promysalin were synthesized using a stereoselective, five-step synthetic route.99 First, Evans' oxazolidinone 54 underwent a diastereoselective hydroxylation (oxidation) upon treatment with sodium hexamethyldisilazane followed by addition Davis oxaziridine 58, then a TBS-protection afforded 59. This was followed by a cross-metathesis reaction with homoallylic alcohol 55, and then hydrogenation of the resulting olefin (not shown) and final ammonolysis of the oxazolidinone moiety with ammonium hydroxide afforded amide 60. EDC-coupling of the alcohol in 60 with acid (−)-62 and then silyl-group removal with tetrabutylammonium fluoride afforded promysalin 53. All four diastereomers of 60 (60a–d) were synthesized and coupled to (−)-62 to afford all possible diastereomers at C2 and C8 of the myristic acid moiety of promysalin (53a–d).</p><p>With the four diastereomers of 53 in hand, NMR comparison and antibacterial assays were utilized in concert to unequivocally identify diastereomer 53a as the natural product. Unfortunately, no optical rotation or authentic sample was available to compare; however, Wuest and co-workers rationalized that the correct enantiomer would exhibit the most potent biological activity.99 Promysalin 53a, which had identical spectral data to those reported in De Mot's isolation paper, proved to have the most potent antibacterial activities against P. aeruginosa strain PA14, which demonstrated an IC50 value of 125 nM (IC50 = 1.8 μM, initial report) and an IC50 value of 1 μM against PAO1. The other diastereomers 53b–d demonstrated >10-to >60-fold less potency against P. aeruginosa strain PA14 than 53a. On the basis of these findings, the absolute stereochemistry of natural promysalin (−)-53 was assigned as (2R,8R,16S).</p><p>By use of the total synthesis of promysalin 53 as a platform, a series of 16 related analogues was generated via diverted total synthesis.96 This approach allowed several structural features to be probed (i.e., ester linker, proline ring size and functionality, hydroxyl group on the myristic acid fragment) to gain structure–activity relationship information regarding 53. From these investigations, several interesting structural features were identified that led to a complete abolishment of antibacterial activity against PA14, including (1) proline ring size and saturation (analogue 63) and (2) substitution of the ester group with an amide (analogue 64). In contrast, removal of the C2 hydroxyl group (analogue 65) and introduction of a vinyl fluoride on the proline moiety (analogue 66) resulted in promysalin analogues with improved antibacterial activities against PA14.</p><p>The SAR insights gained from the diverted synthesis of promysalin analogues enabled the design, synthesis, and utilization of diazirine photoprobe 67, which retained activity, in affinity-based protein profiling (AfBPP) studies.101 Active promysalin photoprobe 67, inactive photoprobe 68 (to identify nonspecific protein binding partners), and promysalin (for competition purposes) were utilized in an elaborate series of experiments (involving irradiation of photoactive probes in cell lysate, subsequent click reaction with biotin azide, pull-down, dimethyl isotope labeling, and LC–MS/MS measurement; Figure 11) that led to the identification of succinate dehydrogenase (SdhC) as the target of promysalin. Further validation that SdhC was the target of 53 was carried out in vitro and via whole genome sequencing of resistant mutants. These findings demonstrate that targeting the tricarboxylic acid (TCA) cycle could be useful in developing narrow, species-selective antibiotic therapies and warrant additional studies regarding SdhC as an antibiotic target.101–103</p><!><p>Bacteria are able to successfully establish an infection when they experience unlimited growth and proliferation within a host.107 In order for bacteria to proliferate, adequate supply of key nutrients is required. Iron is a crucial nutrient that bacteria require to thrive and is known to play critical roles in energy metabolism, stabilization of protein structures, and oxygen transport.107–109 Unfortunately, iron is not membrane soluble and, therefore, is unable to diffuse through bacterial membranes, so acquisition of this key nutrient has its challenges; however, bacteria synthesize and utilize specialized organic molecules known as siderophores to acquire iron from their surroundings.107,108 Bacteria secrete specific siderophores into their environment where they tightly bind iron(III). The resulting iron(III)-siderophore complex is then recognized by specific cell-surface receptors on bacteria, which shuttles the complex inside the bacterial cell. Once within the reductive cytoplasm of the bacterium, iron(III) is reduced to iron(II), resulting in a loss of affinity by the siderophore and subsequent release of iron(II) for unitization by the bacterial cell.107</p><p>Bacteria utilize highly specific siderophore systems that can be exploited for bacterial targeting and drug delivery. For instance, the Gram-positive pathogen S. aureus utilizes two siderophores, staphyloferrin A and staphyloferrin B (69, Figure 12A).110 Enterobactin (71, Figure 12B) is a siderophore produced by multiple Gram-negative pathogens, including E. coli and S. typhimurium.111 P. aeruginosa and other pathogens have effectively developed receptors to recognize and transport iron(III)-siderophore complexes from other bacterial species (xenosiderophores) providing a competitive growth advantage. Although enterobactin is not produced by P. aeruginosa, this siderophore can promote iron uptake in this pathogen.111</p><p>These iron-specific uptake systems have provided a promising platform for targeted antibiotic delivery to select pathogens, especially for Gram-negative pathogens that present challenges regarding compound penetration as a result of two cellular membranes. Miller and co-workers have pioneered this area with much success in recent years with several inspiring examples of potent and targeted antibiotic delivery using a "Trojan horse" strategy that links antibiotics to various synthetic siderophores (sideromycins).111–118 One example is presented in Figure 12C, where Miller and colleagues designed triscatecholate sideromycin 73, inspired by catechol-based siderophore enterobactin 71, which is utilized to target P. aeruginosa.111 Sideromycin 73 was designed to have a linker moiety for attaching an antibiotic agent, in this case amoxicillin (ampicillin was also attached, not shown) to afford "Trojan horse" conjugate 74 (note: acetate groups were installed on catechol moieties to prevent pharmacological side effects and serve as prodrugs while circumventing potential methylation via a catechol O-methyl-transferase that would result in the loss of iron binding capabilities of the siderophore). Trojan horse conjugate 74 demonstrated potent antibacterial activities against P. aeruginosa strains in iron-deficient media, which resulted from the hijacking of energy-dependent active bacterial iron uptake systems required for bacterial growth under these conditions.111 During these studies, MIC values for 74 ranged from 0.05 to 0.39 μM under iron-deficient media compared to MIC values that ranged from 12.5 to 50 μM in iron-rich media (note: amoxicillin was inactive against these P. aeruginosa strains, MIC > 100 μM).</p><p>A more detailed schematic of the Trojan horse strategy to target Gram-negative pathogens can be found in Figure 13A. Recently, Miller's group has published two outstanding examples of Trojan horse conjugates hijacking iron uptake systems that enable Gram-positive antibiotics (an oxazolidinone117 and daptomycin;116 Figure 13B and Figure 13C) to effectively target Gram-negative pathogens, including Acinetobacter baumannii. These impressive examples of Trojan horse antibiotic delivery also showcase "releasable linkers" versus "nonreleasable linkers" that are an important design component in these systems.</p><p>Oxazolidinones target bacterial ribosomes and are effective at treating Gram-positive infections; however, these agents are inactive against most Gram-negative pathogens as they are unable to penetrate their outer membrane, or if they do permeate the Gram-negative bacteria, they are rapidly effluxed.117 This spectrum of activity along with these resistance mechanisms (lack of penetration, efflux) makes oxazolidinones ideal candidates for Trojan horse conjugation. With that, sideromycin 75 was designed as a dual active agent containing siderophore–cephalosporin–oxazolidinone components, which combine the Trojan horse entry of Gram-negative pathogens with β-lactamase triggered release upon destructive ring-opening of the cephalosporin moiety to liberate an active oxazolidinone (76; Figure 13B).117</p><p>In the arsenal of Trojan horse conjugate linkers, it is important to have releasable linker options to avoid conjugate structures that perturb critical drug–target interactions, ultimately preventing antibiotic activities. Early attempts to develop releasable linkers included functional groups that were hydrolyzable (i.e., ester cleavage by esterases112) or reductively cyclized (i.e., trimethyl lock bioreductive activation,115 not shown) and led to problems with premature antibiotic release before iron-uptake dependent penetration of Gram-negative bacteria.117 The cephalosporin linker in 75 requires the enzymatic activity of intracellular β-lactamases for "suicide" antibiotic release, bypassing premature release issues presented by other releasable linker systems. In initial proof-of-concept experiments, 75 underwent rapid and complete hydrolysis by purified ADC-1 β-lactamases, which are ADC enzymes found in A. baumannii that are capable of hydrolyzing cephalosporin antibiotics.117</p><p>The siderophore–cephalosporin–oxazolidinone 75 demonstrated impressive antibacterial activities against multiple Gram-negative pathogens, including A. baumannii (multiple, drug-resistant isolates; MIC = 0.4–6.25 μM), E. coli (MIC < 0.025 μM), and P. aeruginosa (MIC = 0.2–0.4 μM).117 The corresponding oxazolidinone 76, alone or when linked directly to the siderophore moiety (without cephalosporin to release 76), was inactive against these pathogens (MIC from >50 to >500 μM). Interestingly, the siderophore–cephalosporin moiety alone (without oxazolidinone 76; structure not shown) has moderate to good activities against some pathogens but is dramatically enhanced with all three moieties (75) intact against A. baumannii. For instance, against A. baumannii ATCC BAA 1800, the siderophore-cephalosporin components report MIC = 25 μM versus MIC = 0.8 μM for siderophore–cephalosporin–oxazolidinone 75, demonstrating a clear structure–activity relationship for the necessity of all three structural components to display potent antibacterial activities at a level that is clinically relevant.</p><p>In separate work, Miller and co-workers coupled a mixed synthetic ligand analogue of A. baumannii siderophore fimsbactin with daptomycin to give conjugate 79.116 Daptomycin is a large, negatively charged lipopeptide anti-biotic that has significant value as a therapeutic agent against Gram-positive pathogens only. Although daptomycin's mechanism is not fully understood, it has been found to bind and disrupt bacterial cell membranes, resulting in rapid depolarization, concomitant ion efflux and dysregulation of nucleic acid and protein synthesis, and subsequent bactericidal death.119 Prior structure–activity relationship investigations revealed that acylation of the primary amine of the ornithine residue in daptomycin was well-tolerated, and thus the Miller group used this information in the design of daptomycin-siderophore conjugate 79.116 Regarding chemical synthesis, the trichloroethyl chloroformate (Troc) protecting group of 77 was removed using zinc in the presence of glutaric anhydride to afford 78 as a benzyl protected siderophore bearing a (nonreleasable) glutarate linker for subsequent conjugation (Figure 14). A mixed anhydride of 78 was generated in situ before subjecting the siderophore-linker moiety to daptomycin under aqueous reaction conditions for conjugation via amide bond formation. Final hydrogenolytic removal of the benzyl groups provided target daptomycin-siderophore conjugate 79.116</p><p>Siderophore-daptomycin conjugate 79 was shown to bind iron(III) stoichiometrically and demonstrated profound effectiveness against A. baumannii strains (MIC = 0.4–0.8 μM).116 As predicted, daptomycin itself was inactive against A. baumannii strains (MIC > 100 μM) when tested alongside conjugate 79. To demonstrate the importance of iron-binding and uptake regarding conjugate 79, its synthetic precursor bearing bis benzyl group protected catechol moieties (structure not shown) proved to be completely inactive against A. baumannii (MIC > 50 μM). When tested against other Gram-negative pathogens, such as P. aeruginosa, Burkholderia multivorans, and Escherichia coli, conjugate 79 proved to be inactive (MIC > 100 μM), demonstrating the selective targeting of this Trojan horse conjugate toward A. baumannii.</p><p>Following in vitro assessment, Trojan horse conjugate 79 was advanced to testing in mice.116 Intravenous administration of 79 at 250 mg/kg in ICR mice resulted in no observed adverse effects. Following favorable toxicity studies in mice, conjugate 79 was evaluated for in vivo efficacy in a sepsis model of infection in female ICR mice using A. baumannii 17961. Mice were infected with A. baumannii via intraperitoneal (ip) administration, and treatments with conjugate 79 (5, 10, 25 mg/kg) were provided intravenously (iv) 30 min and 24.5 h after infection. In this study, all six mice that did not receive treatment with 79 died after 1 day; however, mice treated with 79 at 10 and 25 mg/kg resulted in survival in 4 of 5 mice (at each test concentration) while only 1 of 5 mice survived with 79 at 5 mg/kg, demonstrating a dose-dependent in vivo efficacy against A. baumannii. This is an outstanding demonstration of the clinical applications that the Trojan horse strategy can be utilized to target Gram-negative pathogens in human patients.</p><p>Cefiderocol (S-649266; structure not shown) is a catechol-bearing siderophore–cephalosporin conjugate that demonstrates potent antibacterial activities against a broad range of Gram-negative pathogens (e.g., carbepenem-resistant Enterobacteriaceae, P. aeruginosa, A. baumannii).120 Cefiderocol has been advanced to clinical studies in human patients, demonstrating the translational potential of the Trojan horse strategy. In a clinical trial for the treatment of complicated urinary tract infection with antibiotic-resistant Gram-negative pathogens, cefiderocol was shown to be noninferior when compared to imipenem–cilastatin treated patients.121 Currently, cefiderocol is being investigated in clinical trials for hospital-acquired pneumonia and carbapenem-resistant infections. Collectively, this body of work aimed at targeting iron uptake mechanisms in bacteria to deliver antibiotics in a Trojan horse fashion is a promising approach to overcome antibiotic-resistant infections.</p><!><p>Isolated from Strepomyces hawaiiensis, the acyldepsipeptides (ADEPs) are a class of natural product antibiotics that have been shown to exhibit potent activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci.122,123 The antibacterial activities for the natural product ADEP1 (80, Figure 15) and related synthetic analogues were evaluated by Brötz-Oesterhelt and co-workers, wherein ADEP1 reported a 8.76 μM IC50 value against MRSA. In addition, synthetic analogues ADEP2 (81) and ADEP4 (82) also reported therein demonstrated markedly improved activity (500 nM and 64.9 nM, respectively).124 Furthermore, ADEP1 and ADEP4 were efficacious in vivo against both S. aureus and E. faecalis infection models. ADEP4 demonstrated the ability to rescue 80% of mice from mortal S. aureus infections following a single dose of 12.5 mg/kg. The group used genomic analyses to identify the target for ADEP analogues as ClpP (caseinolytic protease), a highly conserved, cylindrical bacterial peptidase.124</p><p>The ClpP serine peptidase employs an energy-dependent process implicated in biological functions such as protein quality control, degradation of transient regulatory proteins, and clearance of cellular debris following conditions of bacterial stress.125,126 Under normal homeostatic conditions, ClpP binds to ATP-dependent cochaperones (e.g., ATPases) which help regulate the unfolding and translocation of proteins into the proteolytic sites of ClpP. Crystal structures of ADEP1 (80) bound to Escherichia coli ClpP showed that ADEP analogues occupy the site of ATPase binding.127 The ClpP–ADEP interaction promotes the conversion of the ClpP entrance pore from a closed-to an open-gate form (Figure 16). This allosteric control of the ClpP barrel conformation bypasses the need for an associated cochaperone (an energy dependent process) and permits the unregulated entrance of proteins into the proteolytic active site.128 The resultant increase of ClpP activity diverts proteolysis from native physiological proteins to nascent peptides, leading to inhibition of bacterial cell division.</p><p>In a departure from prior studies (evaluating ADEP-promoted protein degradation against rapidly dividing cells), Lewis and co-workers utilized proteomic analysis to demonstrate that stationary phase MRSA exposed to ADEP4 (82) resulted in a reduced abundance of 24% of cellular proteins compared to the untreated control.129 Since stationary phase populations of S. aureus are typically nondividing (and thus notoriously difficult to treat), this finding unveiled a potential therapeutic intervention against dormant persister cells. To evaluate the effect of ADEP4 against dormant bacteria, a population of persister cells was treated with the agent and it was found that ADEP4 was capable of eradicating persister cells to the limit of detection. Conversely, rifampicin reported no effect on persister cell viability.</p><p>ADEP4 also reported remarkable killing of stationary phase S. aureus following 2 days of treatment, demonstrating a 4 log reduction of viable bacterial cell counts.129 Since ClpP is a nonessential protein for S. aureus, the null clpP (the gene encoding the ClpP protease) mutation frequency is high (∼10−6), and thus, rebounding populations were observed after day 3 of the experiments. It was surmised that formation of resistant mutants could be suppressed via coadministration of ADEP4 with conventional antibiotics. This co-treatment strategy eradicated persister cells to the limit of detection with no concomitant population rebound. It was found that although treatment of a clpP mutant strain with rifampicin demonstrated similar MIC values as that seen against of the wild-type strain, the mutant strains were less capable of producing persister cells (by an order of 10-to 100-fold). The conclusion was that clpP mutations (e.g., those resulting from ADEP treatment) may render the bacteria less fit and thus more susceptible to other antibiotics.</p><p>By use of 96-well plate, non-Calgary Biofilm Device assays, the ability of the ADEP4–rifampicin combination to eradicate bacterial biofilms was also evaluated.129 Following a 24-h S. aureus UAMS-1 biofilm establishment, wells were treated with the ADEP4–rifampicin combination (6.49 μM ADEP4, 10 × MIC; 0.49 μM rifampicin, 10 × MIC), resulting in eradicated biofilms (>4 log-fold reduction) following 3 days of treatment. The ADEP4–rifampicin combination was also shown to be highly efficacious in deep-seated in vivo infection models. Following a 24 h infection period, histopathology was used to confirm the adherence of bacterial biofilms to mouse thigh muscle tissue. Treatment of the infection with the ADEP4–rifampicin combination resulted in an eradication of the biofilm-associated infection within 24 h (representing a >4 log-fold reduction). In contrast, rifampicin, vancomycin, or a combination of both could not eradicate the infection although decreased viable cell counts were observed.</p><p>The LaFleur group was also able to show that the ADEP–antibiotic combination strategy could be expanded to include additional clinically used antibiotics.130 LeFleur and co-workers reported ADEP4 activity against stationary phase multidrug-resistant E. faecalis V583 when treated in combination with ampicillin, ciprofloxacin, daptomycin, oritavancin, or tigecycline. In these cases, the ADEP4–antibiotic combination yielded a 5 log reduction in bacterial cell survival. This was especially interesting as the activity was observed regardless of the mechanistic class of partnered antibiotic. Expression and purification of E. faecium ClpP permitted the use of a fluorometric monitoring assay used to evaluate casein degradation wherein it was reported that ADEP4 activated ClpP with an EC50 of 0.53 μM. A crystal structure was also disclosed, highlighting the binding of ADEP4 to the hydrophobic pockets located between subunits at the apical and distal ends of ClpP. In a murine model of peritoneal septicemia, treatment with ADEP4 (50 mg/kg) and ampicillin (50 mg/kg) yielded a 2 log10 greater reduction in average bacterial burden when compared to antibiotic monotherapy and a 4 log10 reduction of viable bacterial cells relative to the vehicle control.</p><p>Encouraged by the in vivo antibacterial efficacy of ADEP analogues, several groups have put forth medicinal chemistry efforts to improve on this therapeutic strategy. In an attempt to reduce entropic binding penalties, Sello and co-workers disclosed an enhancement of ADEP analogues via introduction of conformational restriction.131 This strategy was realized via replacement of key macrocyclic residues. The result of the rigidified ADEP analogues was a 20-fold improvement of in vitro S. aureus activity from MIC = 649 nM 129 for ADEP4 compared to 30.6 nM for ADEP1g (83). Building upon the library of known antibacterial ADEP analogues, the Duerfeldt group described an attempt to remedy a potential metabolic liability of the ADEP series (i.e., the readily hydrolyzed ester within the lactone core).132 On the basis of the reported crystal structures of ADEPs bound to ClpP, it seemed evident that replacement of the ester with an amide may have no influence on the interaction of the ADEPs with ClpP. Alas, Duerfeldt and co-workers were surprised to learn that amide-containing ADEP analogues 85 and 86 were markedly less active (by up to 100-fold) than the corresponding ester ADEP analogue 84. It was surmised that although the ester oxygen may not participate in molecular interactions with the target, its replacement with an amide nitrogen disrupts intramolecular hydrogen bonds (87, Figure 15) and thus compromises the compact conformation of the active agent (which presumably assists in membrane permeability). Although no biofilm eradication or in vivo assays were conducted as part of these recent studies, the reported novel agents demonstrate that the ADEP-based therapeutic approaches are fruitful pursuits and initial efforts to advance these agents toward clinical use have been very promising.</p><!><p>Cystic fibrosis (CF) patients are plagued with chronic lung infections, which became a source of chemical inspiration for our lab to identify new biofilm-eradicating agents. Many young CF patients are known to experience initial S. aureus lung infections.133 As these patients age, P. aeruginosa subsequently co-infects the lung and is believed to eradicate S. aureus using a series of phenazine antibiotics, including pyocyanin.133–137 We believe that the initial S. aureus infections establish surface-attached biofilms on the inside of lung tissues as CF patients have lung infections that span multiple years.</p><p>In our initial work, we synthesized a diverse series of 13 phenazine compounds, including five naturally occurring phenazines.138 With literature reports of pyocyanin as the main antibiotic of this class responsible for activities against S. aureus, we were sure to include this compound in our initial screening collection. Interestingly, in our initial antibacterial screens against S. aureus and S. epidermidis, we found that 2-bromo-1-hydroxyphenazine, a marine Streptomyces derived phenazine, demonstrated good antibacterial activities with MIC values of 6.25 μM against these pathogens (pyocyanin reported MICs of 50 μM).138 In addition, we found that related synthetic analogue 2,4-dibromo-1-hydroxyphenazine (not shown) demonstrated improved antibacterial activities against S. aureus and S. epidermidis with MICs of 1.56 μM. In later work, we found that 2,4-dibromo-1-hydroxyphenazine was able to eradicate methicillin-resistant Staphylococcus aureus biofilms and reported a minimum biofilm eradication concentration, or MBEC, of ∼100 μM (note: when tested alongside 2,4-dibromo-1-hydroxyphenazine, vancomycin reported an MBEC of >2000 μM in Calgary Biofilm Device assays despite an MBEC of 4 μM against planktonic cells in the same experiment, demonstrating a significant level of biofilm tolerance toward vancomycin).139</p><p>After demonstrating that 2,4-dibromo-1-hydroxyphenazine eradicates MRSA biofilms, we worked to develop modular, convergent synthetic routes to new "halogenated phenazine" (HP; Figure 17A, see structure 95) small molecules.140–143 Chemical synthesis pathways involving phenylenediamine (88) condensation with quinone 89,140,141 a Wohl–Aue reaction142 (aniline 90 condensed with 2-nitroanisole 91), and a Buchwald–Hartwig cross-coupling/reductive cyclization path-way143 (fusing aniline 92 with 2-bromo-3-nitroanisole 93) enabled rapid access to a diversity of 1-methoxyphenazines (94) with various substitutions at the 6–9 positions of the HP scaffold (95). A final boron tribromide (BBr3) demethylation and N-bromosuccinimide (NBS) bromination sequence afforded target HPs 95. These synthetic efforts and subsequent antibacterial studies have led to the identification of several highly potent biofilm-eradicating agents, including HPs 96–98, which demonstrate up to 50-fold more potent biofilm killing activities in Calgary Biofilm Device assays compared to parent 2,4-dibromo-1-hydroxyphenazine (e.g., 98 reported an MBEC of 2.35 μM against MRSA biofilms143).</p><p>These efforts have enabled a detailed structure–activity relationship understanding regarding halogenated phenazine antibacterial agents (see 99, Figure 17B).138–143 In general, substitution at the 6- and 7-positions of the HP scaffold is typically well-tolerated and enhances antibacterial activities compared to the corresponding unsubstituted HP; however, the 8-position of the HP scaffold can influence antibacterial potency in a positive (e.g., halogens improve activity) or negative (e.g., methyl reduces activity) fashion. Interestingly, we found that substitution at the 9-position of the HP scaffold completely abolishes all antibacterial activities. We later discovered the reason for a loss in activity regarding 9- substituted HPs after learning that active HP compounds bind metal(II) cations between the hydroxyl oxygen and adjacent nitrogen (forming a five-membered chelate upon metal binding), which plays a critical role in the antibacterial mode of action. Substituents at the 9-position of the HP scaffold, including a relatively small methyl group, impede the critical metal-chelation event for antibacterial activities.140 Interestingly, general metal-chelating agents (EDTA and TPEN) have been tested alongside HPs and are unable to eradicate bacterial biofilms, highlighting the unique metal-dependent mechanism behind HP small molecules.141–143</p><p>In ongoing efforts to translate HP biofilm-eradicating agents for clinical applications, we are using the SAR knowledge of the HP scaffold to pursue multiple prodrug strategies to functionalize the phenolic hydroxyl group to abolish metal-chelation until selective release within bacterial cells.142,143 This approach provides a platform for extensive developments, including (1) tuning of physicochemical properties (e.g., installation of PEG group to increase water solubility142,143) and (2) diverse functional triggers for HP release (e.g., bioreduction of the quinone in 101 is required to liberate the corresponding HP agent143). Despite initial success with HPs 100 and 101, continued prodrug efforts are ongoing with the goal of developing an effective clinical agent for biofilm infections.</p><p>In separate work, we used HP-14 (96, MBEC = 6.25 μM) as a probe molecule for transcriptomic analysis of treated and untreated MRSA biofilms using RNA-seq technology.144 This platform allowed us to define new cellular targets and pathways critical to biofilm survival, which is challenging to study as these surface-attached bacterial communities are composed of slow-growing or nonreplicating cells. Treating an established MRSA biofilm with HP-14 at low concentration (0.625 μM; 1/ 10 MBEC) for 20 h enabled the identification of >200 gene transcripts that were either up- or down-regulated (≥2.0-fold change in gene expression) compared to vehicle control.</p><p>Using a WoPPER gene cluster analysis tool, we identified 37 gene clusters with alterations in MRSA biofilm gene expression profiles following 20 h treatment with HP-14.144 Upon exposure of HP-14 to MRSA biofilms, six gene clusters involved in iron acquisition were found to be dramatically up-regulated ("activated") via WoPPER analysis, including: hts/sfa (staphyloferrin A; siderophore), sir/sbn (staphyloferrin B), isd (iron-regulated surface determinant; heme iron acquisition), MW0695 (hypothetical protein, similar to ferrichrome ABC transporters), and f hu (ferric hydroxamate uptake; two gene clusters). Following validation of these results using RT-qPCR experiments, a time-course assessment of MRSA biofilms treated with HP-14 (0.625 μM; 1/10 MBEC) revealed that four iron acquisition gene clusters (isd, sbn, sfa, MW0695) were activated in 1 h. The rapid activation of multiple iron uptake gene clusters by low concentrations of HP-14 is profound, as bacterial biofilms are notorious for their dormant phenotypes. Following 4 and 8 h of HP-14 exposure at 0.625 μM (1/10 MBEC), MRSA biofilms demonstrated more significant activation levels of these iron uptake genes (e.g., isdB, 22.3-fold activation after 4 h). Interestingly, EDTA and TPEN (5 μM; general metal-chelating agents) were unable to activate iron acquisition genes in MRSA biofilms,144 which aligns with our previous observations that these metal-chelating agents are unable to eradicate MRSA biofilms (MBEC of >2000 μM) in Calgary Biofilm Device assays.141–143</p><p>On the basis of RNA-seq findings, we believe HP-14 induces rapid iron starvation in MRSA biofilms, which leads to eradication of these surface-attached bacteria.144 With the lipophilic properties of HP-14 (cLogP = 6.25) combined with potent MRSA biofilm eradication activity (MBEC = 6.25 μM), an iron chelating moiety, and rapid activation of multiple iron uptake gene clusters, we believe that this HP rapidly diffuses into biofilm cells and binds iron(II) following the intracellular release of iron(II) from a siderophore or heme. A proposed scheme of HP-14 inducing iron starvation and MRSA biofilm eradication is presented in Figure 18 and aligns with our WoPPER gene cluster analysis and subsequent RT-qPCR results in time-course studies. In addition, we believe that EDTA and TPEN are not able to efficiently penetrate biofilm cells, which is why they are unable to activate iron uptake genes or eradicate biofilms when tested alongside active HP biofilm-eradicating agents. This work not only demonstrates HP-14's ability to rapidly induce iron starvation in MRSA biofilms but highlights an impressive level of sensitivity along with rapid response rates of bacterial biofilms to small molecule threats. In addition, these findings suggest that iron starvation of surface-attached bacterial communities could serve as the Achilles heel to persistent and recurring biofilm infections in the clinic.</p><!><p>Despite increasing concerns regarding antibiotic resistance, antibiotic drug discovery is entering a dynamic and exciting new era. The multidisciplinary approaches of the research programs presented here have, in part, contributed to the reinvigoration of natural-product-based research for antibiotic discovery. Other impactful programs inspired by natural products, yet not included in this Perspectives article, include Genentech's optimized arylomycin analogues for Gram-negative pathogens145 and Boger et al.'s vancomycin work.146–150 The classic tools of synthetic chemistry, microbiology, and chemical biology have been applied in novel ways to answer questions about bacterial pathogenesis and to design innovative therapeutic approaches for combatting infections. Significant challenges lie ahead, but these research efforts along with others have helped pave the way for critical and desperately needed discoveries to address problems presented by antibiotic resistance and tolerance.</p>

PubMed Author Manuscript

Small molecule disruption of B. subtilis biofilms by targeting the amyloid matrix

Summary Small molecule inhibitors of amyloid aggregation have potential as treatment for a variety of conditions. In this issue of Chemistry & Biology, Romero et al. (2013) use amyloid-dependent B. subtilis biofilm formation to screen for amyloid inhibitors, identifying compounds that not only inhibit B. subtilis biofilm formation but also ones that disrupt preformed biofilms.

small_molecule_disruption_of_b._subtilis_biofilms_by_targeting_the_amyloid_matrix

<p>Amyloid fibers are historically associated with diseases such as Alzheimer's, Huntington's and the prion diseases. Many proteins can adopt the amyloid conformation—essentially a β-rich repeating structure where the β strands orient perpendicular to the fiber axis. Ordered amyloid protein polymers upset cellular proteostasis because they can inappropriately interact with membranes or sequester chaperone machinery. Therefore, the search is on for ways to ameliorate amyloid-related diseases by targeting amyloid formation. A major hurdle in this endeavor is that disease-associated amyloid formation is sporadic and difficult to faithfully reproduce in model organisms.</p><p>However, a growing number of 'functional' amyloids have been identified that are assembled by dedicated biogenesis systems. Functional amyloids and their assembly systems have been found in nearly all walks of cellular life, including mammals, fungi and bacteria (Hammer et al. 2008, Fowler et al. 2007). Functional amyloids contribute to cellular biology in various ways, including regulation of melanin synthesis, information transfer, or as a structural component. Furthermore, some of these functional amyloid systems provide a unique platform for understanding how amyloid formation can be directed and controlled so that cellular toxicity is minimized.</p><p>Amyloids are commonly found as the major protein component of the extracellular matrix in bacterial biofilms. Bacteria within the biofilm are protected from environmental insults, including disinfectants and antibiotics, making biofilms a major concern in hospital and industrial settings. Therefore, factors that can disrupt bacterial amyloid formation would be potential lead compounds for targeting bacterial biofilms or amyloid formation in general. The study presented in this issue of Chemistry & Biology by Romero et al. (2013) describes Bacillus subtilis pellicle biofilm as a model system to screen for amyloid inhibitors. The extracellular matrix of B. subtilis biofilms consist of two components, the exopolysaccharide (EPS) and the amyloidogenic protein TasA (Branda et al. 2006). Two small chemical molecules that inhibit biofilm formation by targeting the extracellular amyloid component of B. subtilis biofilms were identified. Importantly, one of the compounds not only inhibited biofilm formation but also disassembled already formed B. subtilis biofilms. Anti-biofilm compounds with distinct modes of action can be used synergistically to increase the potency of inhibition. The two inhibitory compounds described by Romero et al. created an even stronger inhibitory effect when allowed to exert their effect simultaneously. Collaboration between molecules may allow one type of molecule to dissolve pre-formed fibers, while another molecule could discourage new fiber formation (Fig. 1).</p><p>Utilizing functional amyloids as a tool for identifying general amyloid inhibitors has produced several promising leads in recent years. Strategic design of small compounds as amyloid modulators and thorough investigation of their effect on numerous amyloidogenic proteins is a promising approach to battle the threat of amyloid influence on health. One example of strategic design is the use of a small library of fluorescent compounds designed to interact with amyloid pathways based on structure-activity information (Chorell et al. 2012). This approach presents an opportunity to gain specific information on the systems studied by observing the interactions of the molecules and the amyloid protein or the cellular compartment. In the case of E. coli and biofilm formation, molecules have been discovered that inhibit both curli formation and type1-pili assembly, two protein structures that contribute to biofilm formation (Cegelski et al. 2009). Finally, another promising approach to screen for amyloid modulators makes use of the E. coli curli export system to assemble extracellular amyloid fibers of human disease associated Huntington or yeast prion proteins (Sivanathan et al. 2012). This provides an opportunity to screen for molecules affecting formation of the amyloid fibers.</p><p>The two molecules identified by Romero et al. seem thus far to be general amyloid inhibitors, as they were able to inhibit biofilm formation by Bacillus subtilis, Escherichia coli and Bacillus cereus. However, it is interesting to note that a molecule that inhibits amyloid formation by one protein may have a different effect on other proteins, including the ability to stimulate amyloid formation (Horvath et al. 2012). The two inhibitory molecules had no effect on Staphylococcus aureus biofilm even though S. aureus has been shown to produce functional amyloids that stabilize biofilm architecture (Schwartz et al. 2012). This might suggest that the compounds identified by Romero et al. are specific for certain amyloids or that the conditions in which the S. aureus biofilms were grown for this study do not require an amyloid matrix. Fully understanding of the specificity of these compounds and their biological activities will be a fascinating future direction for this work.</p><p>Clearly, Romero et al. have paved the way for capitalizing on a well-understood biofilm system to easily screen and identify new compounds that ameliorate amyloid formation.</p>

PubMed Author Manuscript

Uranyl Speciation in the Presence of Specific Ion Gradients at the Electrolyte/Organic Interface

Uranyl (UO 2+2 ) speciation at the liquid/liquid interface is an essential aspect of the mechanism that underlies its extraction as part of spent nuclear fuel reprocessing schemes and environmental remediation of contaminated legacy waste sites. Of particular importance is a detailed perspective of how changing ion concentrations at the liquid interface alter the distribution of hydrated uranyl ion and its interactions with complexing electrolyte counterions relative to the bulk aqueous solution. In this work, classical molecular dynamics simulations have examined uranyl in bulk LiNO 3(aq) and in the presence of a hexane interface. UO 2+ 2 is observed to have both direct coordination with NO − 3 and outer-sphere interactions via solvent-separated ion-pairing (SSIP), whereas the interaction of Li + with NO − 3 (if it occurs) is predominantly as a contact ion-pair (CIP). The variability of uranyl interactions with nitrate is hypothesized to prevent dehydration of uranyl at the interface, and as such the cation concentration is unperturbed in the interfacial region. However, Li + loses waters of solvation when it is present in the interfacial region, an unfavorable process that causes a Li + depletion region. Although significant perturbations to ion-ion interactions, solvation, and solvation dynamics are observed in the interfacial region, importantly, this does not change the association constants of uranyl with nitrate. Thus, the experimental association constants, in combination with knowledge of the interfacial ion concentrations, can be used to predict the distribution of interfacial uranyl nitrate complexes. The enhanced concentration of uranyl dinitrate at the interface, caused by excess adsorbed NO − 3 , is highly relevant to extractant ligand design principles as such nitrate complexes are the reactants in ligand complexation and extraction events.

uranyl_speciation_in_the_presence_of_specific_ion_gradients_at_the_electrolyte/organic_interface

Introduction<!>Simulation Configurations and Protocols<!>Data Analysis<!>Interfacial Slab and Identification of Truly Interfacial Molecules Analysis.<!>Characteristics of UO 2+<!>Conclusions

<p>Chemical separation and purification of uranium, notably from aqueous solutions, is essential to various environmental [1][2][3] and industrial applications. 4 The highly stable U(VI) exists in the dioxo form, UO 2+ 2 , and can exhibit complicated speciation via complexation by solute anions including nitrate or carbonate. Nitric acid solutions are the most relevant to uranyl separations within the nuclear fuel cycle. Within solvent extraction processes that include Plutonium Uranium Reduction EXtraction (PUREX) 4,5 and Group ActiNide EXtraction (GANEX), 2 hydrated UO 2 (H 2 O) 2+ n and uranyl nitrate complexes 6,7 are the reacting species with extracting ligands. The complexation reaction is presumed to occur at the aqueous/organic phase boundary, and thus the speciation of the metal ions at the interface is of significant importance. 8,9 Although it is well-known that there may exist significant concentration gradients of solutes near the liquid/liquid interface, [10][11][12][13] how this perturbs the speciation of metal ions and their complexes relative to the bulk aqueous phase has not been the topic of significant study.</p><p>An additional complication is that the heterogeneous environment of the liquid/liquid interface 14,15 may lead to a broad ensemble of local chemical environments that have the potential to shift energetic preferences. In bulk nitric acid it is well-known that UO 2+ 2 is on average pentacoordinate and associates with nitrate anions to form UO 2 (H 2 O) m (NO 3 ) n (2 -n)+ where n + m = 5. 16 However the association is weak, as has been measured by a number of experimental methods (UV-Vis, 17,18 IR/ Raman 19 NMR, 20,21 EXAFS [22][23][24] and microcalorimetry 25 ). In the case of the mononitrate complex (n = 1) the reported association constant (K 1 ) varies from 0.05 -0.70, while the second association constant (K 2 ) for the formation of UO 2 (NO 3 ) 2 is generally agreed to be much lower at 0.02 -0.05 at 298 K, depending upon the experimental methodology. Density functional theory (DFT) studies have examined uranyl coordination and nitrate binding modes, [26][27][28] and identified bidentate (η 2 ) nitrate to be significantly more stable than monodentate (η 1 ) in the gas phase. In contrast, the free energy difference between η 2 and η 1 in solution is predicted to significantly decrease, such that an approximate equal population of both coordination modes should be observed in the aqueous phase. 19,26,27 Despite the efficacy of DFT studies of isolated uranyl nitrate complexes, 21,27,29,30 such methods are not able to study the speciation, solvation, and complex ion-ion interactions that occur in bulk electrolytes near industrially relevant conditions, let alone their interfaces.</p><p>Molecular dynamics simulations have emerged as a powerful tool to study multi-component solutions and their interfaces, providing a molecular scale understanding of the complex correlations amongst local solution environments and dynamic equilibria between different chemical species. Yet the applicability of MD simulation is constrained by the fidelity of the potentials that define intra-and inter-molecular interactions. Several non-polarizable pairwise additive potentials have been developed for uranyl cation, and parameterized for aqueous and nitrate containing solutions under dilute conditions. [31][32][33] Unfortunately, as we demonstrate, at appreciable NO − 3 concentrations those models significantly over-predict the degree of uranyl nitrate association and lead to long-range correlations of uranyl nitrate complexes at modest ionic strength. This work begins by optimizing the interaction terms between UO 2+ 2 and NO − 3 using a electrostatic continuum-type correction (ECC). The optimized force fields reproduce the experimentally-determined uranyl nitrate association constants and associated speciation over a wide range of uranyl and nitrate concentrations within bulk LiNO 3(aq) . The electrolyte/hexane interfacial region is then examined at high ionic strength, where significant perturbations to ion hydration across solvation shells, ionion interactions, and the heterogeneity of the interface, all have the potential to alter the association constants of UO 2+ 2 and NO − 3 relative to the bulk. Changes to the association constants would significantly complicate prediction of uranyl speciation, and thus reactivity, at the interface.</p><p>Within the interfacial region, MD simulations predict ion-specific interfacial adsorption that leads to the formation of weak ion double layering, and generates distinct ion concentration gradients approaching the interface. The changing hydrogen bond network and ion gradients significantly influence the water dynamics and organization, while having modest impact upon nitrate fluctuations in the primary coordination sphere of uranyl. Interestingly, a large affect of electrolyte concentration lies within the the timescales associated with species in the first coordination sphere of UO 2+ 2 , as well as its residence within the interfacial region. The timescales of solvent exchange and the residence time of UO</p><!><p>All atom molecular dynamics simulations were performed using the GROMACS 2016.2 software package 34 to study uranyl nitrate speciation in bulk LiNO 3(aq) and LiNO 3(aq) /hexane under varying electrolyte concentrations. Initial system configurations were generated using Packmol, 35 Bulk simulations were performed with a series of concentrations presented in Table 1. These include a 0.05 M and 0.25 M UO 2+ 2 with background electrolyte LiNO 3 from 1 to 5 M so as to compare experimental studies by Suleimenov et al. 36 of uranyl in nitric acid solutions.</p><p>To compare with prior data reported by Ye et. al. 37 , additional simulations of 0.25 M UO 2+ 2 with HNO 3 were also performed. All electrolyte/hexane simulations were performed at 0.25 M UO 2+ 2 and 0.5 M NO − 3 as a function of LiNO 3 concentration from 1 -5 M (Table 1). Non-bonded interactions were modeled using Lennard-Jones and coulombic interactions.</p><p>Lorentz-Berthelot mixing rules were used for obtaining combinations of σ and parameters.</p><p>The UO 2+ 2 and Li + ions were modeled using Wipff et al. 38,39 and Joung et al. 40 force fields respectively, while the NO 3 force fields are derived from from Ye et al. 41 and Benay and Wipff 32 . The UO 2+ 2 force field reproduces the experimentally observed hydration of 5 in the first solvation shell in bulk water, 8,38,42 whereas the Li + potential was parameterized to reproduce the experimental hydration free energies and ion hydration in the aqueous phase. 40,43 The all-atom General Amber Force Field (GAFF) 44 were implemented for n-hexane, with modified Lennard-Jones parameters to reproduce the experimental density and enthalpy of vaporization as developed by Vo et al. [45][46][47] The UO 2+ 2 , Li + , and NO − 3 atom charges were then scaled from 100% to 75% in 5% increments using ECC, 48 which is an indirect correction to account for solvent driven polarization effects on hydrated ions. 49 In this manner, the coulombic interaction were tuned to reproduce the experimentally determined equilibrium constants 18,36 for different uranyl nitrate species and ensure coordination numbers and nitrate denticity that are consistent with experimental studies and prior ab-initio studies. 26,28 As described in the Results and Discussion, the ECC of 90% was observed to best reproduce the experimental uranyl nitrate association constants under ionic strengths similar to Suleimenov et al. 36 and provide reasonable coordination environments. It was employed for all subsequent molecular dynamics simulations. The TIP3P water model 50 was used for the bulk and electrolyte/hexane systems. Optimised force field parameters are given in the Supplementary Information, Table S1.</p><p>All systems were first equilibrated in the isobaric-isothermal NPT ensemble for 40 ns using the Nose-Hoover thermostat 51 and Parrinello-Rahman barostat, 52 followed by isochoricisothermal NVT ensemble for 10 ns. The simulations were performed at 298 K using periodic boundary conditions with a leap frog verlet integrator 53 using a time step ∆t of 2 fs. PME (Particle-Mesh Ewald) summation 54 was used for long range electrostatic interactions. After equilibration, 30 ns production runs were performed in the NVT ensemble and used for data analyses. Sampling frequencies of the production run include 25 fs & 3 ps dump times depending upon the property of interest.</p><!><p>The focus of this work is to understand the variations in uranyl speciation that result from significant changes to ion concentration and changes to solution structure at the interfaces of electrolytes with non-polar media, relative to the bulk electrolyte phase. Toward this end, the macroscopic interfacial properties (interfacial tension and width) were examined alongside analyses that reveal the local structure-including the coordination environments, solvation structure, and molecular speciation. The dynamic properties of molecular interactions, obtained from the relevant time correlation functions, are also reported. Statistical errors were determined using standard deviation of the calculated quantity over the length of the sampled trajectory.</p><p>Interfacial Tension. The Kirkwood and Buff 55 pressure tensor method was used to calculate the interfacial tension, γ, 56 as an integral over the z dimension as</p><p>where L z is the box length, N int is the number of interfaces (N int = 2 in Figure S1) and P zz , P yy , and P xx are the diagonal components of the pressure tensor.</p><p>Local Structure and Speciation. Atom pair distribution functions were first used to examine inter-atomic distance correlations. These were compared to prior experimental and simulation data during force field validation. The composition of the primary coordination sphere about UO 2+ 2 , and the solvation environments about NO 3 and Li + were determined from networks of inter-molecular interactions using the ChemNetworks software package. 57 Distance geometric criterion were employed to define edges between nodes represented by UO 2+ 2 , H 2 O, NO 3 and Li + . These criterion were based upon the first minimum of the associated atom pair correlation functions of interest (including</p><p>as shown in Figure S7). Geometric criterion are listed in Table S2. The distribution of denticities of nitrate complexation to uranyl was determined from the edge</p><p>Dynamic Properties. It is of interest to understand how the presence of the liquid/liquid interface may alter the dynamic behavior of water of solvation and ion-ion interactions. The time of interaction of H 2 O as well as the NO 3 in the primary coordination sphere of UO 2+ 2 were calculated based upon a geometric cutoff r min , that defines the interaction. The probability P(t) associated with the interaction at time t and t+ ∆t is</p><p>and the respective residence time 58 (τ ) is,</p><p>where N (t, ∆t) is the continuous time duration of molecule/ion in the solvation shell or primary coordination sphere about the reference molecule/ion. 59 Nitrate ions are observed to have ∼ 10 × faster dynamic exchange between the primary coordination sphere of uranyl and the second solvation shell, relative to water. Fast dynamic properties have been previously been reported to be sensitive to the geometric cutoff employed to define primary and secondary regions about a solute. To investigate the sensitivity of the nitrate residence time about UO 2+ 2 , the dynamic correction procedure of Ozkanlar et al. 58 was employed to remove the transient breaking and formation of the interaction caused by the U</p><p>Within the correction procedure, a tolerance of 1 ps with average persistence value of 7 ps was used. The computed residence times of nitrate in the uranyl solvation shell without correction was found to be 10 ps, whereas the correction procedure yielded a very similar value of 12 ps.</p><!><p>To identify variations in speciation and solution structure in the interfacial region, two separate analyses were performed. First, a slab of the solution in the interfacial region was analyzed by taking a 10 Å increment in the z direction, consisting of 5 Å on either side of the Gibbs dividing surface of the water, defined as the z -axis position where the density of H 2 O is half of its value in bulk. The speciation, ion concentrations, residence times, and other properties were calculated in each slab and then compared to the analogous metrics of species present the instantaneous surface of the water. The Identification of Truly Interfacial Molecules (ITIM) algorithm 60,61 was employed to define the instantaneous surface of water and ions directly in contact with the organic phase, and for the comparison of the speciation and dynamic properties of the ions in the slabs vs. the instantaneous surface. The density of molecules in the instantaneous surface is fitted to a Gaussian function to obtain the position along z of the mean µ 0 of the distribution. The µ 0 is then used as a reference point (µ = 0) in the interface to define interfacial crest regions (Figure S2) (where the molecular density in the z direction negative to µ (5 Å)) and the trough regions (in the positive direction relative to µ).</p><p>3 Results and Discussion presence of a significant amount of tri-nitrato complex generally not observed in experimental estimations. 37 When nitrate is bound, there is a ∼65% η 2 coordination whereas prior analysis of the relative energetics of η 2 vs. η 1 in solution indicated no significant thermodynamic preference. 19,26,27 Finally, extended organizations are observed in the form of loosely bound intact uranyl nitrate species that appear to be correlated with the presence of UO 2 (NO 3 ) 2− 4 and UO 2 (NO 3 ) 3− 5 as these species have bridging and electrostatic interactions with one another via H 2 O, Li + , and NO 3 -, as observed in the U-U RDF (Figure S3). Although this was noted within the simulation literature, 37,41,62 there lacks strong experimental evidence for such long-range correlations. In combination, these data preclude simple calculation of the equilibrium constants K 1 and K 2 because of the complex equilibria with higher-nitrate containing species and water or nitrate bridged multinuclear U-containing configurations.</p><p>Indeed, using the experimentally measured K 1 and K 2 values of 0.12 and 0.04, 36 it would be predicted that ∼25% of all uranyl species would exist as the mononitrate, and only ∼8% as</p><p>To address these issues the electrostatic continuum correction methodology was em- ployed. This approach scales all ion charges and herein it is optimized to reproduce the experimentally observed equilibrium constants for the formation of the mono-and di-nitrato uranyl complexes. As cross-validation, the ligand denticity and solution organization as a function of LiNO 3 concentration was examined across all ECC values. As the speciation of uranyl-nitrate were examined (Figure 1A), the systematic scaling from 100% was observed to decrease the likelihood of highly coordinated uranyl ions by nitrate, effectively removing the equilibria of the UO</p><p>as well as the loosely organized aggregated species (as demonstrated Figure S3). At a charge scaling of 90% of the original value, the probabilities of over-coordinated uranyl species decreased significantly and loosely bound uranyl nitrate aggregates dissipated (Figure S3). 64 Similarly, the strongly hydrated Li + ions maintain an average hydration number of ∼4.3 at 1 M [LiNO 3 ], using a distance cutoff r min of 3.0 Å in good agreement with the bulk aqueous phase. 65,66 Fitting the equilibrium constants K 1 and K 2 to the simulation data, using the equations S7</p><p>and S9 in the Supplementary Information, yields values of 0.12 and 0.04, respectively, which are well-within the range of experimental observation from spectroscopic measurements and are closest to the values of K 1 = 0.15 ± 0.04 at 6.25 M ionic strength in a solution of NaNO 3</p><p>and HClO 4 , and K 1 = 0.11 in LiNO 3(aq) . 18,19,36 In the system with 3 M LiNO 3 , the η 2 and η 1 modes of uranyl nitrate coordination is observed at 52% and 48%, respectively (Figure 1B), which is in good agreement with prior ab-initio simulations and experimental studies that predicted nearly equal favorability of the two binding nodes in the solution phase. 19,26,27 Charge scaling greater than 10% leads to a significant weakening of the uranyl-nitrate interactions, and minimal concentration of any uranyl nitrate complexes and instead solvent separated ion-pair interactions, as demonstrated by the RDF in Figure S4. In combination, these data indicate that the 90% ECC provides the best representation of uranyl-nitrate interactions in LiNO 3(aq) across a range of concentrations. Using this optimized potential, the experimental K 1 and K 2 are well-reproduced, the ratio of mono-and bidentate NO 3 binding modes are in agreement with ab initio 27 and experimental predictions, 19 and the solution structure as a whole is consistent with experimental observation.</p><p>[LiNO S4).</p><p>Ion solvation exhibits important dynamic properties, characterized by the exchange between first and second solvation shells on the ps to ns timescale. 67 These phenomena are intimately related to complexation reactions that occur via solvent dissociation pathways. 68,69 The residence times of solvating H 2 O about uranyl are generally observed to be high, in the range 40-775 ps depending upon the simulation and experimental techniques, and solution conditions. Consider that NMR which has a distance dependent signal perturbation. 21,67,70 Although the distance at which NMR begins to measure the dynamic exchange of H 2 O is not necessarily known, the computational residence time is generally defined as being strictly between the first and second solvation shells. Strong ion-dipole interactions of UO 2+ 2 with water, 71 and polarization across solvation shells is largely responsible for the long residence times. 72 However, this may be perturbed by long-range competitive interactions with background electrolytes, changes to overall solution-phase dynamic properties, 73</p><!><p>2 at the Electrolyte/Hexane Interface</p><p>The macroscopic and microscopic behavior of liquid/liquid interfaces are deeply intertwined.</p><p>The interfacial tension (γ) increases nearly linearly with LiNO 3 concentration (Table S 3), in a manner consistent with the ion concentration at the electrolyte/hexane interface (Figure 4). The slope corresponding to the change in interfacial tension as a function of electrolyte concentration has been proposed to be a more accurate indicator of ion-specific effects 75,76 than an individual γ value at a specific concentration. 77 The average dγ/dm of 1.69 ± 0.48 mN/mM (mili Newton per meter Molar) is in agreement with the experimental value of 1.23 ± 0.12 mN/mM for the analogous LiNO 3(aq) /vapor system. 77 We now consider the more in-depth molecular scale interfacial chemistry, with an emphasis upon understanding the concentration dependent speciation of uranyl in the interface relative to the bulk.</p><p>Ion Concentration Gradients. It is well-known that ions that reside at the interface perturb molecular-scale behavior as they introduce competitive interactions within an already altered environment relative to the bulk solution. Background electrolytes have been shown to influence metal-ligand chelation and speciation, as well as the rate determining steps in reaction kinetics. These may in turn influence mass-transfer kinetics across liquid interfaces. 68 A standard method to understand the ion concentration approaching the interface is to plot the charge density profiles (shown in Figure 4A). The charge densities as a function of concentration for UO 2+ 2 , NO 3 and Li + are plotted relative to the position of the mean of the water densities (µ 0 ) present in the truly interfacial water layer. The figure shows a sharp negative peak between 0 Å and -5 Å and a positive peak between 0 Å and 5 Å. Collectively, this indicates a weak electric double layer structuring at the liquid/liquid interface. To further understand the distribution of ions in various interfacial regions (the crest and trough) we plotted the number density distribution of ions in the truly interfacial layer (Figure 4B-D) and present the percent distribution of all ions in the truly interfacial layer (layer 1) and subjacent layers in Table 2. The distribution of ions along µ axis reveals that both UO 2+ 2 and NO − 3 predominantly reside in the trough region (positive µ) whereas the number density of Li + is distributed evenly in the crest and trough regions of the truly interfacial layer. From the percentage of ions in the interfacial layer (Table 2) it is apparent that very little Li + exists at the instantaneous surface, although there is significant population within the interfacial region as demonstrated by the density profile. These data are consistent with recent X-ray photo-electron spectroscopy interpretations of the prevalence of Li + in lithium iodide solutions at the electrolyte/vapor interface. 78 Prior work has demonstrated that Li + sheds solvating H 2 O within the instantaneous surface, which disfavors residence therein. 61 When combined with observation of relatively consistent NO − 3 concentration in the instantaneous surface and subjacent layers, the negative charge density in the region directly contacting the organic phase is presumed to be an outcome of Li + cation depletion in the instantaneous surface rather than anionic excess. not lose any H 2 O of solvation within the interfacial region. Nitrate is observed to interact with uranyl in both primary and secondary solvation shells at the electrolyte/hexane interface, as shown in Figure 6C. Indeed, the SSIP form predominates. The concentration of SSIP uranyl-nitrate species is ∼5.5 times that of the complexed mononitrate species at 1 M and ∼6.5 times at 5 M compared in both the bulk and at liquid/liquid interface. The increase in nitrate solvation is observed to be nearly linear in both primary and secondary solvation shells, however the growth of SSIP interactions is significantly more steep.</p><p>Given the changes to the solvation properties of nitrate and uranyl in the interfacial region, and the introduction of competitive interactions at the interface, it is reasonable to question whether the nitrate association constants for uranyl would vary in the interfacial region relative to the bulk. The fractions of uranyl-nitrate complexes within the interfacial region of electrolyte/hexane (shown in Figure 2) reveal that the varying interfacial organization and ion concentrations do not perturb the uranyl-nitrate association constants relative to the bulk region. Within the interface, the mol fraction of UO 2 (NO 3 ) + complex increases linearly from 0.17 ± 0.02 at 1 M to 0.25 ± 0.08 at 5 M [LiNO 3 ]. Even though the fraction of UO 2 (NO 3 ) 2 complexes are less than UO 2 (NO 3 ) + complexes, it also increases from 0.048 ± 0.004 at 1 M to 0.098 ± 0.003 at 5 M [LiNO 3 ]. The K 1 value of 0.13 and K 2 value of 0.06 are obtained by fitting to equations S7 and S9, respectively. A similar coordination behavior is observed in terms of percent denticity changes for all binding modes from 1 M -5 M at the electrolyte/hexane interface compared to bulk. Solvation Dynamics. Although the solvent exchange rate is typically considered a rate limiting process for metal-ligand complexation, when this reaction further depends upon residence in the interfacial region, then both the rate of solvent exchange and the rate of migration in and out of the interface, becomes highly important. Within the interface, the solvent exchange dynamics are significantly faster than in the bulk. At 1 M LiNO 3 , the residence time of water is only a fifth of that in the bulk (75 ps vs. 450 ps). As such,</p><!><p>We present optimized force fields for the interaction of UO 2+ 2 in LiNO 3(aq) that maintains accurate association constants for the formation of uranyl nitrate complexes over a 1 -5</p><p>M electrolyte concentration regime. Under these conditions, the organization and dynamics of the bulk electrolyte solution was investigated. Subsequent biphasic simulation of the electrolyte/hexane system reveal several interesting features of the interfacial region. As anticipated based upon prior work, significant ion concentration gradients are observed for both Li + and NO − 3 , where depletion is observed for the former and excess is observed for the latter. Interestingly, the concentration of uranyl at the interface is the same as in the bulk, presumably because significant populations of solvent separated ion pairs with nitrate prevent uranyl dehydration therein. Second, the timescales of solvent exchange about uranyl are comparable to the residence time of the cation in the interfacial region. Thus, either of these processes may become the rate limiting step for interfacially mediated complexation reactions with extracting ligands. Both processes are also significantly slowed-down as [LiNO 3 ] is increased, nearly doubling over the 1 -5 M regime. Finally, it is shown that despite significant changes to the interfacial organization and dynamics, the uranyl nitrate association constants are unperturbed. Therefore, the knowledge of ion concentration at the interface can be used to predict the changes to uranyl nitrate speciation and thus, the reacting species with extracting ligands like tributyl phosphate as part of the mechanism of solvent extraction.</p>

ChemRxiv

Interactions between pollen tube and pistil control pollen tube identity and sperm-release in the female gametophyte

Flowering plants have immotile sperm that develop within the pollen cytoplasm and are delivered to female gametes by a pollen tube, a highly polarized extension of the pollen cell. In many flowering plant species, including seed crop plants, hundreds of pollen tubes grow toward a limited number of ovules. This system should ensure maximal fertilization of ovules and seed production, however we know very little about how signaling between the critical cells is integrated to orchestrate delivery of two functional sperm to each ovule. Recent studies suggest that the pollen tube changes its gene expression program in response to growth through pistil tissue and that this differentiation process is critical for pollen tube attraction by the female gametophyte and for release of sperm. Interestingly, these two signaling systems, called pollen tube guidance and pollen tube reception are also species preferential. This review focuses on Arabidopsis pollen tube differentiation within the pistil and addresses the idea that pollen tube differentiation defines pollen tube identity and recognition by female cells. We review recent identification of genes that may control pollen tube:female gametophyte recognition and discuss how these may be involved in blocking interspecific hybridization.

interactions_between_pollen_tube_and_pistil_control_pollen_tube_identity_and_sperm-release_in_the_fe

Introduction<!>Pollen tubes differentiate in response to growth through the pistil<!>Pollen tube differentiation for sperm release is controlled by three MYBS<!>Pollen tube reception signaling by the female gametophyte<!>Do MYB target genes interact with the pollen tube reception signaling components?<!>MYB-mediated pollen tube differentiation is important for species recognition during pollen tube reception<!>Pollen tube S18 MYB transcription factors respond to growth through the pistil<!>S18 MYB regulated gene classes and their putative roles during pollen tube receptions<!>

<p>The pollinated pistil is a system that integrates hundreds of individual cell:cell interactions to achieve maximal seed production. Consider the journey of a single pollen tube: it interacts with several female cell types is it germinates on the stigma, enters the transmitting tissue of the style, turns out onto the ovary surface, grows up a funiculus, enters an ovule micropyle, contacts a synergid cell, and bursts to release its cargo of two sperm(1, 2)(Figure 1, Figure 2A). The two sperm cells than interact with the degenerated synergid cell before fusing with the female gametes (the egg and central cell) to produce an embryo and endosperm within a developing seed (3).</p><p>The complexity of the pollen-pistil system becomes apparent when you consider a model like the Arabidopsis thaliana (At) that contains 50-60 ovules and hundreds of pollen tubes (Figure 1). It is likely that each of the cell:cell interactions listed above (and others) is mediated by multiple pollen tube and female cell proteins and other molecules (e.g. carbohydrates). It is also becoming clear that these signaling systems are not discrete or linear, but feedback on each other to optimize reproductive success. For example, it was recently shown that the fusion of sperm cells with female gametes prevents attraction of more pollen tubes to an already fertilized ovule (4-6). Integration of gamete fusion and pollen tube attraction systems ensures that only a single pair of sperm cells is delivered to the female gametophyte and that gamete fusion occurs successfully before an ovule becomes incapable of attracting another pollen tube. These findings suggest the interesting possibility that steps in the flowering plant reproductive process that were thought be distinct from each other are integrated in ways that significantly enhance reproductive success.</p><p>Pollen tube reception is the cell:cell signaling system that results in cessation of pollen tube growth in the female gametophyte, synergid degeneration, and pollen tube rupture/sperm release. Substantial progress has been made in At toward identifying female components of this signaling system (reviewed in (7)), however, relatively little is known about how the female gametophyte senses pollen tube arrival or about how the pollen tube prepares for rupture and sperm release. We review recent studies that suggest the pistil induces a specific pollen tube gene expression program that is critical for pollen tube reception. We discuss this as an example of how distinct cell:cell interaction systems are integrated to maximize reproductive success and how differentiation of the pollen tube within the pistil may be essential for species-preferential recognition of the pollen tube by the female gametophyte.</p><!><p>Pollen tube physiology changes when it grows through floral tissue. For example, pollen tubes must grow through pistil explants to be able to respond to attractants in vitro (8-10). These experiments use a semi-in vitro (SIV) pollen tube guidance assay in which pollen tubes grow through the stigma and a portion of style before growing onto the surface of pollen growth media toward ovules(10). In Torenia, where LURE pollen tube attractants were first described(11), the ability to respond to attractants in vitro increases with prolonged growth through pistil tissue(12). However, when LURE binding sites were assessed by incubating pollen tubes with LUREs followed by detection with anti-LURE antibodies, it was observed that, while growth the pistil was required to generate LURE binding sites, prolonged exposure to the pistil did not increase detection of binding sites further (12). These data suggest that the pollen tube senses the pistil environment, that LURE receptor (not yet identified) expression increases as a consequence, and that prolonged exposure to the pistil environment may activate the receptor.</p><p>Interestingly, a pair of At pollen tube expressed membrane-associated receptor-like cytoplasmic kinases (LIP1 and LIP2) were recently shown to be induced by growth in the pistil and required for the full response to purified LURE proteins in the SIV assay and for pollen tube guidance in the pistil(13). It is not yet clear whether LIP1 and LIP are directly involved in LURE perception, however, these findings underscore the idea that pollen tubes differentiate during growth in the pistil resulting in expression of proteins required to respond to pollen tube guidance cues.</p><!><p>Over 1000 genes were detected in the transcriptome of pollen tubes grown in the SIV assay that were not detected in pollen tubes grown in vitro (14). To identify regulators that control pollen tube gene expression in response to the pistil, 26 SIV-induced transcription factors were identified (14). This list included three closely related R2R3-MYB type transcription factors (Subgroup18 (S18): MYB97, MYB101 and MYB120) (14); single and double myb mutants did not display seed set defects, however triple mutants had a ∼70% reduction in seed production (15). myb triple mutant pollen grains and tubes develop normally, grow through the pistil, and target ovules nearly as efficiently as wild type. These data suggest that MYB97, MYB101 and MYB120 do not regulate expression of the LURE receptors. However, upon interaction with the female gametophyte, myb triple mutants fail to arrest their growth and ∼72% of ovules contain coils of pollen tubes within the female gametophyte(15). Synergid cells targeted by these mutant pollen tubes fail to degenerate normally suggesting a loss of communication between pollen tube and synergid cells during pollen tube reception (15). Furthermore, myb triple mutant pollen tubes fail to burst and release sperm. These data suggest that MYB97, MYB101 and MYB120 are critical for the pollen tube to exchange signals with the female gametophyte required for successful fertilization.</p><!><p>Pollen tube reception requires a number of synergid-expressed genes including FERONIA (FER) (16), NORTIA (NTA) (17) and LORELEI (LRE) (18) (Figure 2A). Loss-of-function mutations in of each of these genes cause the same phenotype: wild type pollen tubes coil in mutant ovules and fail to release sperm. FER is a transmembrane receptor-like kinase of the CrRLK1L family predicted to have pollen tube or female-gametophyte ligand(s) important for pollen tube reception. FER has a malectin-like extracellular domain, so its ligand could be a carbohydrate or glycoprotein (19) (Fig 2A). FER is localized to the filiform apparatus, and is required for NTA, a 7-pass transmembrane Mildew Resistance Locus O family protein, to be re-localized from the secretory system to the filiform apparatus upon pollen tube arrival (17) (Figure 2A-2B). NTA may interact with synergid- or pollen tube-expressed proteins upon relocalization and also contains a calmodulin-binding domain in its cytoplasmic C-terminus, possibly allowing synergid cell perception of calcium oscillations during reception (20) (Fig 2B-2D). LRE encodes a glycosylphosphatidylinositol-anchored protein predicted to be associated with the synergid membrane (18) (Figure 2A). The mechanisms by which FER, NTA, and LRE direct pollen tube reception are unknown and it will be important to define how they sense pollen tube arrival to initiate synergid degeneration and pollen tube burst.</p><p>Relatively little is known about the pollen tube-expressed genes involved in pollen tube reception and no direct connections between pollen tube and synergid genes have been made. Interestingly, a pair of pollen tube-expressed members of the FER family members [CrRLK receptor-like kinases, ANXUR1 (ANX1) and ANXUR2 (ANX2)] may be negative regulators of pollen tube burst. anx1, anx2 double mutants produce pollen tubes that burst very soon after germination (21, 22). Like FER, the ligands for ANX1 and 2 are not yet known and the nature of ANX contribution to pollen tube reception is unclear because the double mutant phenotype is not informative about their role during synergid interactions.</p><p>Auto-inhibited Calc ium ATPase9 (ACA9) encodes a pollen-specific calmodulin-binding calcium pump localized to the pollen tube plasma membrane(23). aca9 pollen tubes have growth defects, however, they can reach ovules in the upper portion of the pistil and ∼50% of these enter the ovule micropyle and arrest, but fail to burst (23). This result suggests that ACA9 is important for regulating pollen tube calcium dynamics required for pollen tube burst. Recent live-imaging experiments have shown that calcium concentrations spike in the pollen tube and the synergids as pollen tube burst occurs(20) (Figure 2B-D). It will be very interesting to determine how aca9 pollen and nta (which contains a predicted calmodulin-binding domain) synergid mutants affect these dynamics.</p><!><p>myb97, myb101, myb120 triple mutant pollen tubes fail to stop growing and burst within the female gametophyte, so the genes regulated by these transcription factors are candidates for pollen tube components of the pollen tube reception mechanism. By comparing the transcriptomes of pistils pollinated with either wild-type or myb triple mutant pollen, three main categories of MYB-regulated genes were identified: transporters, small proteins and peptides, and carbohydrate active enzymes (15) (Figure 2A). Many of these genes were found to be in large gene families, potentially explaining why extensive genetic screening in At has not identified male pollen tube reception mutants.</p><p>Several sugar/proton symporters in the major facilitator family (24) were identified as MYB regulated. AtSUC7, AtSUC8, and AtSUC9 are highly induced in wild-type pollen tubes grown in SIV conditions, but are absent in myb triple mutants (15). myb triple mutants have no pollen tube growth defect in vitro or in the pistil, suggesting these sucrose transporters are not required to important sugars to fuel growth, but may have a specialized function in regulating the pollen tube osmotic state during pollen tube reception.</p><p>Multiple small proteins and peptides were found to be potential targets of MYB activation during pollen tube growth (Figure 2A). One of these is a thionin, an 87 amino acid, secreted, cysteine-rich protein (CRP2460, [Silverstein, 2007 #1975}). Thionins have been shown to nucleate membrane pores in artificial lipid bilyers and rat neuronal cells (25); this is an intriguing potential function for pollen tube-expressed thionins given that myb triple mutant pollen tubes fail to express these peptides and also have defects in pollen tube burst and initiating synergid degeneration (15). Interestingly, small (130-152 aa) secreted proteins with domains homologous to stigmatic Papaver rhoeas self-incompatibility (SI) protein PrsS1(26) were also found to require pollen tube MYBs for expression in the pollen tube (15). In Papaver PrsS1 is secreted from the stigma and is bound by PrpS, its pollen-expressed receptor (27). Ligand:receptor interaction blocks germination of self pollen through a mechanism involving calcium as a second messenger and programmed cell death (PCD) (28). The function of At S-protein homologues (SPHs) is unclear (29) and it is surprising to find that they are expressed in pollen tubes in response to growth through the pistil. It will be interesting to test whether a signaling module similar to that described in Papaver is involved in gametophyte interactions that lead to synergid degeneration and pollen tube burst.</p><p>The third category of pollen tube MYB-regulated genes encode proteins that interact with carbohydrates, many of which are many are predicted to be extracellular (Figure 2A). These include hydrolases (i.e. Glycoamylase, O-glycosylhydrolase, p-1,3-glucanase, Pectin lyase-like), and pectin methylesterase inhibitors (15). These proteins may be expressed by the pollen tube to modify the cell wall in preparation for pollen tube reception. Alternatively, these proteins may modify the cell wall or other pollen-derived carbohydrates for recognition by the female gametophyte. As mentioned above, FER contains an extracellular malectin domain that could interact with a pollen tube-derived carbohydrate and since myb triple mutant pollen tubes behave as if FER signaling is defective, it will be interesting to determine whether pollen tube MYBs regulate production of FER ligands.</p><!><p>Pollen tube reception fails in interspecific crosses of Rhododendron (30) or At (16); pollen tubes of one species overgrow without releasing sperm in ovules of the exotic species. These data suggest that pollen tube reception is an important pre-zygotic barrier to interspecific hybridization. When At myb triple mutant pollen tubes enter a wild-type At ovule, they overgrow and fail to release sperm (15). These observations lead to the hypothesis that pollen tube MYBs may control expression of the determinants of pollen tube identity during interspecific recognition.</p><p>To begin to assess the role of pollen tube MYBs in determining pollen tube recognition, pollen tube reception was investigated in inter-accession and interspecies crosses (Figure 3). The rates of pollen tube overgrowth when myb triple and myb double mutants were used to pollinate At pistils were higher than in any of the inter-accession crosses tested (Figure 3M). Arabidopsis korshinskyi (Ak) and Olimarabidopsis pumila (Op) pollen tubes overgrew in At ovules more frequently than any of the accessions tested, but the At myb triple mutant phenotype was even more pronounced (Figure 3M). These results are consistent with the idea that pollen tube MYBs control pollen tube identity as recognized by the female gametophyte.</p><p>When Ak or Op were used as females in crosses with At pollen, increased rates of pollen tube overgrowth were not observed because the percentage of untargeted ovules (likely due to incongruity in pollen tube attraction) was so high that pollen tube reception could not be adequately assessed (Figure 3I,J,M). However, crosses of At myb triple mutant pollen onto Ak or Op pistils showed increases in rates of pollen tube coiling as well as increases in the number of untargeted ovules (Figure 3K-L, M). This finding suggests that MYBs are required for two levels of pollen tube identity factors: 1) core pollen tube identity recognized by exotic female gametophytes and 2) species-preferential identity recognized by the native female gametophyte.</p><p>Future work will need to address the mechanisms by which individual MYB target genes confer these identities. One possibility is that MYB-mediate pollen tube differentiation fails when pollen tubes grow through an exotic pistil. Alternatively, MYBs may be properly activated by the exotic pistil and may in turn activate their targets, but the products of these target genes are not recognized by the exotic female gametophyte. In either case, exploration of how the pistil induces MYB97, MYB101, and MYB120 and how the products of their target genes mediate gametophyte interactions provides a path to understand how suitable mates are identified during flowering plant reproduction.</p><!><p>A schematic of an Arabidopsis thaliana ovary with 12 ovules (instead of actual 50-60). Pollen grains (red) are germinating pollen tubes on the stigma. Pollen tubes target the female gametophyte (gray). Mature pollen grains (represented in black with two sperm and a nucleus [gray] express MYB101, and lower levels of both MYB97 and MYB120. Transcription of MYB targets is low or absent in pollen grains. Growth through the stigma and style activate transcription of MYB97 and MYB120; the three MYBS activate target genes in the pollen tube. The pollen tube factors that sense the pistil environment and activate MYB expression are not yet known.</p><!><p>(A) Schematic of the pollen tube and female gametophyte at the beginning of pollen tube reception. Pollen tube contact with one of the synergid cells is indicated (dotted line). Proteins with roles in pollen tube reception and MYB-regulated gene products are highlighted. Abbreviations: sn - synergid nucleus, vn - vegetative nucleus. (B-C) Schematic shows pollen tube reception between a pollen tube and the synergid that will degenerate. Calcium concentrations are symbolized with a color spectrum (red is high calcium, green is low calcium). (B) FER dependant-NTA relocalization occurs after pollen tube arrival. (C) Synergid degeneration is accompanied by loss of synergid nuclear integrity. (D) Pollen tube burst, Sperm release and double fertilization.</p><!><p>Pollen tube reception is defective in inter-accession and inter-species crosses. (A-H) Arabidopsis thaliana (At) male sterile 1 (ms1) pistils from the Landsberg accession were pollinated with various pollen donors for 24 hours before imaging pollen tube growth patterns using aniline blue staining and fluorescence microscopy (as in (31)). m - micropyle, arrowheads indicate pollen tubes. (A) At Col-0 accession, normal entry. (B) At myb120-3, coiling. (C) At myb97-1,120-3, coiling and attraction of two pollen tubes. (D) At myb97-1,101-4,120-3, coiling. (E) At Cvi-0 accession, two pollen tubes. (F) At Lc-0 accession, coiling, two pollen tubes. (G) Ak, coiling, two pollen tubes. (H) Op, coiling, two pollen tubes. (I-L) Emasculated Ak pistils were crossed with At Col-0 (I-J) or myb triple mutant (K-L). At Col-0 tubes show (I) wild-type pollen tube entry, and (J) wild-type entry & one tube failing to enter the micropyle; the majority of ovules were untargeted in interspecific crosses (M). myb triple mutants show (K) pollen tube coiling and (L) failure to enter the micropyle (3 tubes) in interspecific crosses. (M) Quantification of pollen tube reception. Arabidopsis accessions: Col-0 (CS1092), Ler-0 (CS20), Lc-0 (CS28443), Cvi-0 (Cs902), Pn-0 (CS28645), Ove-0 (CS28590). Related species: Arabidops is korshinskyi (Ak) (CS4653) or Olimarabidopsis pumila (Op) (CS3700). Asterisks denote statistically significant differences from controls by a students t-test, p-value <0.05.</p>

PubMed Author Manuscript

Magnesium impacts myosin V motor activity by altering key conformational changes in the mechanochemical cycle

We investigated how magnesium (Mg) impacts key conformational changes during the ADP binding/release steps in myosin V and how these alterations impact the actomyosin mechanochemical cycle. The conformation of the nucleotide binding pocket was examined with our established FRET system in which myosin V labeled with FlAsH in the upper 50 kDa domain participates in energy transfer with mant labeled nucleotides. We examined the maximum actin-activated ATPase activity of MV FlAsH at a range of free Mg concentrations (0.1\xe2\x80\x939 mM) and find that the highest activity occurs at low Mg (0.1\xe2\x80\x930.3 mM), while there is a 50\xe2\x80\x9360% reduction in activity at high Mg (3\xe2\x80\x939 mM). The motor activity examined with the in vitro motility assay followed a similar Mg-dependence and the trend was similar with dimeric myosin V. Transient kinetic FRET studies of mantdADP binding/release from actomyosin V FlAsH demonstrate that the transition between the weak and strong actomyosin. ADP states is coupled to movement of the upper 50 kDa domain and is dependent on Mg with the strong state stabilized by Mg. We find that the kinetics of the upper 50 kDa conformational change monitored by FRET correlates well with the ATPase and motility results over a wide range of Mg concentrations. Our results suggest the conformation of the upper 50 kDa domain is highly dynamic in the Mg free actomyosin. ADP state, which is in agreement with ADP binding being entropy driven in the absence of Mg. Overall, our results demonstrate that Mg is a key factor in coupling the nucleotide- and actin-binding regions. In addition, Mg concentrations in the physiological range can alter the structural transition that limits ADP dissociation from actomyosin V, which explains the impact of Mg on actin-activated ATPase activity and in vitro motility.

magnesium_impacts_myosin_v_motor_activity_by_altering_key_conformational_changes_in_the_mechanochemi

<!>EXPERIMENTAL PROCEDURES<!>Myosin V cDNA Construction, Expression, and Purification<!>Stopped-flow Measurements and Kinetic Modeling<!>Thermodynamic analysis<!>FRET Measurements<!>Time Resolved Anisotropy<!>Mg Dependence of ADP release rate constants<!>ATPase Assays<!>In Vitro Motility Assays<!>Mg alters steady-state ATPase and in vitro motility<!>mantdADP binding to acto-MV FlAsH with and without Mg<!>Conformation of the nucleotide binding pocket as a function of temperature with and without Mg<!>Dissociation of mantdADP from acto-MV FlAsH with and without Mg<!>Mg concentration dependence of mantdADP release from acto-MV FlAsH<!>Correlation of the strong-to-weak ADP isomerization with the maximum ATPase rate<!>Thermodynamic analysis<!>Time-resolved anisotropy<!>DISCUSSION<!>Structural mechanism of MgADP coordination in myosin V<!>Mg is required for formation of the strong ADP binding state<!>Kinetic mechanism of Mg dependent ADP release<!>Implications for Mg dependent regulation of MV

<p>The family of P-loop nucleotide triphosphatases (NTPases) which includes G-proteins, kinesins, and myosins contain a highly conserved and well characterized nucleotide binding region 1–3. A common thread within this protein family is that the nucleotide bound to the activesite modulates the affinity of the NTPase for its track in the case of motors or effector protein in the case of G proteins 1, 2. In motor proteins conformational changes in the active-site are also coupled to structural changes that produce force and motion. Three main structural elements are known to coordinate nucleotide binding and hydrolysis in the active-site of P-loop NTPases; P-loop, switch I, and switch II4. These elements coordinate an active-site magnesium ion (Mg) associated with the bound nucleotide that is thought to be central to high-affinity nucleotide binding and hydrolysis 5. Studies with myosins have demonstrated that variations in physiological Mg concentrations can modulate motor activity 6–8. In addition, coordination of the active-site Mg is also critical for the motor activity of kinesin suggesting a common structural mechanism may exist between these two motor protein families 9. In G-proteins, Mg exclusion plays a critical role in mediating GDP dissociation 10–12. In order to fully understand the potential role of metal-ion regulation in P loop NTPases it is critical to determine how Mg regulates key structural changes in their NTPase cycles.</p><p>Myosin motors generate force by coupling small conformational changes in the nucleotide binding region to a large swing of the light chain binding region ("lever arm") during a cyclic interaction with actin filaments. The actomyosin ATPase cycle (scheme 1) consists of nucleotide-states that correspond to either "weak" (bold) or "strong" actin binding conformations. In the absence of nucleotide myosin binds to actin with very high-affinity. ATP binding to actomyosin (K'1K'2) induces dissociation of the complex and the following ATP hydrolysis step (K3) stabilizes the pre-force generating conformation. The hydrolysis products are released slowly from the active-site of myosin until actin binding accelerates the release of phosphate (K'4) and triggers the force generating conformational change in the lever arm. The resulting actomyosin. ADP states have high actin affinity. The release of ADP is thought to occur in two steps, with the first step associated with an isomerization of the nucleotide binding pocket (NBP) (K5A or Kpocket) and the second step involving local active-site rearrangements during the release of ADP (K5B or Kligand) 5–8, 13. The active site isomerization is thought to be associated with a transition from a "weak" ADP affinity state to "strong" ADP affinity state 7. An additional swing of the lever arm is thought to occur in many myosins during one or both of the ADP release steps 13–15.</p><p>Mg is required for ATP to bind to myosin with high-affinity and for the ATP hydrolysis step 16. However, these steps are not likely important in altering the in vivo actomyosin motility as Mg concentrations in the physiological range (0.8–1.2 mM free Mg,17) do not significantly alter these steps. One common theme in the myosin motor family is that the ADP release step is critically important in mediating the contractile velocity and load dependence. Strain dependent ADP release limits the maximal sliding velocity of skeletal muscle myosin 18, 19 and allows for mechanical gating and processive walking of dimeric myosin V 20.</p><p>Several biochemical and structural studies have demonstrated that Mg can alter the kinetics of ADP release in myosin I, II, and V 6, 7, 21. A study by Kintses et. al.22 found that Mg shifts the equilibrium from the weak to the strong ADP binding state of Dictostelium myosin II. However, they could not determine if Mg was required to form the strong ADP binding conformation as is known to be the case in G-proteins 12. Previous studies with myosin V have been particularly revealing in examining the actomyosin. ADP states and Mg dependence. Two studies examined the mantADP fluorescence in the active site, which provided evidence for two ADP states in the presence and absence of actin6, 7. These studies found that Mg can impact the equilibrium between the weak and strong ADP states6, as well as the rate of nucleotide release from the weak state 6, 7. The results were in agreement with the crystal structure of myosin V in the presence of ADP, which contains no active site Mg and suggested Mg can be released from the active site prior to ADP5. Based on these results, Rosenfeld et al. 6predicted varying free Mg concentrations would alter the motile properties of myosin V. Nagy et al.8 followed up on these studies by demonstrating that free Mg can alter the actin-activated ATPase rate, which correlated with the overall rate of ADP release. Nagy et al.8 also determined that mutating a residue associated with Mg coordination in the active site can impact the Mg dependence, suggesting that the Mg in the active site and not another allosteric binding site is responsible for altering ADP release kinetics. However, no studies have directly correlated the actin-activated ATPase rate, in vitro motility, and specific structural transitions in the actomyosin. ADP states. Furthermore, it is unknown how Mg alters the structural transitions at the active site and how these transitions impact the conformation of functionally important regions of myosin, such as the actin-binding and lever arm regions.</p><p>We have developed a spectroscopic approach to examine structural changes in the actomyosin V. ADP states using FRET between mant labeled nucleotides and FlAsH labeled in the upper 50 kDa (U50) domain23–25. Our results are consistent with the FRET measurements being sensitive to the two structural actomyosin. ADP states, which we suggested to be modulated by the conformation of switch I23, 24. We determined that the equilibrium between the two switch I conformations in the presence of ADP is sensitive to temperature, since a shorter average distance was found at low temperature and a longer average distance was found at higher temperatures. Based on these results we hypothesize that the position of the U50 domain correlates with the position of switch I, and that the switch I movement may be coupled to the movement of the U50 domain. Communication between switch I and the U50 domain is thought to be responsible for ATP-induced dissociation of actomyosin as ATP binding induces a switch I closed/actin binding cleft open state with weak affinity for actin. We also found that mutations in the switch II region can impact the equilibrium between the two switch I conformations in the presence of ADP24. In the current study we examined the impact of Mg on specific structural transitions in the active site and how Mg alters the dynamics of the U50 domain. We also determined how Mg alters the kinetics and thermodynamics of structural changes associated with the actomyosin ADP release steps. Finally, we correlated the Mg dependence of the ADP release steps with the actin-activated ATPase and in vitro motility properties of myosin V. Our results demonstrate that Mg is a key factor in mediating the structural and chemo-mechanical properties of myosin V.</p><!><p>All reagents were of the highest purity commercially available. ATP and ADP were prepared fresh from powder. N-Methylanthraniloyl (mant)-labeled 2′-deoxy-ADP (mantdADP) was purchased from Jena Bioscience (Jena, Germany). The mantdADP concentration was determined from absorbance measurements at 255 nm using ɛ255 of 23,300 M−1·cm−1. ATP and ADP concentrations were determined by absorbance at 259 nm using an ɛ259 of 15,400 M−1·cm−1. FlAsH (fluorescein biarscenical hairpin-binding dye) was generously provided by Roger Tsien and Stephen Adams (University of California, San Diego).</p><!><p>Two chicken myosin V constructs were used for this study. One contained a single IQ motif (MV) (residues 1–792) and the other was a heavy meromyosin (MV HMM) construct containing an N-terminal FLAG tag and C-terminal YFP 26. In the MV 1IQ construct residues 292–297 were substituted with a tetracysteine motif (Cys-Cys-Pro-Gly-Cys-Cys) for FlAsH labeling23–25, 27. MV contained a C-terminal Myc tag (EQKLISEEDL) followed by a FLAG tag (DYKDDDDK). The G440A mutation was introduced as described previously 24. Both myosin V constructs were coexpressed with chicken calmodulin and purified by anti-FLAG affinity chromatography. The purity was greater than 95% based on Coomassie-stained SDS gels. Myosin concentrations were determined using the Bio-Rad microplate assay using bovine serum albumin (BSA) as a standard or by absorbance (ɛ280 = 103,600 M−1·cm−1). MV labeled with FlAsH, referred to as MV FlAsH, was generated as previously described 23–25, 27. Actin was purified from rabbit skeletal muscle using an acetone powder method 28. All experiments were performed in K50TCEP buffer (50 mM KCl, 1 mM EGTA, 1 mM TCEP, and 10 mM imidozole-HCl, pH 7.0, 25°C) supplemented with the appropriate amount of MgCl2. Free Mg concentrations were calculated using MaxC 2.5 and the stability constants for ADP and ATP (http://www.stanford.edu~cpatton/).</p><!><p>Transient kinetic experiments were performed in an Applied Photophysics (Surrey, UK) stopped-flow apparatus with a dead time of 1.2 ms. A monochromator with a 2-nm band pass was used for fluorescence excitation, and cutoff filters were used to measure the fluorescence emission. All optical filters were provided with the stopped-flow instrument. The mantdADP was excited at 365 nm, in the presence and absence of MV FlAsH or unlabeled MV, and the FRET emission was measured with a 515 nm long pass filter. Nonlinear least-squares fitting of the data was done with software provided with the instrument or Kaleidagraph (Synergy Software, Reading, PA). Uncertainties reported are standard error of the fits unless stated otherwise. All concentrations mentioned in the stopped-flow experiments are final concentrations unless stated otherwise.</p><p>Kinetic modeling and simulations were performed with Pro-K (Applied Photophysics) or Kintek Explorer (Kintek Corp.) software using schemes 1 and 2, also used in kinetics studies of myosin V 6, 7, 23, 24. The mantdADP binding and dissociation transients were normalized prior to fitting to the kinetic model. The mantdADP binding data were fit to a two-step binding model described in previous reports 7, 23, 29, where the slow and fast exponential rates, and the amplitude of the slow phase from the double exponential fits are described by equations 1–3. The equation (Eq. 3) defining the amplitude of the slow phase was slightly modified from our previous report 23, since the component associated with the unbound mantdADP does not contribute to the fluorescence enhancement.</p><p> (Eq. 1)kslow=(k+ligand∗[mantdADP](k−pocket+k+pocket)+k−ligand∗k+pocket)(k+ligand∗[mantdADP]+k−ligand+k+pocket+k−pocket) (Eq. 2)kfast=k+ligand∗[mantdADP]+k−ligand+k+pocket+k−pocketwhere k+ligand was determined from the linear dependence of mantdADP binding to actomyosin V (fast phase of mantdADP binding to actomyosin). (Eq. 3)Aslow=(k+pocket/k−pocket)/(1+k+pocket/k−pocket)The mantdADP dissociation transients were also fit to a two state model described by Hannemann et al.7 and used in our previous report 23. Equations 4–6 were used to describe the fast and slow exponential rate constants, as well as the amplitude of the slow phase of the double exponential transient. (Eq. 4)kfast=k−ligand+k+pocket (Eq. 5)kslow=k−pocket[k−ligand/(k−ligand+k+pocket)] (Eq. 6)Afast=[k−pocket/(k+pocket+k−pocket)]∗[k−ligand/(k−ligand+k+ligand)]The overall affinity can be calculated with equation 7. (Eq. 7)KD=1/Kligand∗{(1/Kpocket)/[1+(1/Kpocket)]}</p><!><p>The thermodynamic parameters were determined by examining the temperature dependence of each of the ADP-binding steps as described 23. The enthalpic and entropic contributions to the free-energy associated with each step were calculated in the presence of 2 mM and 10 mM MgCl2 as well as 4 mM EDTA.</p><!><p>FRET was used to measure the distance between donor fluorophore, mantdADP, and the acceptor fluorophore, FlAsH-labeled MV, using the Förster energy transfer theory 23. The energy transfer efficiency (E) was measured from the increase in acceptor emission. We mixed acto-MV FlAsH with mantdADP (donor mantdADP+ acceptor), acto-MV unlabeled with mantdADP (donor only), and acto-MV FlAsH and ADP (acceptor only) and monitored the stopped-flow fluorescence (excitation 365 nm) transients with a 515 nm long pass filter. The efficiency of energy transfer and distance between the donor and acceptor probes was calculated using equations described in our previous work 23–25, 27. The only difference from our previous work was that we used the fluorescence intensity determined from the stopped-flow mixing transients instead of the fluorescence spectra examined in a spectrofluorimeter. We observed no difference in the quantum yield of mantdADP bound to actomyosin V in the presence and absence of Mg, while differences in the mantdADP quantum yield at different temperatures were taken into account as previously described 23, 24.</p><!><p>Time-resolved anisotropy was measured using a custom built single-photon counting (SPC) spectrophotometer 30. The G-factor for this instrument is 1.0. Polarized time-resolved fluorescence waveforms were acquired with the emission polarizer orientation set to 0°, 54.7°, and 90° relative to the vertically polarized excitation source and analyzed globally according to Eq 8–12 31. The observed fluorescence, I(t), is fit by convolving the measured instrument response function, IRF, determined from light scatter of the excitation source, with the polarization-dependent fluorescence decay function, f(pol, t) (Eq. 8). The polarized fluorescence decay model depends on the emission polarization with the magic-angle emission (54.7°, Eq. 9) described by a sum of exponentials with discrete amplitude, Ai, and lifetime, τi, terms. The vertically (0°) and horizontally (90°) polarized emissions depend on the magic-angle emission and the time-resolved anisotropy function, r(t), according to Eq. 10 and 11. The time-resolved anisotropy function was assumed to be a single exponential with initial, ri, and infinite, r∞, anisotropies, and rotational correlation time, τRi (Eq. 12). The initial anisotropy, ri, infinite anisotropy, r∞, and rotational correlation time, τRi, were global parameters shared by all lifetime species τi in Eq. 9. The total anisotropy was calculated from the time-resolved fluorescence, and time-resolved anisotropy functions according to Eq. 13.</p><p> (Eq. 8)I(t)=∫−∞∞IRF(t−t′)f(pol,t)(t′)dt (Eq. 9)f(54.7°,t)=∑i=12Aie−tτi (Eq. 10)f(0°,t)=(1/3)f(54.7°,t)[1+2r(t)] (Eq. 11)f(90°,t)=(1/3)f(54.7°,t)[1−2r(t)] (Eq. 12)r(t)=r∞+riexp(−t/τRi) (Eq. 13)ro=∫−∞∞f(54.7°t)r(t)dt∫−∞∞f(54.7°′t)dt</p><!><p>We used the equation described in Rosenfeld et al.6 to determine the Mg affinity for acto-MV FlAsH in the weak and strong ADP binding states. This equation also allows for extrapolating the ADP release rate constants to Mg free and saturating conditions, (Eq. 14)k-pocket.Obs=k−Mgpocket∗([Mg]/KD.Mg)+k−pocket([Mg]/KD.Mg)+1where k−pocket.obs is the rate constant determined at each Mg concentration, k−pocket is the rate constant extrapolated to Mg free conditions, and k−Mg.pocket is the rate constant at saturating Mg conditions, [Mg] is the free Mg concentration, and KD.Mg is the affinity of Mg for the actomyosin. ADP strong state. A similar analysis was performed with the k−ligand rate constant.</p><!><p>ATPase assays were performed in the stopped-flow at 25°C using the NADH coupled assay 25, 32. The steady-state ATPase rate at each Mg concentration (0.5–10 mM MgCl2) of 0.05–0.1 μM MV FlAsH or MV HMM in the presence of 20 μM actin and 1 mM ATP was determined.</p><!><p>Actin filament motility was measured using the in vitro motility assay 33 used previously to measure MV FlAsH 25 and MV HMM 26 motility. MV FlAsH was specifically attached to the nitrocellulose-coated surface with an anti-Myc antibody, while MV HMM was directly adhered to the nitrocellulose-coated surface. The surface was blocked with bovine serum albumin at a concentration of 1 mg/ml. The motility of F-actin labeled with rhodamine-phalloidin was observed using an activation buffer consisting of K50 supplemented with the appropriate amount of Mg, 3.4 μM calmodulin, 0.35% methylcellulose, and 1 mM ATP. Phosphoenol pyruvate (2.5 mM) and pyruvate kinase (20 units/ml) were added as an ATP regeneration system. Glucose oxidase (0.1 mg/ml), glucose (3 mg/ml), and catalase (0.018 mg/ml) were added to reduce photobleaching. After the addition of the activation buffer, the slide was promptly viewed using a NIKON TE2000 microscope equipped with a 60x/1.4NA phase objective. Images were acquired at 2–5 second intervals for a period of 3–5 minutes. We utilized a shutter controlled Coolsnap HQ2 cooled CCD digital camera (Photometrics) binned 2x2 for all imaging. To measure velocity, the video records were transferred to Image J and analyzed with the MtrackJ program 34.</p><!><p>We examined the steady-state ATPase activity of MV FlAsH (Figure 1A) and MV HMM (Figure 1B) in the presence of 20 μM actin using the NADH coupled assay at 25°C. The ATPase activity was measured with K50 buffer containing varying concentrations of MgCl2 (0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0 mM). The actin-activated ATPase results were plotted as a function of free Mg concentration. We found that the highest ATPase activities were observed at low free Mg concentrations (0.1–0.3 mM) and there was a 60% reduction in activity at high free Mg concentrations (3–9 mM). We also examined the sliding velocity with MV FlAsH (Figure 1A) and MV HMM (Figure 1B) in the in vitro motility assay and found the Mg-dependence was very similar to the ATPase results (Figure 1). We analyzed 20–30 filaments per condition and the mean and standard error of the mean was calculated for each Mg concentration. In K50 buffer containing 4 mM EDTA, there was no actin-activated ATPase activity (rate = 0.06 s−1) or in vitro motility observed.</p><!><p>We examined the impact of Mg on the conformational changes associated with ADP binding to acto-MV. The kinetics of nucleotide binding were examined by mixing mantdADP with acto-MV FlAsH in the stopped-flow at 4, 10, 15, and 25°C in K50 buffer with 2 mM MgCl2 or 4 mM EDTA (Figure 2). The FRET signal was examined by exciting at 365 nm and measuring the fluorescence emission through a 515 nm long pass filter. In the presence of 2 mM MgCl2, the fluorescence transients were best fit to a bi-exponential function. The fast phase was found to be linearly dependent on mantdADP concentrations (Figure 2A) while the slow phase was independent of mantdADP concentrations (Figure 2B). Thus, the data were fit to a kinetic model described previously 23, in which mantdADP first forms a "weak or open" (Kligand) complex with actomyosin and transitions into a "strong or closed" (Kpocket) conformation (scheme 2). The slope of the linear dependent fast phase was used to determine k+ligand and the transients were fit to kinetic equations 1–3 to determine k+pocket, k−pocket, and k−ligand (scheme 2)</p><p>The fast and slow phases of mantdADP binding to acto-MV FlAsH were both dependent on temperature. The relative amplitudes of the fast and slow components were similar over the range of mantdADP concentrations measured but varied at each temperature (Figure 2D). Overall, our kinetic results from mantdADP binding to acto-MV FlAsH in the presence of 2 mM MgCl2 were similar to our previous results with 1 mM MgCl2 23 except the relative amplitudes demonstrated a higher fractional distribution of the slow component. Identical experiments were performed in the presence of 10 mM MgCl2 and the rates and relative amplitudes of the fast and slow components were similar to 2 mM MgCl2 (Table 1). These results indicate that the strong ADP conformation and corresponding "high-FRET" position of the U50 domain is more favorable at higher MgCl2 concentrations. Under Mg free conditions (4 mM EDTA K50 buffer) we found that the fluorescence transients were dominated by a fast fluorescence increase that fit to a single exponential and was linearly dependent on the mantdADP concentrations (Figure 2C). The second-order rate constant determined from the slope of the single exponential fits as a function of mantdADP concentrations was faster than determined with Mg, especially at 25°C where it was more than 2-fold faster (Figure 2C, Table 1). The y-intercept of the linear fit of the fast component was about 10-fold higher without Mg than in the presence of 2 mM MgCl2.</p><!><p>We determined the maximum FRET efficiency of the acto-MV FlAsH.mantdADP complex by stopped-flow mixing and measurement of acceptor enhancement (Figure 3). Compared to conventional titrations, stopped flow mixing reduces inner filter effects arising at high mantdADP concentrations and at the same time yields fluorescence transients from which amplitudes and kinetic rates can be determined.</p><p>We determined the amplitude of the FRET signal in the stopped-flow after mixing acto-MV FlAsH (0.5 μM actin, 0.25 μM MV FlAsH) with varying concentrations of mantdADP (0–30 μM mantdADP) in the presence of 2 mM MgCl2 (Figure 3A) or 4 mM EDTA (Figure 3C) at 4, 15, and 25°C. The amplitudes of the donor only and acceptor only were also determined at 30 μM mantdADP concentration, which allowed for determination of the FRET efficiency and calculated distances, as summarized in Table 2. In the current study, the trend of distance change as a function of temperature at 2mM MgCl2 was similar to our previous work at 1mM MgCl2 23. (The distance change is 4.5Å at 2mM MgCl2 and 2–4Å at 1mM MgCl2 23).</p><p>At low temperature (4°C) we found the average distance was similar in the presence (2 mM MgCl2) and absence of Mg (4 mM EDTA). Interestingly, at 25°C the FRET distance increased dramatically in the absence of Mg, while in the presence of Mg there was a much smaller increase in distance relative to 4°C. Therefore, the temperature-dependent change in FRET is much more dramatic in the absence of Mg.</p><!><p>We examined mantdADP dissociation from acto-MV FlAsH in K50 buffer containing 2 mM MgCl2 and 4 mM EDTA (Figure 4). A complex of acto-MV FlAsH.mantdADP was mixed with saturating ATP (final concentrations: 0.25 μM MV FlAsH, 0.5 μM actin, 10 μM mantdADP, 1 mM ATP) and the resulting fluorescence decrease was monitored as described above (ex = 365 nm, em = 515 nm long pass filter). As observed in our previous work 23 the fluorescence transients followed a biexponential function in the presence of Mg (Figure 4A and C). The fast component was equivalent to the y-intercept from the mantdADP binding experiments (Figure 2), and therefore the slow component was modeled to be the conformational change in the U50 domain associated with the transition from the "strong" to "weak" actomyosin. ADP nucleotide state. We fit the fluorescence transients to kinetic equations 1–6 defined previously 7 and used in our previous report 23, which allowed us to determine rate constants k−pocket, k+pocket, k−ligand at each temperature. The values for these rate constants are reported as the average from the association and dissociation experiments (Table 1), demonstrating relatively good consistency between both sets of experiments. The fluorescence transients were also fit to kinetic schemes using Kintek explorer and the fits yielded rate constants that matched well with fits performed using analytical equations. The fluorescence transients in the absence of Mg (4 mM EDTA) were dominated by a fast fluorescence decrease that was ~10-fold faster than in the presence of Mg (Figure 4B). We observed a slow fluorescence decrease (~0.5 s−1) that was a small component of the total fluorescence change (~5%) at lower temperatures (4–15°C). We attribute the slow transition to an off-pathway intermediate since the predicted rate constant for k−pocket in the absence of Mg is much faster (14.8±0.2 s−1) (see Figure 6B). The slow fluorescence decrease could also be due to non-specific interactions of mantdADP and MV FlAsH.</p><!><p>We examined conformational changes in acto-MV FlAsH during mantdADP release as described above in the presence of the entire range of Mg concentrations (0.5 – 10 mM MgCl2) at 25°C (Figure 5). A complex of acto-MV FlAsH.mantdADP was mixed with saturating ATP (final concentrations: 0.25 μM MV FlAsH, 0.5 μM actin, 10 μM mantdADP, 1 mM ATP) in K50 buffer containing differing amounts of MgCl2. The fluorescence transients of the FRET signal were fit to a bi-exponential function at all Mg concentrations (Figure 5A & C). We found that the fast phase and slow phases of mantdADP release were both altered by Mg concentration, with the fast phase more steeply dependent on Mg. The relative amplitudes of the fast and slow phases were also dependent on Mg, with the fast phase more populated at low Mg concentrations and the fast/slow amplitude distribution similar at free Mg concentrations 0.9 mM and above. We determined the rate constants k−pocket, k+pocket, k−ligand at each Mg concentration (25°C) as described above (Figure 6 A & B).</p><p>To examine the role of the switch II region in Mg-dependent ADP release, similar experiments were performed with the mutant MVG440A FlAsH 24 in the presence of actin. The G440A mutation disrupts the rotation of switch II which prevents closure of the NBP and uncouples the nucleotide and actin binding regions in myosin V 13. The fluorescence transients of mantdADP release from MVG440A FlAsH showed two phases, while the slow phase was 10-fold slower than wild-type MV FlAsH (Figure 5 B & D). It is unclear if the slow phase represents the strong-to-weak ADP transition, an off pathway intermediate, or non-specific interactions. The release from the weak actomyosin. ADP (k−ligand) state was similar to wild-type in the absence of Mg but was accelerated 3-fold at saturating Mg. Thus, the ability of Mg to slow the rate constant for ADP release from the weak state is attenuated in MVG440A FlAsH.</p><!><p>Kinetic analysis allowed for determination of the rate and equilibrium constants associated with the formation of the weak actomyosin. ADP state (Kligand) and transition into the strong actomyosin. ADP state (Kpocket) 7, 23. Figure 6A demonstrates the dependence of Kpocket on the Mg concentration which increases to a value of 0.33 at 0.9 mM free Mg and above. The ADP release rate constant for myosin V was found to be similar with mantdADP and unlabeled ADP 32, 35 and this is the rate limiting step in the actin-activated ATPase35 and in vitro motility36 assays. Therefore, it is reasonable to examine the correlation between the steady-state assays (Figure 1) done with unlabeled ATP to the transient kinetic results performed with mantdADP. A comparison of the k−pocket rate constant and the kcat for ATPase as a function of Mg demonstrates these rates are very similar in the range of MgCl2 concentrations analyzed (0.5 – 10 mM). We also demonstrate that k−ligand is at least two-fold faster than k−pocket in this Mg concentration range (Figure 6B). Our results demonstrate that the k−pocket step correlates well with the rate limiting step in the ATPase cycle which we also concluded from our temperature dependent studies 23. By fitting the Mg dependence of k−pocket to the equation (Eq. 14) described by Rosenfeld et al. 6 we determined the value of k−pocket in the absence of Mg (k−pocket = 14.8 ± 2.0). This equation also allows estimation of the Mg affinity in the actomyosin V strong (KD.Mg = 0.9 ± 0.4 mM) and weak (KD.Mg = 0.6 ± 0.3 mM) ADP binding conformations, while the affinity of Mg for free ADP (KD = 0.35±0.3 mM) was previously determined7. By the principle of detailed balance, the value for Kpocket and k+pocket in the absence of Mg was estimated to be 0.28 and 4.2 s−1, respectively. The estimated values for Kpocket and k+pocket are not in agreement with our results that demonstrate the slow phase was not present in the dissociation and binding experiments. Thus, it is likely that there are multiple biochemical states in the absence of Mg.</p><!><p>The thermodynamic parameters associated with the Kligand and Kpocket steps were calculated from the van't Hoff plots (Figure 6C and Table 3). It should be noted that the values for Kligand are reported under standard state conditions (1M concentration of reactants) and therefore the value for k+ligand is likely an overestimate. In addition, previous studies demonstrated that Kligand inlcudes two steps, the formation of the collision complex between actomyosin and mantdADP followed by the transition into the weak actomyosin. ADP state7, 37. Therefore, the calculated free energy values for Kligand should be interpreted as a relative comparison, while the thermodynamic analysis provides information about the role of Mg in this biochemical transition. The free energy change associated with Kligand in the presence of Mg was dominated by the enthalpic component in the presence of 2 and 10 mM MgCl2. Interestingly, in the absence of Mg (4 mM EDTA) we found that the enthalpic component was dramatically reduced while there was a large positive entropic component associated ADP binding. We found that the thermodynamics of the Kpocket step were entropy driven and quite similar at 2 and 10 mM MgCl2, as well as similar to our previous measurements at 1 mM MgCl223.</p><!><p>The time-resolved anisotropy measurements demonstrate that the FlAsH fluorophore has a rotational correlation time on the ns time scale (Table 4). There results suggest that although the FlAsH fluorophore is fairly restricted in is dynamics because of its attachment to the tetracysteine motif, it has some dynamic motion that will randomize the orientation factor. We also determined that there was no difference in the time-resolved anisotropy parameters in comparing the acto-MV. ADP and acto. MV states, which suggest the local environment of the FlAsH fluorophore does not change in the strong and weak ADP binding states.</p><!><p>Many studies have focused on the ADP release steps of the actomyosin ATPase cycle, as these steps have been linked to strain dependent processive walking in myosin V 38, 39 and the detachment limited model of muscle contraction in myosin II 19. The current study focuses on how Mg impacts the structural changes associated with the ADP release steps in actomyosin V. We find that the actin-activated ATPase and motile properties of both monomeric and dimeric myosin V are similarly dependent on Mg. We demonstrate that high Mg impacts ADP release by slowing the rate-limiting isomerization of the NBP, which is associated with movement of the U50 domain. Additionally, we find that the release of ADP from the "weak" ADP affinity state is Mg dependent as was reported previously 6, 7. FRET results indicate a more dramatic temperature dependent distance change in the absence of Mg, suggesting Mg is essential for stabilizing the "strong" ADP affinity conformation. Overall, our results indicate that active-site Mg coordination can impact key structural changes that are critical for the motile and force generating properties of myosin V.</p><!><p>Mg is coordinated in the NBP with highly conserved structural elements. S218 of switch I and T170 of the P-loop coordinate Mg directly while D437 of switch II coordinates the Mg via a water molecule 5 (Figure 7). Our results suggest Mg coordination by these structural elements is likely important for stabilizing the "strong" ADP affinity NBP conformation. It has been proposed that strong actin binding causes switch I to favor an open conformation, which perturbs the coordination of Mg and initiates structural rearrangements that lead to the release of ADP 22. The crystal structure of myosin V in the presence of ADP has no Mg present and both switch I and II adopt an open conformation 5. Such an active-site conformation is also seen in the crystal structure of nucleotide free myosin V, which fits well into to the rigor actomyosin complex determined by cryo-electron microscopy 40. Our results show that the conformation of the U50 domain, which is proposed to be dependent on the position of switch I 23, is in turn dependent on Mg. Our previous work has demonstrated that switch II also plays a role in stabilizing a closed NBP conformation and is involved in the allosteric communication between the nucleotide and actin binding regions 24. Nagy et al 8 found that mutations in switch II can disrupt the Mg dependent ADP release mechanism. Our results further demonstrate the role of switch II in ADP release, since the G440A mutant 41 disrupts formation of the strong ADP affinity state even at 10 mM MgCl2 (Figure 5B&D). Because the G440A mutation prevents a closed switch II conformation it may indirectly impact the coordination of Mg by preventing D437 of switch II from properly coordinating Mg through a water molecule. Moreover, the G440A mutation may also impact the hydrogen bond between Y439 of switch II and R219 of switch I 8 that could potentially alter the direct Mg coordination by S218 of switch I (Figure 7). Also, in kinesin the interaction between switch I and switch II and their coordination of Mg and surrounding water molecules is critical for Mg and ADP release. Switch II in kinesins has been shown to undergo a large movement during Mg release 42. Since the G440A mutation precludes the rotation of switch II, we expected a complete insensitivity to Mg in the ADP dissociation experiments (Figure 5B&D). However, we only see a partial insensitivity, and we attribute this observation to the fact that apart from the indirect coordination of Mg via switch II, Mg is also directly coordinated via residues of P-loop and switch I which may play a role in the modest suppression of ADP release at higher Mg in the G440A mutant. The current study and previous work8, 24 that examined the impact of switch II mutations implies that the Mg-dependence of the structural changes we observe are attributed to Mg binding in the active site, while we cannot rule out that Mg binding to another allosteric site in myosin could alter key structural transitions.</p><!><p>Our earlier work found that the strong-weak ADP binding equilibrium monitored by FRET is temperature dependent and provides an indication of the distance change associated with the two conformations 23. Scheme 2 demonstrates the pathway for ADP release from actomyosin, which indicates the Mg free (lower) and Mg saturated (upper) pathways. In current study we investigated the impact of Mg on FRET as a function of temperature. In the presence of Mg, the kinetic studies fit well to Scheme 2 (top) in which Kpocket determines the population of the weak (low FRET) and strong (high FRET) ADP binding states. In the absence of Mg, the kinetic measurements of both mantdADP binding and release indicate that actomyosin V is dominated by a single conformation at all temperatures (Figures 2C, 3C&D, 4B&C). At lower temperatures the FRET distance in the presence and absence of Mg is similar, while at higher temperatures the difference dramatically increases (~5Å). These results can be explained if the weak ADP binding conformation is highly dynamic in the absence of Mg. This highly flexible state allows some high FRET conformations, relatively close distances between the mant-FlAsH fluorophores, to be populated on the nanosecond timescale even though the NBP is in a weak ADP binding conformation. The transitions into the high FRET conformations are not seen in the kinetic measurements because they are too rapid and ensemble averaged. Interestingly, the high FRET conformations are more populated at low temperature indicating the structure is more flexible at low temperature. It is possible that there is a hinge region that becomes more flexible at lower temperatures, similar to what is found in cold-denaturation 43–45. As temperature increases in the absence of Mg the flexible region becomes more rigid and only the low FRET states are populated. The thermodynamic analysis supports the hypothesis that the weak ADP binding state is highly flexible in the absence of Mg. We find that ADP binding occurs with a large positive entropy, suggesting enhanced conformational entropy, only in the absence of Mg. Hence, Mg coordination is required for formation of the strong ADP affinity state and may be coupled to the flexibility of the U50 domain.</p><!><p>Two previous studies examined the influence of Mg on the kinetics of the ADP release steps in myosin V 6–8. These studies examined the FRET signal from internal tryptophan residues to mantADP or mantdADP. Our results are in good general agreement with these studies in that they concluded that Mg strongly influences the rate constant for ADP release from the weak ADP binding state 6, 7. Our FRET results with the mant-FlAsH pair suggest that Mg coordination enhances the formation of the switch I closed NBP conformation, as originally proposed by Rosenfeld et al.6. In addition, our results demonstrate directly that the Mg dependent change in the NBP is coupled to the conformation of the U50 domain. Interestingly, Hannemann et al.7 also found that there was a structural change in the actin binding region detected by a difference in the degree of pyrene actin quenching in the two actomyosin. ADP states. The probability of Mg being released prior to the release of the cation-free ADP species was also proposed by Rosenfeld et al. 6. Our results are in agreement with Rosenfeld et al.6 in that Mg exchange can occur in both the strong and weak ADP binding states, which allows Mg to influence both the Kpocket and Kligand steps.</p><p>Our current results indicate that Mg can alter the rate-limiting conformational step wherein the NBP goes from a strong to weak ADP binding state (k−pocket) prior to the release of ADP (Figure 6A). The maximal ATPase rate and in vitro motility sliding velocities as a function of Mg follow a similar trend as the k−pocket rate constant (Figures 1 & 6B). Therefore, our results favor a mechanism in which Mg can dissociate from the strong ADP binding state, which accelerates the transition into the weak ADP binding state (k−pocket) and results in faster ADP release, maximal ATPase activity, and in vitro motility.</p><!><p>Many enzymes and cellular functions are known to be dependent on Mg, the second most abundant cation in the cell. The concentration of Mg inside cells is tightly regulated while large fluxes of Mg across the cell membrane have been reported 17, 46. The cytosolic free Mg levels are different in different cell types and they range between 0.8–1.2 mM. A recent in vitro study 47 with dynein demonstrated a reduction in its processivity at higher Mg concentrations while, at lower Mg concentrations, there was an enhancement in dynein processivity. This work speculated on the in vivo role of Mg as a switch to regulate the processivity of motor proteins like dynein. Our study shows significant changes in the functional properties of both monomeric and dimeric myosin V in the physiological Mg concentration range. The processive mechanism of dimeric myosin V is tightly coupled to strain sensitivity and mechanical gating between its two heads plays a critical role in this process. It is hypothesized that the ~50-fold difference in the rates of ADP release from the lead and trail heads of myosin V is associated with the pre- and post-powerstroke conformation of the lever arm 48, 49. Mg may play an important role in a strain dependent communication pathway between the lever arm and the NBP which alters the ADP release rates in response to strain. Interestingly, a recent study found that in kinesin the metal ion binding site can be altered to allow manganese binding which allows the enzymatic and motile properties of kinesin to be modulated by the presence of manganese9. Therefore, understanding the mechanism of metal ion regulation of motor proteins could be utilized as a mechanism for specifically altering the in vivo activity of motors and for designing motor-based nanodevices. Future studies on myosin will focus on the impact of Mg on lever arm swing and its impact on strain dependent ADP release. Overall, the current study suggests a central role for Mg in mediating the force generating and motile activities of myosin V, which provides a framework for revealing the conserved structural mechanism of the load dependent ADP release in myosin motors.</p>

PubMed Author Manuscript

Synthesis of Multilamellar Walls Vesicles (MLWV) Polyelectrolyte Surfactant Complexes (PESCs) from pH-Stimulated Phase Transition Using Microbial Biosurfactants

Multilamellar wall vesicles (MLWV) are an interest class of polyelectrolyte-surfactant complexes (PESCs) for the wide applications ranging from house-care to biomedical products.If MLWV are generally obtained by a polyelectrolyte-driven vesicle agglutination under pseudoequilibrium conditions, the resulting phase is often a mixture of more than one structure. In this work, we show that MLWV can be massively and reproductively prepared from a recentlydeveloped method involving a pH-stimulated phase transition from a complex coacervate phase (Co). We employ a biobased pH-sensitive microbial glucolipid biosurfactant in the presence of a natural, or synthetic, polyamine (chitosan, poly-L-Lysine, polyethylene imine, polyallylamine). In situ small angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) show a systematic isostructural and isodimensional transition from the Co to the MLWV phase, while optical microscopy under polarized light experiments and cryo-TEM reveal a massive, virtually quantitative, presence of MLWV. Finally, the multilamellar wall structure is not perturbed by filtration and sonication, two typical methods employed to control size distribution in vesicles. In summary, this work highlights a new, robust, non-equilibrium phase-change method to develop biobased multilamellar wall vesicles, promising soft colloids with applications in the field of personal care, cosmetics and pharmaceutics among many others.

synthesis_of_multilamellar_walls_vesicles_(mlwv)_polyelectrolyte_surfactant_complexes_(pescs)_from_p

Introduction<!>Chemicals<!>Preparation of stock solutions<!>Preparation of samples<!>Polarized Light Microscopy (PLM)<!>Cryogenic transmission electron microscopy (cryo-TEM)<!>Results<!>Quantitativity and size control

<p>Polyelectrolytes and surfactants may assemble into complex structures known as polyelectrolyte-surfactant complexes (PESCs). When these compounds are oppositely charged, their self-assembly process is mainly driven by electrostatic interactions and it results in the formation of aggregates, which have a broad range of applications in biological materials, [1][2][3][4][5] drug delivery, [6][7][8] surface modifications, 9 colloid stabilization 10 and flocculation 11 and consumer health-care products. The rich mesoscopical and structural organisation of surfactants combined with the electrostatic interactions with polyelectrolytes give rise to a wide range of structures and phases. [12][13][14][15][16][17] Many works reported cubic or hexagonal mesophases 15,16 but also a number of micellar-based structures: pearl-necklace morphologies, 2,18,19 interpenetrated polyelectrolytewormlike/cylindrical micelles network, 18,[20][21][22] spheroidal clusters composed of densely packed micelles held by the polyelectrolyte, the latter known as complex coacervates when they form a liquid-liquid phase separation. 18,23,24 Very interesting PESCs structures are formed when the surfactant forms low curvature vesicular morphologies. It is in fact generally admitted that modifying vesicles by the addition of polyelectrolytes is an interesting, cheap and simple approach to obtain nanocapsules, 22 which are good candidates to be used as versatile delivery systems, 18,22 like gene delivery, 1,21,25,26 or as MRI contrast agents. 27 One of the first PESCs vesicular systems has been reported more than 20 years ago in DNA-CTAB (cetytrimethylammonium bromide) systems, which were the precursors of a number of carriers for gene transfection and often referred to as lipoplexes, when cationic lipids replace surfactants in DNA complexation. 28,29 If the term lipoplexe supposes the use of nucleic acids as complexing agents, similar structures, often addressed to as onion-like structures 30 or multilamellar vesicles, 13 were observed using both lipids and surfactants complexed by a wide range of polyelectrolytes. However, multilamellar, or onionlike, vesicles are rather characterized by single-wall membranes concentrically distributed from the outer to the inner core of the vesicle. Lipoplexes, on the contrary, are vesicular objects with a large lumen and a dense multilamellar wall. For this reason, in this work we employ the name multilamellar wall vesicles (MLWV).</p><p>The mechanism of formation of MLWV was addressed by several authors, but a common agreement is not achieved, yet. Several works propose that the lipid:polyelectrolyte ratio controls the fusion of single-wall vesicles MLWV, 18,28,[31][32][33][34] while others rather observe vesicular agglutination under similar conditions. [35][36][37] In fact, a general consensus has not been found and a multiphasic system including agglutinated vesicles and MLWV are actually observed. 38 The question whether or not MLWV, and PESCs in general, are equilibrium structures and how they DOI: 10.26434/chemrxiv.12058929 are formed is still open, especially when they are prepared under non-equilibrium conditions. 18 To the best of our knowledge, the only works exploring a stimuli-induced approach in the synthesis of MLWV in particular, and PESCs in general, were proposed by Chiappisi et al.. 20,39 However, the pH variation in these work was still performed under pseudo-equilibrium conditions with equilibration times ranging from 2 to 15 days for each pH value.</p><p>In a recent work, we have explored a Co-to-MLWV phase transition under nonequilibrium conditions using a continuous variation in pH, 40 as illustrated by Figure 1. We could show that in the presence of G-C18:1, an acidic microbial glycolipid biosurfactant, 41,42 and poly-L-lysine (PLL), a cationic polyelectrolyte (PEC), the pH-stimulated micelle-to-vesicle phase transition of the lipid drives a continuous, isostructural and isodimensional, transition between complex coacervates and multilamellar wall vesicles. In the present work, we generalize the method of preparing MLWV through a phase transition approach performed under non-equilibrium conditions and we show its performance in comparison to the more accepted method of vesicular agglutination. We show that this method can be applied to a broader set of polyelectrolytes and we explore in more detail the structure and size control of MLWV.</p><!><p>In this work we use microbial glycolipids G-C18:1, made of a single β-D-glucose hydrophilic headgroup and a C18 fatty acid tail (monounsaturation in position 9,10). From alkaline to acidic pH, the former undergoes a micelle-to-vesicle phase transition. 41 The syntheses of glucolipid G-C18:1 is described in Ref 43 and 42 , where the typical 1 H NMR spectra and HPLC chromatograms are given. The compound used in this work have a molecular purity of more than 95%.</p><p>The polyelectrolytes used in this work are chitosan, obtained from the deacetylation of chitin from crusteans' shells, poly-L-lysine, widely used in biomedical field, and polyethylenimine. Chitosan oligosaccharide lactate (CHL) (Mw ≈ 5 KDa, pKa ~6.5) 44 with a deacetylation degree >90%, poly-L-lysine (PLL) hydrobromide (Mw ≈ 1-5 KDa, pKa ~10-10.5), 45 polyallyllamine hydrochloride (PAH) (Mw ≈ 1-5 KDa, pKa ~9.5), 45 polyethylenimine (PEI) hydrochloride (linear, Mw≈ 4 KDa, pKa ~8) 46 and gelatin (type A, from porcine skin, Mw ≈ 50-100 KDa, isoelectric point 7-9) are purchased from Sigma-Aldrich. All other chemicals are of reagent grade and are used without further purification.</p><!><p>G-C18:1 (C= 5 mg.mL -1 , C= 20 mg . mL -1 ), CHL (C= 2 mg . mL -1 ), PLL (C= 5 mg . mL -1 , C= 20 mg . mL -1 ), PEI (C= 5 mg . mL -1 ), PAH (C= 2 mg . mL -1 ) and gelatin (C= 5 mg . mL -1 ) stock solutions are prepared by dispersing the appropriate amount of each compound in the corresponding amount of Milli-Q-grade water. The solutions are stirred at room temperature (T= 23 ± 2 °C) and the final pH is increased to 11 by adding a few μL of NaOH (C= 0.5 M or C= 1 M).</p><!><p>Samples are prepared by mixing appropriate volume ratios of G-C18:1 stock solutions at pH 11 and cationic polyelectrolyte (PEC) stock solutions, as defined in Table 1. The final total volume is generally set to V= 1 mL or V= 2 mL, the solution pH is about 11 and the final concentrations are given in Table 1. The pH of the mixed lipid-PEC solution is eventually decreased by the addition of 1-10 µL of a HCl solution at C= 0.5 M or C= 1 M. The rate at which pH is changed is generally not controlled although it is in the order of several µL . min -1 .</p><p>Differently than in other systems, 47,48 we did not observe unexpected effects on the PESC structure to justify a tight control over the pH change rate. employed using an energy of E= 12 keV and a fixed sample-to-detector (Eiger X 4M) distance of 1.995 m. For all experiments: the q-range is calibrated to be contained between ~5. 10 -3 < q/Å -1 < ~4.5 . 10 -1 ; raw data collected on the 2D detector are integrated azimuthally using the inhouse software provided at the beamline and so to obtain the typical scattered intensity I(q)</p><p>profile, with q being the wavevector (𝑞 = 4𝜋 sin 𝜃 𝜆 , where 2θ is the scattering angle and λ is the wavelength). Defectuous pixels and beam stop shadow are systematically masked before azimuthal integration. Absolute intensity units are determined by measuring the scattering signal of water (Iq=0= 0.0163 cm -1 ).</p><p>The same sample experimental setup is employed on both beamlines: the sample solution (V= 1 mL) with the lipid and PEC at their final concentration and pH ~11 is contained in an external beaker under stirring. The solution is continuously flushed through a 1 mm glass capillary using an external peristaltic pump. The pH of the solution in the beaker is changed using an interfaced push syringe, injecting microliter amounts of a 0.5 M HCl solution. pH is measured using a micro electrode (Mettler-Toledo) and the value of pH is monitored live and manually recorded from the control room via a network camera pointing at the pH-meter located next to the beaker in the experimental hutch. Considering the fast pH change kinetics, the error on the pH value is ± 0.5.</p><!><p>PLM experiments are performed with a transmission Zeiss AxioImager A2 POL optical microscope. A drop of the given sample solution is deposited on a slide covered with a cover slip. The microscope is equipped with a polarized light source, crossed polarizers and an AxioCam CCD camera.</p><!><p>Cryo-TEM experiments are carried out on an FEI Tecnai 120 twin microscope operated at 120 kV and equipped with a Gatan Orius CCD numeric camera. The sample holder is a Gatan Cryoholder (Gatan 626DH, Gatan). Digital Micrograph software is used for image acquisition.</p><p>Cryofixation is done using a homemade cryofixation device. The solutions are deposited on a glow-discharged holey carbon coated TEM copper grid (Quantifoil R2/2, Germany). Excess solution is removed and the grid is immediately plunged into liquid ethane at -180°C before transferring them into liquid nitrogen. All grids are kept at liquid nitrogen temperature throughout all experimentation. Images were analyzed using Fiji software, available free of charge at the developer's website. 49</p><!><p>In recent publications, 40,50 we have explored the complex coacervation between microbial glycolipids and cationic polyelectrolytes (PEC). For this reason, this aspect is only briefly shown in here. Cryo-TEM images presented in Figure 2 show the structure of PECcomplexed G-C18:1 complex coacervates above pH 7. Irrespective of the selected PEC, all systems show spheroidal colloids of variable size in the 100 nm range. One can identify two types of structures, both typical of complex coacervates: 23,24,50,51 dense aggregated structures, shown in Figure 2a,c and very similar to what was found by us 40,50 and others, 23 are attributed to dehydrated, densely-packed, micelles tightly interacting with the polyelectrolyte; a biphasic medium composed of spheroidal, poorly-contrasted, colloids embedded in a textured medium describe hydrated structures of less defined composition, probably describing an intermediate of coacervation step. The latter were also reported by us 40,50 and others. 51,52 In all cases, the complex coacervate phase (Co) is a PESC forming in the micellar region of the surfactant's phase diagram and having the specificity of a liquid-liquid phase separation, 18,24 compared to other supramicellar PESCs undergoing a solid-liquid phase separation. 18 The difference between dense and poorly-contrasted structures is PEC-independent and it is more related to the stage of coacervation. At an early stage, colloids with a relatively low electron density form and coexist with a rich micellar phase. Free micelles progressively interact with residual polymer chains. At a later, entropy-driven (dehydration and counterion release), 53 stage of coacervation, droplets with a higher electron density massively form.</p><p>Unfortunately, neither the texture of the particles nor their internal structure can be easily controlled as they strongly depend on the type of PEC, its stiffness, charge density, stage of coacervation and even kinetics. For these reasons, isolating a specific structure in a Co phase can be challenging and we have ourselves found coexisting dense and poorly-contrasted structures, 40 thus preventing any reasonable structure-composition generalization concerning the images presented in Figure 2. At pH below 7, vesicular structures with multilamellar walls (MLWV phase) are observed by cryo-TEM for all PEC samples (Figure 3). These structures are closely-related to a lipoplexe-type phase rather than to an onion-like phase, whereas the latter is composed of concentric single-wall vesicles, while the former keeps a free lumen and a thick multilamellar wall. 28 Figure 3 also shows a strong packing of the multilamellar walls as well as a strong interconnection between adjacent vesicular objects, in agreement with lipoplexes and other multilamellar wall vesicles reported in the literature. 22 The walls are constituted of alternating sandwiched layers composed of tightly packed polyelectrolyte chains and interdigitated layers of G-C18:1. 40 d-spacing can be directly estimated from cryo-TEM images (Figure 3e,f) and we find a set of values of d= 33.7 ± 4.95 Å for the PLL system and d= 31.6 ± 3.00 Å, 25.3 ± 4.60 Å and 41.1 ± 0.30 Å respectively for CHL, PAH and PEI systems. Within the error, these values are compatible with interdigitated G-C18:1 layers, [40][41][42] of which the thickness can be estimated to be around 30 Å by applying the Tanford relationship, 54 but also close to what is classically recorded for lipoplexes. 21,22,32 One should note an interesting feature on Figure 3d: the multilamellar walls of the PECSs involving PEI appear less tightly packed and more disordered DOI: 10.26434/chemrxiv.12058929 9 than for other PESCs. This effect may be a consequence of the freezing protocol, although all samples have been frozen in the same way, or related to the specific use of PEI. Another hypothesis, which does not exclude the previous one, could be that the local disorder results from electrostatically induced undulations of the membrane, as already reported on lamellar DNA-lipid complexes. 55 Cryo-TEM images recorded on the Co (Figure 2) and MLWV (Figure 3) phases show that the Co-to-MLWV transition is a general property of G-C18:1 PESCs: it strictly depends on the lipid phase behavior, while the polyelectrolyte only guarantees the cohesion between the lipid membranes. We highlighted elsewhere 40 4a), a broad correlation peak is observed at about q= 0.17 Å -1 for all lipid:PLL ratios, where the peak can be more pronounced either with concentration (B profile) or lipid:PLL ratio (A profile). SAXS profiles B and C were previously assigned to complex coacervates, and more details on the structure of the Co phase can be found in Ref. 40 .</p><p>In similar systems, the slope at low q was shown to be indicative of the shape of the PESC; 39 here, the slope is below -3. If such values are typical of fractal interfaces, 56,57 we cannot unfortunately draw any conclusion on the structure of the complex coacervates, most likely because the Co phase in these systems is heterogeneous. 40 Below pH 7 (Figure 4b), a sharp diffraction peak and its first harmonics are visible respectively around q1= 0.17 Å -1 and q2= 0.34 Å -1 , characteristic of the (100) and ( 200) reflections of a lamellar order in the walls, described previously and shown in Figure 3. The dspacing of 37 Å is in agreement with the ones deduced from cryo-TEM (Figure 3e,f). Similar results are obtained at different lipid:PLL ratios (Figure 4c,d) but also for other PEC. All pH-resolved in situ contour plots in Figure 4 show three common features: 1) the Co-to-MLWV transition between pH 8 and 7, where q1 and q2 refer to the first and second order peaks of the lamellar wall; 2) a low-q shift of q1 and q2 when pH decreases to about 4.5, indicating a swelling of the lamellar period, and 3) a loss of the signal between about pH 4.5 and pH 3.5, below which a constant peak at higher q-values (generally around q= 0.2 Å -1 ) appears. These phenomena were quantitatively described in more detail in Ref. 40 and will only be summarized hereafter.</p><p>When fully deprotonated at basic pH, G-C18:1 is in a high curvature, micellar, environment (Co phase) at basic pH. This state, represented by the drawing superimposed on Figure 4d, is proven by both cryo-TEM and the broad correlation peak at about q0= 0.17 Å -1 .</p><p>After crossing the transition pH range between 8 and 7, the number of negative charges decreases and G-C18:1 is entrapped in a low-curvature, interdigitated layer, environment. The continuity between q0 and q1 strongly suggest an isostructural and isodimensional transition between the micelle and membrane configutations, without any loss of the interaction with the polyelectrolyte. This is also sketched on Figure 4d. When the pH is decreased further, the COOH content increases and thus the membrane charge density decreases. The interlamellar distance consequently increases due to the repulsive pressure exerted by the charged polyelectrolyte, which undergoes hydration and increase internal electrostatic repulsion. 2,59,60 When hydrogenation of carboxylate groups reach a certain extent, attractive interaction with PLL can no longer hold the membranes together and MLWV then lose their long-range lamellar order, which results in their complete disruption and the concomitant expulsion of PLL. Below pH 3, this mechanism leads to the precipitation of a polyelectrolyte-free lamellar, L, phase, which is also observed for PEC-free G-C18:1 solutions.</p><p>A closer look at the experiments in Figure 3 indicates two additional features. The pH stability domain of the MLWV phase seems to vary with the lipid:PLL ratio. Comparison of Figure 3c and Figure 3d, respectively recorded at lipid:PLL= 1:1 and 1:2 reveal that the q1 peak of the MLWV phase is observed between pH 8 and 7. At the 1:2 ratio the MLWV phase starts at about pH 8 while at the 1:1 ratio the MLWV phase is only visible at pH is below 7. At higher concentrations (C= 10 mg . mL -1 ), but still for a 1:1 ratio, the stability frontier seems to be shifted at pH of about 7.5. 40 Although we do not have enough data to draw a general trend, it is wellknown that the lipid:polyelectrolyte ratio reflects the negative:positive charge ratio and for this reason it has a direct impact on the electroneutrality, thus affecting a number of structural features of PESCs: the wall thickness of the multilamellar structure, 20,61 the PESC morphology and colloidal stability. 18 For instance, order is noticeably improved when the charge ratio approaches 1:1, 62 and micelle-polyelectrolyte complex coacervation can be favoured or not. 63 This ratio is particularly crucial to control the properties of the lipoplexes and thus their applications: lipid/DNA ratio was reported to influence both the formation of lipoplexes and the release of DNA 64 and gene transfer activity. 65 Many authors have shown that the lipid:polyelectrolyte ratio actually controls the formation of MLWV structures 18,28,[31][32][33][34] over agglutinated single-wall vesicles, [35][36][37] but in fact it is more likely that a general consensus has not been found, yet, and reality often consists in a mixtures of MLWV and agglutinated DOI: 10.26434/chemrxiv.12058929 12 vesicles, 38 although many authors do not specify it. One of the reasons that could explain such discrepancy is the parallel influence of several other parameters like the charge density on both the lipid membrane and in the polyelectrolytes, the rigidity of the latter, the bending energy of the lipid membrane, the ionic strength and so on. 14,18 In the present case, it is important to note that: 1) G-C18:1 forms a stable MLWV phase with all PEC tested in this work and of different origin (biobased vs. synthetic) and rigidity. 2) Multilamellar wall vesicles are stable in the neutral pH range, which can be a good opportunity for applications in the biomedical field, for</p><p>instance.</p><p>An interesting remark concerns the long-range order inside the vesicular multilamellar walls. The width of the lamellar peak around q ~0.2 Å -1 is more than ten times larger for the CHL (Figure 4e, Δq ~3.10 -2 Å -1 ) than the PLL (Figure 4c,d, Δq ~2.10 -3 Å -1 ) system, either suggesting an average smaller size of the lamellar domains or a poorer lamellar order in the case of the MLWV obtained from CHL. The reason behind such difference could be the bulkiness and stiffness of CHL with respect to PLL, 31 but one should recall from Figure 2 and related discussion that [G-C18:1 + CHL] solutions do not form an extensive Co phase. We have already made the hypothesis that the Co phase is necessary to form the MLWV phase, 40 and we will reinforce this assumption in the next part of this work. The data collected so far show that G-C18:1 interacts with all polyelectrolytes tested in this work and that its micelle-to-vesicle phase transition drives the Co-to-MLWV transition. As one could reasonably expect, the strong electrostatic interactions between the positively-charged PEC and negatively-charged G-C18:1 drives the PEC formation across the entire pH range. To test the solidity of the PESCs synthesis using G-C18:1 and polycations, we employ gelatin as a possible alternative polyelectrolyte and which could be interesting to prepare biobased PESCs. We use a commercial (Aldrich) source of gelatin type A, a natural protein of isoelectric point between 7.0-9.0, below which the charge becomes positive. Figure 5 shows pH-resolved in situ contour plots of gelatin and [G-C18:1 + gelatin] samples. The control gelatin sample in Figure 5a shows no specific contribution across the entire pH range between 0.1 < q / Å -1 < 0.4. Interestingly, the [G-C18:1 + gelatin] sample presented in Figure 5b does not show any signal either in the same pH and q range, except for the systematic signal of the lamellar, L, phase of G-C18:1 below pH 4. 40,41 Despite an expected positive charge density of gelatin, the in situ SAXS experiment shows no sign of the Co phase above pH 7, indicating that the charge density is probably too low to interact with negatively-charged G-C18:1 micelles. Although somewhat unexpected because interactions with negatively-charged sodium dodecyl sulfate micelles across a wide compositional and pH range were reported in other studies, 66 this result is not a surprise. What it is more interesting from a mechanistic point of view is the lack of the MLWV phase below pH 7. Given its isoelectric point, type A gelatin is positively charged below pH 7 and it is then expected to interact with G-C18:1 negative membranes.</p><p>In this work we have used a broad set of polyelectrolytes, of which the different chemical nature let us explore various aspects of their interactions with G-C18:1. If the nature of the polyelectrolyte (stiffness, charge density, …) is known to strongly affect the morphology and structure of PESCs, 14,31 in this work we show that: 1) when the Co and MLWV phases are formed, the structure of the corresponding colloidal structures is very similar, whichever the polyelectrolyte used, even if local phenomena like swelling or long-range order may vary from one polyelectrolyte to another.</p><p>2) The Co and MLWV phases are only obtained with polyelectrolytes with a net positive charge, that is polycations. 3) The MLWV phase is always preceded by the Co phase, which seems to be a necessary condition to drive the isostructural and isodimensional Co-to-MLWV transition. This phenomenon does not occur when gelatin is employed and where the MLWV phase is not observed. On the contrary, the MLWV phase is obtained for the CHL system, despite the fact that we do not have a proof by SAXS of the Co phase. To this regards, we must outline that the SAXS signal for the [G-C18:1 + CHL] system at basic pH is dominated by the precipitated CHL phase, which we think to be in major amount but not the only phase. Cryo-TEM shows the presence of an unknown fraction of complex coacervates, which we believe to be source of the MLWV phase at pH below 7. We also believe DOI: 10.26434/chemrxiv.12058929 14 that the higher disorder of the MLWV phase in the [G-C18:1 + CHL] system (broader first order diffraction peak compared to the PLL-derived MLWV in Figure 4e) could be attributed to the smaller fraction of the initial Co phase. In other words, the presence of a less ordered MLWV phase in the CHL system could then the indirect proof that probably a small fraction of the Co phase forms in the CHL system.</p><!><p>If the synthesis of PESCs involving vesicles and polyelectrolytes, and eventually forming MLWV, has long been addressed in the literature, 36,67,68 very few studies, if none, address the issue of quantitativity in relationship to the mechanism of formation. In particular, the synthesis of MLWV from a continuous isostructural phase transition from a coacervate phase has not been addressed before, because MLWV are generally obtained by mixing vesicles and polyelectrolytes in solution. 18,28,[31][32][33][34]36 If some authors state that the formation of MLWV is driven by the lipid:polyelectrolyte ratio, other authors show that a mix of agglutinated vesicles and MLWV are actually obtained. 37,38 Other procedures could probably be followed to increase this control when working with pre-formed vesicles, such as the insertion of the polymer into the hydrophobic vesicle bilayer, which was reported in the case of polycations bearing pendant hydrophobic groups. 36,69 However, it was found that such interaction could be accompanied by lateral lipid segregation, highly accelerated transmembrane migration of lipid molecules (polycation-induced flip-flop), incorporation of adsorbed polycations into vesicular membrane as well as aggregation and disruption of vesicles. 69 To evaluate the amount of MLWV with respect to agglutinated vesicles, we compare the sample obtained by continuous Co-to-MLWV phase transition with a sample obtained by the more classical approach consisting in mixing G-C18:1 single-wall vesicles and polyelectrolyte, the main one employed in the literature of MLWV. If SAXS can prove the presence of a multilamellar structure, it cannot be easily employed to quantify and discriminate between the two structures. For this reason, instead of SAXS, we evaluate the content of MLWV between the two methods of preparation by combining cryo-TEM with optical microscopy using crossed polarizers. If cryo-TEM can differentiate between agglutination and MLWV, its high magnification is poorly compatible with good statistics, unless a large number of images are recorded. On the contrary, optical microscopy using cross polarizers is the ideal technique to differentiate, on the hundreds of micron scale, between MLWV and agglutinated vesicles: multilamellar structures (but not single-wall vesicles) show a characteristic maltese cross DOI: 10.26434/chemrxiv.12058929</p><p>pattern 70 under crossed polarizers, found both in concentric lamellar emulsions 71 and in spherical lamellar structures. 72 Cryo-TEM of a samples obtained from a Co-to-MLWV phase transition was shown in Figure 3 and, as already commented above, they show a massive presence of vesicular structures having multilamellar walls, as also confirmed by the corresponding SAXS data presented in Figure 4. Figure 6 shows two representative microscopy images of a typical sample prepared with the same approach; images are collected under white (a,d) and polarized light with polarizers at 0°-90° (b,e) and 45°-135° (c,f). The system is characterized by a large number of vesicles highly heterogeneous in size but all below ~10 μm. Under polarized light and crossed polarizers the entire material displays a typical maltese cross colocalized with each vesicle.</p><p>Despite the aggregation of the vesicles, also observed with cryo-TEM, maltese crosses are welldefined and nicely separated and each identifying single multilamellar wall vesicles. The entire material displays such a characteristic birefringency, strongly suggesting a quantitative presence of MLWV. The experiment consisting in mixing acidic solutions (pH 3.8) of pre-formed G-C18:1 single-wall vesicles and PLL is shown in Figure 7. A preliminary investigation by optical microscopy results in a different behavior and distribution of signal with respect to the sample obtained through the Co-to-MLWV phase transition. Figure 7a shows representative images of a sample being constituted of aggregated objects, each of size below 1 μm, expected for G-C18:1 vesicles. 42 The corresponding images recorded using crossed polarizers (Figure 7b,d)</p><p>show a broad, undefined, birefringency associated to the aggregates with little, if no, content of maltese crosses. The featureless, generalized, birefringency signal suggests that MLWV are either not formed or they form in small amounts, in good agreement with the data presented by others. 37,38 This assumption is confirmed by cryo-TEM images recorded on the same system and showing a mixture of structures including agglutinated vesicles but also "cabbage-like" and multilamellar structures (Figure 7e-f).</p><p>The massive presence of MLWV structures obtained through the phase transition process compare to the mixture of structure obtained from a direct mixing of preformed vesiclespolyelectrolyte solutions confirms the crucial role of the complex coacervates in the formation of MLWV: coacervation seems to be a requirement to the extensive formation of vesicular structures with multilamellar walls. 40 This is also in agreement with the data obtained from the [G-C18:1 + gelatin] system presented in Figure 5 and prepared using the pH variation approach.</p><p>Also in that case, the absence of a complex coacervate phase had as a consequence the absence of the MLWV phase. An additional piece of evidence comes from the CHL system, in which the limited amount of the Co phase generates a more disordered MLWV phase. Combination of the data obtained with gelatin and employing the in situ pH variation with the data obtained by mixing vesicle and polyelectrolyte solutions at a given pH demonstrates the importance of the precursor Co phase during the phase change method in order to obtain a massive presence of MLWV structures. If the Co-to-MLWV phase transition is able to quantitatively produce MLWV, its main drawback is the poor control over their size distribution, as shown both by TEM and optical microscopy. To improve this point, we employed filtration (Figure 8a-c) and sonication (Figure 8d-f), these methods being known to efficiently control vesicles size distribution, 73 but unclear whether or not they have any deleterious impact on the MLWV structure. According to the cryo-TEM data in Figure 8a-c, filtration (pore size, φ= 450 nm) promotes the stabilization of colloidally-stable spherical MLWV, of which the diameter seems to be contained between 50 nm and about 300 nm, in agreement with the filter pore size. Concerning the effect of sonication, Figure 8d-f also shows a large number of spherical, un-aggregated, MLWV colloids, although the diameter appears to be bigger of several hundred nanometers if compared to the filtered sample. The cryo-TEM results are confirmed by intensity-filtered DLS experiments, presented in Figure 8g. The as-prepared sample (black curve) shows a MLWV distribution centered at 716 nm, while the filtered sample shows a distribution centered at 460 nm. To better evaluate the impact of sonication, we tested the influence of sonication time and according to DLS data (Figure 8g) we find that at t= 30' the size distribution is centered at higher diameter values and it is even broader than the as-prepared sample. Applying the same sonication conditions, but over a longer period of time (t= 1' or t= 1'30''), it is possible to reduce the MLWV diameter even if the size distribution is broader than the filtration approach, in agreement with the cryo-TEM data.</p><p>These experiments show that control of the size distribution of MLWV is possible using standard methods employed in liposome science without perturbing the multilamellar wall structure. Finally, when the membrane reaches neutrality, polymeric repulsion becomes strong enough to disassemble the lamellae. The polyelectrolyte will most likely be entirely solvated and at sufficiently low pH (< 3) the G-C18:1 precipitates in the form of a lamellar phase, possibly free of the polyelectrolyte, a behavior characteristic of the control lipid solution at the same pH.</p><p>We employ four polyelectrolyte, synthetic and natural and with different characteristic of rigidity and charge density (chitosan, poly-L-Lysine, polyethylene imine, polyallylamine); however, the nature of the polyelectrolyte does not seem to be a relevant parameter concerning the fate of the transition, as otherwise found for most PESCs. This may be explained by the strong proximity between the lipid and the polyelectrolyte throughout the isostructural Co-to-MLWV transition. If the method described in this work does not allow a tight control over the size distribution of MLWV, we also find that the multilamellar wall structure is stable against filtration and sonication, two common methods employed to control the size of vesicles. Last but not least, we show that if we employ the classical approach consisting in mixing pre-formed vesicles with a cationic polyelectrolyte solution at a given pH, we find a much broader structural diversity, including agglutinated single-wall vesicles, multilamellar but also cabbage-like structures, in agreement with previous literature studies.</p>

ChemRxiv

Visualizing 3D molecular structures using an augmented reality app

We present a simple procedure to make an augmented reality app to visualize any 3D chemical model.The molecular structure may be based on data from crystallographic data or from computer modelling. This guide is made in such a way, that no programming skills are needed and the procedure uses free software and is a way to visualize 3D structures that are normally difficult to comprehend in the 2D space of paper. The process can be applied to make 3D representation of any 2D object, and we envisage the app to be useful when visualizing simple stereochemical problems, when presenting a complex 3D structure on a poster presentation or even in audio-visual presentations. The method works for all molecules including small molecules, supramolecular structures, MOFs and biomacromolecules.

visualizing_3d_molecular_structures_using_an_augmented_reality_app

INTRODUCTION<!>THE HOW TO GUIDE: STEP 1: MAKING A 3D MODEL OF THE MOLECULE<!>NOTE : PERFORMANCE IMPROVEMENTS<!>Remove excess strutures:<!>CONCLUSION

<p>Conveying information about three-dimensional (3D) structures in two-dimensional (2D) space, such as on paper or a screen can be difficult. Augmented reality (AR) provides an opportunity to visualize 2D structures in 3D. Software to make simple AR apps is becoming common and ranges of free software now exist to make customized apps. AR has transformed visualization in computer games and films, but the technique is distinctly under-used in (chemical) science. 1 In chemical science the challenge of visualizing in 3D exists at several levels ranging from teaching of stereo chemistry problems at freshman university level to visualizing complex molecular structures at the forefront of chemical research. Visualization can be especially challenging since molecules are getting larger and more complex and span 3D. An elegant way to visualize molecules in 3D is to 3D print the desired structure, and protocols of how to do this starting from molecular structures have recently been described. 2 To describe the geometry and symmetry of complex molecules chemists are often forced to draw molecules in simplified or schematic ways and thus neglect information. One example of a highly complex molecule that is difficult to display in 2D is the Molecular Borromean Rings prepared by Stoddart and co-workers (Figure 1a). In their structural representation some atoms and labelling of atoms are omitted to simplifying the structure. 3 In the simplified 2D image bonds and atoms are overlapping and thus still make it difficult to visualize the geometry and symmetry. Many chemists even draw the same molecule twice in different formats in the same paper to better explain the connections of the different elements and its geometry. This is illustrated with the porphyrin nano-ball by Anderson (Figure 1b) and the supramolecular complex between biotin [6]uril and the iodide anion (Figure 1d). [4][5] It can be challenging to come up with a new synthetic route for complicated molecules such as Paclitaxel (Figure 1c) because it is hard to visualize how sterically congested regions effects each other. 6 For these types of problems described above, a simple way to visualize molecules in 3D would be beneficial. to view these structures in AR. The app can be found via the QR code or the link in Figure 2. a) The Borromean rings are shown in two different ways to emphasize the geometry and symmetry of it. b) The porphyrin nanoball is presented in two different ways to highlight the geometry and the connection of the different elements in the nanoball. c) Paclitaxel is complex molecule with many stereo centers. A 3D view of it helps to appreciate the complexity and may aid retrosynthetic analysis. d) A topview and sideview of the crystal structure of the biotin [6]uril. Download the app by following the link below (Figure 2) and see how the AR works.</p><p>In this contribution we describe how to make a simple AR app for a mobile device (e.g. phones and tablets) to visualize molecules in 3D. It is important to emphasize that this guide is made in such a way, that no programming skills are needed, and that only free software is used. When the app is made it is free to transfer the app via an USB cable from the computer to the mobile devices. However, if you want to publish the app in Google Play it requires a one-time payment to Google Play of 25 $. This guide must be followed tightly, because a series of programs are needed. When the AR app is made and transferred to a mobile device, a camera opens and it recognises an image. The image may be on a poster, in a book or on a screen. The recognition leads to a 3D model (of one or more molecules of your Journal 5/18/21 Page 4 of 14 own choice) is opening as a part of the real world through your mobile device. When all the software is installed correctly, the app is simple to make and can be used at a poster session or in classroom.</p><p>Download an example of the AR app (follow the link or QR code below) on your android mobile device and see how it works and what it looks like (in the real augmented world). Once the app is downloaded and opened, then point the camera at Figure 1 and 2 and a 3D model of the molecules will appear in AR.</p><p>Link to the AR app: https://play.google.com/store/apps/details?id=com.UniCPH.Android.MoleculAR P Figure 2. A 2D image of Biotin [6]uril. Download the app from the link below or the QR code and point the mobile device to see the structure in 3D through the camera. https://play.google.com/store/apps/details?id=com.UniCPH.Android.MoleculAR</p><!><p>If you don't have a specific molecule or just want to make the app to visualize a common molecule, then skip this step and go to "Step 2: Working with Jmol".</p><p>There are many programs that allow you to draw molecules in 3D. The most realistic 3D models for the given molecule are either from a crystal structure or a high level optimization calculation by using software such as "Gaussian". It is also possible to draw the molecule in chemical drawing software, such as "Chem3D" in the "ChemDraw" software package.</p><p>When the geometry of your molecule is satisfying, then save it at the desktop as a "mol"-file. Only the geometry of the molecule is needed to be correct at this state. The format of your molecule is added in the next step. If you want all the nitrogen atoms to be blue, then write "color nitrogen blue" or if you want the bonds to be black then write "color bonds black" etc.</p><p>When To publish an app to Google play a developer account is needed, this requires a one-time payment of 25 $ (2019). Find the website by searching "Google play developer console", pay for the account and log in.</p><p>A new app is created by clicking the "Create application" button and filling in the name and default language, finish by clicking "Create" and the "Store listing" menu opens. In this menu fill in the following information: 1. Short description, 2. Full description, 3. Upload an image to create the icon for the app, this image must have a resolution of 512x512. 4. Upload at least two screen shots. 5. Upload a "feature graphic" this image must have a resolution of 1024x500. The left hand navigation menu contains the two mandatory steps "Content rating" and "App content" both of these are questionaires pertaining to the nature of the content, complete these by filling in the required information. Now navigate to the "Pricing & distribution" and perform the following steps. 1. Choose "FREE" app. 2.</p><p>Select the countries that you want the app to be avaliable in. 3. Verify that you app complies with content guidelines and US export laws by checking the boxes in the "Consent" section. Finalise by pressing "save draft".</p><p>To publish the app navigate back to the App eleases section, press the "Edit release" button in the "Production track" section. Finalise by clicking the review button. Google play will now review that all the necesary information have been provide and the app complies with registration. This can take up to seven days.</p><!><p>If very large structures or slower android devices are used performance of the app can be a little sluggish. It is not withing the scope of this paper to describe the full set of rendering optimizations one can do in Unity but the following two hints should get you a good portion of the way.</p><p>Journal 5/18/21 Page 13 of 14</p><!><p>When Jmol is used to generate the obj-file, an addition sphere is created for each atom, even though these spheres is located at the same spot as the original atom sphere unity takes up processing power calculating positions of these. To remove these extra sphere open the unity project again. In the Hierarchy panel expand the hierachy until the structure is expanded, right click at the structure and select "Unpack prefab". Now scroll down to the children named "SprereXXX" where XXX is a integer, select them all and press "delete" this should not affect the 3D structure of the molecule but signigicantly increase the performance.</p><!><p>We have described how to make a simple augmented reality app to visualize any 3D chemical model using free softwares. The method works for all molecules including small molecules, supramolecular structures, MOFs and biomacromolecules.</p>

ChemRxiv

Non-Invasive, Real-Time Reporting Drug Release In Vitro and In Vivo

We developed a real-time drug-reporting conjugate (CPT-SS-CyN) composed of a near-infrared (NIR) fluorescent cyanine-amine dye (CyN), a disulfide linker, and a model therapeutic drug (camptothecin, CPT). Treatment with dithiothreitol (DTT) induces cleavage of the disulfide bond, followed by two simultaneous intramolecular cyclization reactions with identical kinetics, one to cleave the urethane linkage to release the NIR dye and the other to cleave the carbonate linkage to release CPT. The released CyN has an emission wavelength (760 nm) that is significantly different from CPT-SS-CyN (820 nm), enabling easy detection and monitoring of drug release. A linear relationship between the NIR fluorescence intensity at 760 nm and the amount of CPT released was observed, substantiating the use of this drug-reporting conjugate to enable precise, real-time, and non-invasive quantitative monitoring of drug release in live cells and semi-quantitative monitoring in live animals.

non-invasive,_real-time_reporting_drug_release_in_vitro_and_in_vivo

Introduction<!>Results and discussion<!>Conclusions

<p>Accurate assessment and monitoring of the concentration of functional agents in biological tissues is important in biology and medicine.1-4 In anticancer drug development and clinical cancer treatment specifically, the therapeutic benefit of drugs is a function of the concentration-time profile in tumor tissues.5-8 Therefore, it is crucial to know the change of drug concentration in the plasma and local tissues over a certain time-course.1 As the vast majority of therapeutic agents used in pro-drug and conjugated nanomedicine are in their inactive form, it is also essential to ensure the conjugated, inactive drug can actually be released in vivo and become therapeutically active.9-15 However, conventional methods to assess the drug release and track the dynamics of active drug concentrations are very complicated.2,16-21 Harvested tissues generally need to be homogenized and cells need to be lysed in order to release the agents from certain intracellular compartments.22-29 During these processes, especially in the process of cell lysis, the intact drug carriers (e.g., micelles or liposomes) will also be disrupted, posing great difficulty in differentiating the released active agents from those inactive agents remaining in the carriers at the point of tissue harvesting. As such, development of minimally invasive or non-invasive methods to confirm the drug release and determine the active drug concentration kinetics in localized tissues is of great benefit.30</p><p>Near-infrared (NIR) fluorescence imaging has been widely used for monitoring biological processes in living objects because of its deep tissue penetration capability of NIR signal and the effective elimination of tissue auto fluorescence interference.31-37 Compared to other molecular imaging techniques such as magnetic resonance imaging (MRI), positron emission tomography (PET), and computed tomography (CT), NIR fluorescence imaging has great potential to track some biological processes noninvasively, in real time, and with sequential, longitudinal monitoring capability.38-42 Using NIR fluorescence imaging as a readout tool, which has been used in drug delivery applications, would be of particular interest for tracking the kinetics of drug release in vivo.43-46 Here, we report the design and use of CPT-SS-CyN to enable precise, real-time, and non-invasive monitoring of drug release quantitatively in live cells and semi-quantitatively in vivo. CPT-SS-CyN has a built-in disulfide bond, which can be cleaved through reduction in the cells. The cleavage of the disulfide bond controls the two subsequent elimination reactions that are responsible for the concurrent release of the drug and reporting dye molecule.</p><!><p>CPT-SS-CyN is composed of a near-infrared fluorescent cyanineamine dye (CyN), a reductive-responsive disulfide linker, and a model therapeutic drug (camptothecin, CPT). The disulfide linker between the drug and the NIR dye is responsive to intracellular reducing agents such as glutathione (GSH), cysteine (Cys) and thioredoxin (Trx).47-50 Treatment with these reducing reagents induces cleavage of the disulfide bond, followed by two subsequent intramolecular cyclization reactions, one to cleave the urethane linkage to release the NIR dye and the other to cleave the carbonate linkage to release CPT with 1,3-oxathiolan-2-one as the same byproduct of the two cyclization reactions. The released NIR dye has a maximum emission wavelength (760 nm) that is significantly different from CPT-SS-CyN (820 nm), and the release of the NIR dye can thus be used to monitor the release of CPT in vitro and in vivo (Scheme 1).</p><p>Synthesis details of CPT-SS-CyN can be found in the Electronic Supporting Information (Scheme S1-5 and Fig. S1-5†). The chemical structure of CPT-SS-CyN was confirmed by 1H NMR, 13C NMR and ESI-MS analyses (Fig. S4-5†). RP-HPLC analysis showed over 95% purity of the obtained CPT-SS-CyN (Fig. S6†). Following the same method, we synthesized CPT-CC-CyN as the noncleavable control (Scheme S1). We first used RP-HPLC and ESI-MS to confirm the trigger-induced degradation of CPT-SS-CyN upon the addition of dithiothreitol (DTT). As shown in the RP-HPLC spectrum, the single peak of CPT-SS-CyN at 40.6 min, corresponding to the major peak of 1220.5 m/z in the ESI-MS spectrum, decreased significantly upon treatment with DTT (Fig. S6c-6d†). Released CPT (16.2 min) and CyN (44.2 min) were detected (Fig. S6†), demonstrating the successful disulfide cleavage of CPT-SS-CyN followed by the two concurrent elimination reactions as shown in Scheme 1.</p><p>We then used a combined RP-HPLC and fluorimetric analysis to further confirm that the release of CPT occurred concurrently with the observed fluorescence changes (Fig. S7-S8†). At different time points, amount of CPT released was measured by RP-HPLC and fluorescence intensity of degraded products was measured at 760 nm on a fluorescence spectrometer. As shown in Fig. 1, when CPT-SSCyN was treated with DTT, the percentage of the released CPT was found to have a linear correlation with the normalized increase in fluorescence intensity at 760 nm (R2 = 0.9989). In the absence of DTT, however, neither CPT release nor enhanced fluorescence intensity at 760 nm was observed. In comparison, CPT-CC-CyN without a disulfide linker (10 μM) showed no release of CPT or CyN in the presence of 100-fold DTT concentration (1 mM) (Fig. S9b†).We thus consider that the change in fluorescence emission at 760 nm can act as a direct on-off signal to determine CPT release.</p><p>To demonstrate our hypothesis that the active drug release and kinetics could be quantitatively assessed by monitoring the NIR fluorescence signal changes in live cells, Hoechst pre-stained HeLa cells in serum free DMEM medium was incubated with CPT-SS-CyN (0.5 μM), and NIR fluorescence imaging was collected in-situ with an emission filter of 680-750 nm on a GE In-Cell Analyzer at different time points (Fig. 2a). Significant increase in fluorescence signal was observed in HeLa cells after 2-h incubation, indicating the uptake of CPTSS-CyN and release of CyN and CPT in cells. The gradual increase in the NIR fluorescence intensity in the range of 680-750 nm was observed over time. In comparison, no fluorescence signal in the range of 680-750 nm was observed in HeLa cells after treatment with CPT-CC-CyN (0.5 μM) for 5 h (Fig. 2c, and Fig. S9c†). To demonstrate that the fluorescent enhancement in HeLa cells was due to reductive degradation of CPT-SS-CyN, L-buthionine sulfoximine (BSO, 100 μM) and diethylmaleate (DEM, 300 μM) were used as GSH inhibitors to investigate whether inhibition of GSH levels in HeLa cells would reduce or slow down the degradation of the reporting conjugate.51 As shown in Fig. 2c, fluorescence signal in the range of 680-750 nm greatly decreased in BSO/DEM treated HeLa cells after 5-h incubation with CPT-SS-CyN (0.5 μM).</p><p>To evaluate whether CPT-SS-CyN enables quantitative assessment of released CPT via following the fluorescence changes in live cells, we measured the concentrations of the released CPT in lysed HeLa cells via liquid chromatography-multiple-reaction monitoring-mass spectrometry (LCMRM/MS) and the fluorescence intensity change at 680-750 nm per cell via GE In-Cell Analyzer (Fig. S10-S14†), and then sought inter-correlation of these two parameters. As shown in Fig. 2b, a linear relationship (R2 = 0.98) between the average fluorescence intensity per cell and the amount of released CPT per cell was observed, demonstrating that the NIR fluorescence intensity change can be utilized as a diagnostic tool for quantitative CPT release studies. This calculation was further validated by correlating the average fluorescence intensity with the amount of released CyN in the living cells. Fig. S12† showed the average fluorescence intensity enhancement at Cy5 channel (excitation at 640-660 nm, emission at 680-750 nm) analyzed by GE In-Cell Analyzer correlates linearly (R2 = 0.98) with the amount of CyN released per cell, as determined by fluorescence spectrometer. Fig. S13† showed a linear relationship between the average amount of CyN per cell analyzed by fluorescence method and average amount of CPT per cell analyzed by LC-MRM/MS. The real-time tracking of active CPT release in the living cells was further investigated. As shown in Fig. 2d, the amount of actively released CPT increased from 0.094 × 10-17 mole/cell at 10 min post incubation to 5.223 × 10-17 mole/cell at 5 h post incubation. However, negligible enhancement of the average fluorescence intensity per cell was observed when HeLa cells were incubated with CPT-CC-CyN, indicating minimal release of CyN and CPT. These studies and experimental results demonstrated the potential of CPT-SS-CyN prodrug as a diagnostic tool for real-time tracking of CPT release quantitatively in live cells.</p><p>We next assessed the drug-reporting capability of CPT-SSCyN in vivo. Athymic nude mice bearing human A375 melanoma were intratumorally injected with CPT-SS-CyN (5 nmol of CyN, in 25 μL DMSO/PBS (1:10 v/v)) and in vivo whole-body fluorescence imaging was taken on an in vivo imaging system (Maestro, CRi, Inc., excitation filter: 615-665 nm, emission filter: 680-950 nm). Image collection started simultaneously as the intratumoral injection of CPT-SS-CyN and continued for 5 h. Fig. 3a showed the fluorescence spectra of tumor tissues and the whole-body images of mice at 10 min, 2 h, and 5 h post injection of CPT-SS-CyN. During the entire imaging acquisition period (5 h), strong and steady fluorescence signal enhancement was observed at 10 min post injection, and the fluorescence spectra of tumor tissues showed a noticeable shift from ~820 nm (CPT-SS-CyN emission) to ~ 740 nm (CyN emission). The increase in the fluorescent intensity of CyN at 740 nm over time in tumor tissues was showed in Fig. 3b. The fluorescence intensity at 5 h post injection increased by 5 times compared to that at 10 min post-injection. Based on the obtained linear relationship of CPT release and CyN fluorescence intensity enhancement from the in vitro CPT-SS-CyN (Fig. S13†), the amount and kinetics of active CPT release thus can be monitored semi-quantitatively in vivo. We evaluated the cytotoxicity of CPT, CyN, CPT-SSCyN, and CPT-CC-CyN using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Fig. S15†). Both the NIR dye (CyN) and the control prodrug (CPT-CCCyN) showed negligible cytotoxicity against HeLa cells. While CPT-SS-CyN showed comparable cytotoxicity to free CPT due to the intracellular GSH induced CPT release.</p><!><p>In summary, we developed a real-time drug-reporting system (CPT-SS-CyN) composed of a disulfide bond as a cleavable linker, a cyanine-amide moiety as a near-infrared (NIR) fluorescence reporter, and camptothecin (CPT) as a model therapeutic agent. Treatment with DTT induces cleavage of the disulfide bond, followed by two simultaneous intramolecular cyclization reactions, which leads to concurrent release of CPT and NIR dye. We have demonstrated the possibility of real-time monitoring drug release via reading the NIR fluorescence change in vitro and in vivo. Given the quantitative assessments in the live cells and semi-quantitative measurements in live animals, this developed drug reporting system may enable more precise, real-time, and non-invasive monitoring of active drug release in the development and application of chemotherapeutic treatments. This technique may also be broadly used for monitoring the in vitro and in vivo release of other substrates, such as small molecule inhibitors and activators. The conjugate may be encapsulated or conjugated (e.g., via the oligo(ethylene glycol) terminal) to a controlled release device for drug delivery applications.</p>

PubMed Author Manuscript

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.

bruton's_tyrosine_kinase_regulates_tlr7/8-induced_tnf_transcription_via_nuclear_factor-κb_recruitmen

<!>Introduction<!>Materials and methods<!><!>TNF production following TLR7/8 stimulation requires Btk<!>Effect of Btk depletion on TLR4-and TLR7/8-mediated TNF transcription<!><!>Effect of Btk depletion on TLR4-and TLR7/8-mediated TNF transcription<!>NF-κB sites in TNF promoter and a 252bp region of TNF 3′UTR are required for Btk effects<!><!>NF-κB sites in TNF promoter and a 252bp region of TNF 3′UTR are required for Btk effects<!><!>Discussion<!>Transparency document

<p>Btk is required for TLR7/8 signalling in primary human macrophages.</p><p>R848-induced TNF mRNA is more Btk dependent than LPS-induced TNF mRNA.</p><p>Btk transcriptional control of TNF following R848 requires the promoter and 3′UTR.</p><p>Btk knockdown reduces p65RelA translocation to the nucleus upon TLR7/8 stimulation.</p><!><p>TNF production is precisely regulated at both the gene and protein expression level [1]. Toll-like receptors (TLRs), by recognising ligands as diverse as bacterial cell wall components and nucleic acids, are important inducers of TNF production in disease. In addition, recognition of endogenously derived damage-associated molecular patterns (DAMPs) makes them key players in the induction and maintenance of autoimmune inflammation [2].</p><p>Non-receptor tyrosine kinases play a major role in TLR signalling [[3], [4], [5]], and in particular, Bruton's Tyrosine Kinase (Btk), a member of the Tec family of non-receptor protein tyrosine kinases (PTKs), is a crucial regulator of TLR induced TNF production [6,7]. In humans, a lack of functional Btk leads to X-linked agammaglobulinemia (XLA), a condition characterised by both B cell deficiency and ineffective immune responses to bacterial and viral challenge [8]. XLA patient monocytes show reduced production of TNF and IL-1β in response to TLR2 and TLR4 ligands [9,10] and stimulation of XLA-derived dendritic cells with siRNA results in significantly decreased production of both TNF and IL-6 [11]. Btk deficiency in B cells reduces TLR9-induced production of IL-10, leading to elevated levels of TNF, IL-6 and IL-12p40 [12,13] a finding that may explain the increased levels of cytokines present in XLA serum [14].</p><p>In HEK293 cells Btk physically interacts with the cytoplasmic Toll/IL-1 receptor (TIR) domains of TLRs 4, 6, 8 and 9 as well as the adaptor molecules Myd88 and Myd88-adapter-like (Mal) [15]. Following stimulation, TLR receptors (except TLR3) recruit Myd88 via its cytoplasmic Toll/IL-1 receptor (TIR) domain. Various other molecules including IL-1 receptor-associated kinases 1 and 4 (IRAKs 1 and 4), TNF receptor associated factor (TRAF) 6, TAB2/3 and TAK1 then associate with the receptor complex. IκB is phosphorylated by the TAK1-activated IκB kinase (IKK) complex, ubiquitinated and degraded by the 26S proteasome. Following NFκB release from the inhibitory IκB complex, p65RelA is phosphorylated on a number of serines to regulate p65RelA nuclear translocation and gene transactivation [16]. NFκB is considered to be essential for TNF transcription, and over-expression of IκBα decreases TNF production from LPS-stimulated human primary macrophages [17].</p><p>Here we provide evidence for Btk in TLR7/8 signalling in human primary macrophages. Btk regulates TLR7/8-induced TNF production at early time points via the 3'enhancer region of the TNF gene. Moreover, we show that Btk controls the initiation of TNF transcription through NF-κB recruitment. Interestingly, TNF transcription in response to LPS was less affected, revealing a previously unreported distinction between TLR4 and TLR7/8-mediated TNF gene transcription.</p><!><p>Reagents and Antibodies. R848 and LPS were from Alexis Biochemicals and macrophage colony-stimulating factor (M-CSF) was from Peprotech. Polyclonal rabbit anti-Btk antibody for immunoprecipitation was a gift from M. Tomlinson (University of Birmingham, U.K.), mouse anti-Btk antibody (clone 10D11) for Western blotting was from BD Bioscience and anti-GAPDH (ab9484) and rabbit isotope control antibodies were from Abcam. The phosphotyrosine clone 4G10 was from Millipore. Cell Signaling provided anti-IκBα (#9242), anti-phospho-p65RelA (Ser536) (for Western blot; #3036), and anti-p65RelA (for confocal; #3033). Anti-p65RelA (sc-372) for Western blotting was from Santa Cruz Biotechnology.</p><p>Monocyte isolation and adenoviral infection. Following ficoll-hypaque centrifugation, monocytes were elutriated from PBMC as previously described [17]. Monocytes were treated with M-CSF (100 ng/ml) for 3–4 days prior to counting and re-seeding. Creation of adenoviral constructs and method of infection as previously described [17]. For double infections, cells were first infected with the luciferase adenovirus at multiplicity of infection (moi) 50:1 for 2h, rested for 4h in serum containing medium prior to secondary infection at moi 100. Luciferase reporter assays were performed as previously described [9,18].</p><p>Gene knockdown by siRNA. 5 × 106 primary human monocytes were transfected with targeting siRNA or control oligunucleotides (siControl D-001206-13 and human Btk SMARTpool M-003107-01, Dharmacon, IL) at concentrations ranging from 100 to 300 nM using Human Monocyte Nucleofector Kit (Amaxa Biosystems, Germany) according to manufacturer's instructions. After 24 h, monocytes were cultured in 5% HIFCS phenol red free RPMI with 100 ng/ml M-CSF for a further 72h. STAT1 phosphorylation by Western blot following siRNA nucleofection was assessed after a further 24h in the absence of M-CSF and stimulation ± IFN (1 ng/ml) for 5 min.</p><p>Immunoprecipitation and Western Blotting. M-CSF-differentiated macrophages were plated on 10 cm2 petri dishes and serum starved for 2h prior to stimulation. Cells were lysed in ice-cold lysis buffer (20 mM Tris-Base pH 7.6125 mM NaCl, and 1% Nonidet P-40), containing freshly added 10 mM DTT, 100 μM Na2VO3, 5 mM NaF, 1x Protein Inhibitor Cocktail (Sigma). Debris was removed by centrifugation, and supernatants were pre-cleared with protein G-sepharose. Btk was precipitated with polyclonal rabbit anti-Btk anti-sera and protein G-sepharose for 1.5 h. Immunoprecipitated complexes were washed in lysis buffer before resolving on 10% SDS-PAGE gel and transferring to nitrocellulose membrane (Millipore). The membrane was blocked for 1h in TBS-Tween (0.1%) with 2% BSA for the detection of phosphorylated proteins or in 5% skimmed milk for other proteins.</p><p>Immunocytochemistry. After siRNA transfection, monocytes were differentiated in M-CSF for 72h. Macrophages were plated on glass coverslips (ECN 631-1578, VWR) and stimulated with R848 (1 μg/ml). Cells were fixed with 4% (w/v) paraformaldehyde in PBS for 15 min at 37 °C, quenched with 50 mM NH4Cl/PBS for 10 min, and permeabilised with 0.1% (w/v) Triton X-100 in PBS for 5 min. Samples were blocked with 3% (w/v) BSA in PBS for 30 min at room temperature followed by incubation with anti-phospho-p65RelA (Ser536) diluted in 3% (w/v) BSA in PBS for 1 h at room temperature. After washing, secondary antibody was added; phosphorylated p65RelA (Ser536) with Alexa Fluor 488 (A11034, Invitrogen), actin cytoskeleton with Alexa Fluor 546 Phalloidin (A22283, Invitrogen) and the nucleus with DAPI (D1306, Invitrogen). Samples were mounted with ProGold antifade mounting media (P36934, Invitrogen). Confocal 'z' stack were used to quantify the intensity of staining by measuring the respective brightness of the pixels for each of detection channels using Fiji image analysis software.</p><p>Cytokine measurements by ELISA. TNF concentration in supernatants was determined by ELISA (BD Biosciences), according to the manufacturer's instructions. Absorbance was read and analysed at 450 nm using a Fluostar Omega (BMG Labtech, Aylesbury, UK) plate reader and analysed using MARS data analysis software.</p><p>Real-time RT-PCR. RNA was extracted from macrophages using Blood RNA extraction kit (QIAGEN), and genomic DNA removed using TURBO DNA-free kit (Applied Biosystems). cDNA was subjected to real-time PCR analysis using SYBR Premix Ex Taq (Lonza) on a Corbett Rotor-Gene 6000 (Qiagen). Primers for measuring primary human TNF transcripts were 5′-GCAGTCAGATCATCTTCTCG-3′ and 5′-GGTACAGGCCCTCTGATGGCAC-3'. Mature human TNF transcripts were 5′-CCTGCTGCACTTTGGAGTGATCGG-3' & 5′-GTACAGGCCCTCTGATGGCACCACC-3′, respectively. Primers for actin-related protein transcripts (ARP) were 5′- CGACCTGGAAGTCCAACTAC-3′ and 5′- ATCTGCTGCATCTGCTTG-3'. Relative quantification of gene expression was expressed as fold mRNA/ARP as determined using the comparative ΔΔCT method.</p><p>Statistical analysis. Values correspond to mean ± SEM or SD. In experiments with multiple groups, differences were first evaluated using repeated-measures ANOVA and then Dunnett's test to compare group means. Unpaired Student's t-test was used when comparing differences between two groups.</p><!><p>Btk is required for cytokine production in TLR4 and TLR7/8 signalling. M-CSF-differentiated macrophages were (A, B) serum starved for 2h prior to stimulation with 10 ng/ml LPS or 1 μg/ml R848 up to 20 min. Btk was immunoprecipitated using anti-Btk antibody followed by western blotting with 4G10 antibody. Anti-rabbit IgG as isotope control. (C) Macrophages infected with adenovirus at moi 10 to 150 were left for 48 h. Btk expression in cell lysates by Western blot using GAPDH as a protein loading control. (D, E) Following infection, cells were stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h and TNF production assessed by ELISA. Btk over-expression was normalised to control (black bar; empty adenovirus) for each moi. (F) Monocytes transfected with Btk siRNA (siBtk#9 and #11) or control siRNA (siCon) at 200 nM then M-CSF-treated for 4 days and Btk expression assessed by Western blot. (G, H) TNF or IL-6 in supernatants was measured by ELISA from siRNA manipulated cells after 18hr with either no stimulus or 1ug/ml R848. (I) Monocytes were transfected with Btk siRNA (siBtk) or control siRNA (siCon) at 100, 200 or 300 nM and then M-CSF-treated for 4 days. B- was no transfection; B+ transfection with buffer alone. Cell lysate expression of Btk was assessed by Western blot. (J) Monocytes were incubated with no oligo (nil), control oligo or BTK oligo at 200 nM prior to nucleofection. After 24 h of M-CSF treatment, media was replaced, and after a further 24 h cells were plated and stimulated ± IFN (1 ng/ml) for 5 min. Cell lysates were used to determine the phosphorylation status of STAT1 and levels of BTK by SDS-PAGE and western blotting. (K, L) Monocytes were transfected with 200 nM siBtk or siCon, M-CSF-treated for 4 days, and then stimulated with either LPS (10 ng/ml) or R848 (1 μg/ml) for 18h; TNF concentration was assessed by ELISA. Cytokine levels are expressed as means of triplicate repeats ± SEM. Results shown combined data from 3 to 6 separate donors. Statistical analysis: student's t-test, ***p < 0.001, **p < 0.01, *p < 0.05.</p><!><p>To determine whether Btk modulates TLR7/8 driven TNF, Btk was over-expressed as previously described [9] as confirmed by western blot (Fig. 1C). Cells were then stimulated with either LPS or R848 and TNF production after 18 h was measured. Over-expression of Btk resulted in significantly increased TNF production in response to both LPS and R848 (Fig. 1D and E). To test the effect of Btk down-regulation on TNF production two siRNA duplexes; siBtk#9 and siBtk#11 were tested and produced similar decreases in Btk expression (Fig. 1 F). In the absence of TLR stimuli (NS), siRNA nucleofection alone did not induce any detectable cytokine production (Fig. 1G and H). In the presence of R848, siBtk#9 showed the greatest effect on cytokine production and hence was used for the remaining experiments (Fig. 1G and H). Three doses of siRNA were tested (100, 200 and 300 nM) and 200 nM was chosen as the concentration for subsequent experiments. Additionally, there was no basal increase in STAT1 phosphorylation following RNAi knockdown indicating that subsequent findings are attributable to Btk knockdown as opposed to the induction of an interferon response (Fig. 1J). Following Btk inhibition, TNF release was significantly reduced from 4 to 10h after LPS stimulation and 2–18h after R848 stimulation (Fig. 1K and L). Interestingly the effect of Btk ablation was more pronounced and longer lasting for R848-induced TNF production than for LPS.</p><!><p>TNF is controlled at a number of different stages in its production. Our previous work showed that message stability is an important control point for TNF production [9]. TNF is also controlled at the transcriptional level and data from other workers in HEK293s and immortalised monocyte lines have suggested that this is how Btk mediates its effect on TLR7/8 induced TNF [19].</p><!><p>Btk regulates TLR7/8-induced TNF transcription. (A) Schematic representation of the TNF gene indicating primer pair positions used for transcriptional start site (TSS) and downstream regions of the gene. Monocytes were transfected with 200 nM Btk siRNA (siBtk) or control siRNA (siCon), M-CSF-treated for 4 days, and then stimulated with either LPS (10 ng/ml) (B and D) or R848 (1 μg/ml) (C and E) up to 10h. TNF mature (B, C) and primary (D, E) mRNA levels measured by RT-PCR using primers for downstream or TSS regions of the TNF gene. Graphs show mean values ± SD of triplicate measurements for a single donor: representative of 3 independent experiments using different donors.</p><!><p>In response to Btk knockdown, primary TNF transcripts (analysed using primers over an intron-exon boundary of TNF; Fig. 2A) showed a similar pattern demonstrating that the reduction in transcript level is due to deficient transcription rather than RNA processing (Fig. 2D and E). Taken together, these data reveal fundamental differences between the way that Btk controls TNF production downstream of TLR4 and TLR7/8 stimulation with R848-induced TNF mRNA being more Btk dependent than that induced by LPS.</p><!><p>Transcription of TNF is mediated by a number of transcription factors including AP-1, Oct-1 and NF-κB [20]. 4 distinct NF-κB binding regions have been described within the TNF locus; 3 sites (κB1, κB2/ζ/2a, κB3) within the promoter region and a fourth cluster (sites 4, 4a and 4b) in the 3′UTR. Site 4 plays an essential role in LPS-mediated TNF transcription in macrophages and DCs [18,21] and our previous studies have highlighted the importance of a 252bp region in the 3′UTR of TNF for TLR4-induced transcription [18].</p><!><p>Btk regulates TLR7/8-induced TNF via both promoter NFkB sites and downstream regions of the gene. (A) Adenoviral luciferase reporters based on the human TNF gene. Positions of NFκB sites (2/ζ/2a, 3 and 4/4a/b) are shown. AAA denotes site of poly-A tail. X indicates position of point mutations used to destroy NFκB site(s). (B) M-CSF-differentiated macrophages were infected with luciferase reporter adenovirus at moi 50 followed by AdBtk or AdCon at moi 100. After 24h cells were stimulated with R848 (1 μg/ml) and lysed after 6h. Values are normalised to non-infected controls and represent combined data from >3 separate donors (means ± SEM).</p><!><p>In contrast, the 5'only construct, the 4X NF-κB construct (all NF-κB sites destroyed by point mutations), and the 5′785 construct, which lacks a 252bp region in the 3′UTR, were not able to increase luciferase production after Btk over-expression (Fig. 3B). Thus demonstrating the need for both the NF-κB sites in the TNF promoter (site 1, cluster 2, site 3), and the 252bp region in the 3′UTR to mediate the effect of Btk on TLR7/8 driven TNF production.</p><!><p>Btk down-regulation decreases R848-induced p65 phosphorylation. (A) Monocytes were transfected with 200 nM Btk siRNA (siBtk) or control siRNA (siCon), M-CSF-treated for 4 days, and then stimulated with R848 (1 μg/ml) for 90min. p65 RelA phosphorylation and degradation of IκBα was assessed by western blotting with anti-p-p65 (Ser536) and anti-IκBα antibodies. Blot represents one of 3 independent experiments. (B) siRNA transfection of monocytes was performed as described in (A). Primary human macrophages were plated on glass coverslips, stimulated with R848 (1 μg/ml) for 30min, fixed and stained for phosphorylated p65RelA (green), actin (red) and nucleus (blue) (scale bars = 10 μm). Images are representative of 3 independent experiments. (C) Staining intensity of p-p65 in the nucleus. Mean values from 2 to 3 nuclei per each condition, for each of 3 independent experiments (means ± SEM). Statistical analysis: student's t-test. ***p = 0.0003. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</p><!><p>Understanding how TNF expression is controlled is crucial for the next generation of anti-inflammatory therapies [22]. TLR7 and 8 both recognise single stranded RNA, which is central to the recognition of both viral and bacterial pathogens [23,24]. The ability of TLR7/8 to recognise endogenously released RNA has implications for autoimmune diseases where TLR7/8 contributes to inflammation in RA and SLE [25,26].</p><p>Btk is a critical signalling component of a wide variety of immune receptors, including Fc receptors, gp130 containing cytokine receptors, and the B cell receptor where it is required for the activation of NF-κB [6,27]; the cumulative effect of Btk depletion is an impaired immune response. Downstream of TLR7/8 engagement numerous signalling pathways are activated, including the NF-κB, MAPK, AP1 and the IRF family [23,28]. Doyle et al. showed that TLR8 stimulation activates Btk in THP1 cells via phosphorylation of tyrosine 223 and increased autokinase activity [19]. TLR8-mediated Ser536 phosphorylation of p65RelA in murine BMDM was significantly reduced in Xid mice [19]; our data agrees with these findings and extends them to show that TLR7/8 mediated activation of Btk occurs in primary human macrophages. Additionally, we reveal distinct differences in the requirement for Btk in TLR4 and TLR7/8 signalling within the same cell type. In TLR7/8 signalling Btk acts upstream of NF-κB activation, regulating the serine phosphorylation of p65RelA and thereby its nuclear localisation - consequently, TNF transcription is significantly inhibited without Btk. In contrast, despite similar reductions in p65RelA binding to NF-κB sites in Btk depleted cells after LPS (TLR4) stimulation, transcription of TNF was largely un-affected. Btk-independent TNF transcription following LPS suggests that additional transcription factors/regulatory mechanisms may be active for TLR4 signalling that are absent for TLR7/8.</p><p>Phosphorylation of p65RelA occurs after release from the inhibitory IκBα complex and the importance of Ser536 phosphorylation of p65RelA is demonstrated by the mutation of Ser536 to alanine which abolishes the interaction of p65RelA with the transcriptional coactivators p300 and CREB-binding protein (CBP) thereby decreasing transcriptional activation [29,30]. Btk lies upstream of this phosphorylation event in a number of systems, including signalling via TLR4 and the B cell receptor [30,31], but the mechanism is unknown. p65RelA can be phosphorylated on Ser536 by IKKα and IKKβ for example, but neither molecule has been described to interact with Btk.</p><p>Non-receptor tyrosine kinases are attractive targets for therapeutic intervention as small molecule inhibitors are readily synthesised [32]. Btk inhibitors have demonstrated considerable success in clinical trials, particularly in combating B cell malignancies [33,34] and in the treatment of autoimmune disorders such as lupus and inflammatory arthritis in animal models [[35], [36], [37]]. Btk inhibition acts not only via reduced B cell development and function, but also on other cell types such macrophages limiting cytokine and chemokine production [38,39].</p><p>We have used loss of function and overexpression manipulation technologies to demonstrate the importance of Btk in response to different TLR ligands in primary human macrophages. Btk plays an important role in the TLR7/8 mediated induction of TNF and there are clear differences between TLR4 and TLR7/8-mediated TNF production. These data add important insight into the control of TNF production in primary human cells and may suggest molecular targets for the development of more efficient anti-inflammatory therapeutics.</p><!><p>Online dataOnline data</p>

PubMed Open Access

A theorized new class of polyhedral hydrocarbons of molecular formula CnHn and their bottom-up scaffold expansions into hyperstructures

We address the use of Euler's theorem and topological algorithms to design 18 polyhedral hydrocarbons of general formula C n H n that exist up to 28 vertexes containing four-and six-membered rings only; compounds we call "nuggets". Subsequently, we evaluated their energies to verify the likelihood of their chemical existence. Among these compounds, 13 are novel systems, of which 3 exhibit chirality. Further, the ability of all nuggets to perform fusion reactions either through their square faces, or through their hexagonal faces was evaluated. Indeed, they are potentially able to form bottom-up derived molecular hyperstructures with great potential for several applications. By considering these fusion abilities, the growth of the nuggets into 1D, 2D, and 3D-scaffolds was studied. The results indicate that nugget 24a (C 24 H 24 ) is predicted to be capable of carrying out fusion reactions. From nugget 24a , we then designed 1D, 2D, and 3D-scaffolds that are predicted to be formed by favorable fusion reactions. Finally, a 3D-scaffold generated from nugget 24a exhibited potential to be employed as a voxel with a chemical structure remarkably similar to that of MOF ZIF-8. And, such a voxel, could in principle be employed to generate any 3D sculpture with nugget 24a as its level of finest granularity.On a very thought-provoking article in New Scientist, entitled "Why think up new molecules?", Prof. Roald Hoffman presented reasons to justify why he thinks that this is a worthwhile venture 1 . Speculative, inventive and somewhat risky predictions to either confront or make an exquisite use of a theory, are, by their very nature, scientific endeavors. As Prof. Roald Hoffman concludes, "The predictor leaves the safety of known molecules and properties for the unknown. He or she takes a risk. And, in a way, flirts-in a game of interest and synthesis-with the experimentalist. " 1 . In this article, we do indeed take this path and present a new subclass of hydrocarbons we call nuggets.Polyhedral hydrocarbons of general formula C n H n comprise a class of organic compounds that can exhibit unique properties, such as: tensioned bonds in rings that may be formed by three, four or more carbon atoms 2 ; energy storage capability 3 ; high density 3 ; aromaticity or antiaromaticity 4 ; magnetism 5 ; and symmetry such as the ones exhibited by platonic solids and regular prisms 5 . However, due to their sometimes strongly stressed bonds, syntheses of polyhedral hydrocarbons are hardly easy. In this sense, Eaton et al. 6 reported a synthetic strategy for the polyhedral hydrocarbon cubane (C 8 H 8 ), which is a tetraprism system. Further, Katz et al., synthesized the C 6 H 6 compound, which is a triprism system 7,8 . In particular, this compound exhibits a more tensioned structure than cubane 7,8 . In addition, the C 10 H 10 polyhedral hydrocarbon was also synthesized 8,9 .From a structural perspective, the bond angles of polyhedral hydrocarbons, that are either platonic or prismanes, are of smaller values (60°-90°), when compared with the most common bond angles of carbon atoms (109.5°). These small bond angles introduce a structural tension, which tends to energetically destabilize the system.An interesting aspect of the polyhedral hydrocarbon cubane is its ability to store a large quantity of energy 10 . Based on the cubane synthesis, a set of derivatives was prepared that presented potential to be applied to materials science due to their cube fusion abilities. Examples of the cubane derivatives are the octamethylcubane 11 and octacyclopropylcubane compounds 12 . In addition, Moran et al. evaluated the viability of carbon and hydrogen

a_theorized_new_class_of_polyhedral_hydrocarbons_of_molecular_formula_cnhn_and_their_bottom-up_scaff

<!>Results and discussion<!>Energetics of nugget-nugget face-fusion reactions.<!>Growth of nuggets into patterns.<!>Conclusions

<p>www.nature.com/scientificreports/ formed cages with ions, in which these systems have the potential to be applied in magnetic resonance, acting as contrast agents, with semiconductive and ferromagnetic properties 13 . Cubane derivatives can also be employed as additives, for example, in fuel, due to their tensioned structures 14 . In addition, 4-methyl-cuban-1-amine and 4-methyl-cuban-1-methylamine compounds exhibited antiviral biological activity 15 . Finally, if synthesized in larger amounts, heptanitrocubane would perhaps be one of the most effective non-nuclear explosives possible 16 .</p><p>Poater et al. studied several structural and energy aspects of a class of packed carbon nanoneedles, that were conceptualized by stacking up units of 4, 6, and 8 carbons with potential applications to nanomedicine by acting as drug carriers through nonpolar biologic media 17 . The ability of the polyhedral hydrocarbons to be structurally fused was further examined by Katin et al. 18 The authors studied a material based on polyprismanes and concluded that these systems are similar to the carbon nanotube 18 . In addition, the interactions of the orbitals between the parallel rings of these materials seem to be the main component associated with the stability of the systems 19 .</p><p>Karpushenkava et al. 20 , studied both structural and vibrational properties of a set of polyhedral hydrocarbons of the C n H n cage class in gas phase. The authors concluded that when the energy associated with the cage tension is either negative or slightly positive, the corresponding compounds could be synthesized. An unique exception was verified for a triprism compound with a cage energy of + 55.2 kJ mol −1 (ref 20 ).</p><p>Wang et al., reported three stable isomers of the type C 24 H 24 . In their article, G3(MP2) calculations revealed that the optimized geometries of these systems have a positive value for Δ f H 21 . These geometries are unstable when compared to their fullerene isomers. In addition, one of the structures formed with Si has the potential to be a semiconductor material and, by replacing the CH groups with nitrogen atoms, high-energy density materials can be prepared 21 .</p><p>On the other hand, DFT methods were also employed by Shamov et al. 22 to predict both structural and energy properties of a set of C n H n compounds, with n being 12, 16, 20, and 24. Both C 12 H 12 and C 20 H 20 compounds were synthesized, and the energetic properties indicated that C 16 H 16 and C 24 H 24 could be prepared. In this sense, Ohno et al., investigated both dimers and trimers of the regular prisms, with 6, 10, 12, 14, 16, 18 and 20 faces, connected by cubane-shaped bridges 23 . Their results also revealed that these compounds are able to be formed in organic reactions at low temperatures. Moreover, due to the metastable nature of the regular prismatic compounds, they could be potentially employed, for example, in energy storage 23 .</p><p>In this article, we employ Euler's theorem to deduce polyhedra containing four-and six-membered rings that exist up to 28 vertexes, that we call "nuggets". We then evaluate their energetics in order to conjecture the likelihood of their existence. Finally, because all nuggets can be fused together in several manners, either through their square faces, or through their hexagonal faces, we investigated the fusion abilities of this set of nuggets to investigate the perspectives for their growth into 1D, 2D, and 3D-scaffolds.</p><!><p>The nuggets structural possibilities from Euler's theorem. Our intention was to design hydrocarbon polyhedra that could be potentially stable. Although there are polyhedral hydrocarbons of the type C n H n with triangular faces, such as the tetrahedron 24 and the triprism 25,26 , as well as ones with pentagonal faces, such as the dodecahedron and the pentaprism 9,26 , we decided to restrict our work to polyhedra whose faces are polygons with an even number of vertices. Such systems can have alternating double bonds, thus potentially displaying energy stabilization due to electronic delocalization.</p><p>Let us first consider polygonal hydrocarbons of formula C n H n . The smallest polygon with this formula is triangular C 3 H 3 . However, C 3 H 3 is a radical system. The same happens with C 5 H 5 , as shown in Fig. 1. Actually, all neutral polygonal C n H n hydrocarbons with n being an odd number must be radical systems.</p><p>On the other hand, when n is an even number with n ≥ 4, the C n H n polygonal hydrocarbons are neutral systems, with cyclobutadiene, C 4 H 4 , and benzene, C 6 H 6 , displaying planar structures and thus being the most important members of this class. But, when n is equal to or larger than 8, the compounds become non-planar 27 . Figure 2 shows images of these polygonal compounds up to n = 10.</p><p>Because we intend to grow the polyhedra into 1D, 2D, and 3D-scaffolds by fusing together their polygonal faces, we will restrict the polyhedra in this work to those with square and hexagonal faces only, since it would be very difficult, if not impossible, to fuse together two significantly non-planar and twisted faces. In these polyhedral compounds, each carbon atom must be bound to a single hydrogen atom as well as to three other carbon atoms as well.</p><p>Euler's theorem 28 defines a relation between the numbers of faces, edges and vertices for any simple polyhedron: the polyhedra of our interest. Simple polyhedra are topologically equivalent to a sphere, that is, these systems are polyhedra that have no central cavities as "donuts". Therefore, if inflated, in the limit, these systems would become spheres. There are two possibilities for a hydrogen atom bonded to a carbon atom in a carbon polyhedron: either it is located inside or outside the polyhedron. If the hydrogen atoms appear in the interior of the polyhedron, steric effects would be very significant due to the congestion between other hydrogen or carbon atoms, especially for the smaller polyhedra. Moreover, if all hydrogen atoms always point inwards, at least one hydrogen atom would have an HCC angle less than 90°, which is not reasonable from the point of view of chemical bonds. Therefore, to be chemically realistic in applying Euler's theorem, we will focus on carbon polyhedra with the hydrogen atoms of the CH bonds always pointing outwards.</p><p>Euler's theorem for simple polyhedra relates the number of faces (F), edges (E), and vertices (V) by the formula:</p><p>where V is the number of vertices, E is the number of edges and F is the number of faces.</p><p>(1)</p><p>where F 4 is the number of square faces, and F 6 is the number of hexagonal faces.</p><p>Of course, each square face of the polyhedron delimits four edges, and each hexagonal face delimits six. However, if the edges are counted from each polyhedral face, they would be counted twice, since each and every edge of the polyhedron is shared by exactly two faces. Accordingly, the relation between the number of edges, E, and the number of square and hexagonal faces of such a polyhedron is given by the following equation:</p><p>For our polyhedra, the number of vertices is represented by the union of three edges. That is, each carbon atom is chemically bonded to exactly three other carbon atoms, i.e. V = V 3 ; the fourth bond being to a hydrogen atom. And each edge is bounded by two distinct end points: the vertices. Therefore, the relation between the number of edges and the number of vertices is given by: From Euler's formula, Eqs. (1), and (4): From Eqs. (2), (3), and (5), we obtain:</p><p>(2) www.nature.com/scientificreports/ By simplifying the term 6F 6 on both sides of Eq. ( 7), we finally obtain that F 4 = 6. This reveals that any simple polyhedron that has only square and hexagonal faces must always have 6 square faces for an arbitrary number of hexagonal faces, except one. This exception is because Euler's formula is a necessary, but not sufficient condition for a polyhedron to exist. As can be intuited from Fig. 3 a configuration of one hexagon and six squares cannot be possibly closed into a polyhedron without forming at least a second hexagonal face. Consequently, the number of hexagonal faces must be either 0 (for the cube), or equal or greater than 2 for a constant number of six square faces.</p><p>Design and computational details. The software Blink 29 developed by the research group of one of us (SL) was used to generate a set of unique nuggets from the cube up to the three different solids with 28 vertexes, all with 6 square faces and up to 10 hexagonal faces. From graph encoded manifold and UNIVs data 30 the Blink software is capable of generating several representations of graphs, only formed by faces with even numbers of vertices-squares, hexagons, octagons, etc. In this article, the Blink software was employed to map the topologically different and possible shapes of up to n = 28 vertices. Among all possibilities generated, we selected, according to chemical criteria, a subclass that we call nuggets that is composed of those that have structural forms containing six squares and an arbitrary number of hexagons, either equal to zero, or greater than or equal to two, generating a set of three-dimensional representations of the nuggets. From this class, we selected the first 18 that led to chemically different structures 29,30 .</p><p>Hence, we generated a set of all different such polyhedra, starting with the cube, C 8 H 8 , up to those containing 28 vertices, of empirical formula C 28 H 28 , a number which we found to be reasonable to explore from a chemical point of view. Figure 4 shows the chemical structures of all 18 nuggets obtained, identified by the number of vertices, that is of carbon atoms, which is identical to the number of hydrogen atoms, and an additional letter in case there are more than one such nuggets for a given number of vertices.</p><p>Being fully aware that predicting the properties of unusual molecules is risky, in order to calculate structural, vibrational and energy properties of the set of 18 nuggets, we needed to choose a quantum chemical model chemistry that would be at the same time both accurate enough and workable, given the size of the systems that we want to study, to be able to make educated inferences on the prospects of their chemical realities. We thus chose the ωB97XD functional by Chai and Head-Gordon because of its inclusion of a version of empirical Grimme's D2 dispersion as well as long-range correction with superior results 31 , together with the 6-31G* basis set of Petersson et al. 32 , for ease of computation of the larger hyperstructures formed by the molecular building blocks. Accordingly, all geometries of the designed nuggets, as well as the more complex 1D, 2D, and 3D systems were fully optimized by ωB97XD/6-31G* calculations via both Spartan'14 33 and Gaussian09 34 softwares. All structures have been characterized to be minima with frequency calculations.</p><p>Nuggets exhibiting polyhedral chirality. From Fig. 4, nugget 24b , nugget 26b , and nugget 28b exhibit polyhedral chiral properties, as can be seen, in an illustrative manner, in Fig. 5, below, where we represent their respective pair of enantiomers. www.nature.com/scientificreports/ Nuggets as voxels. Voxels are the three-dimensional (3D) equivalents of pixels. Analogously to pixels, which can be used to generate any 2D images by juxtaposition, voxels can be likewise used to generate any 3D sculptures. Voxels can be virtual as in computer 3D graphics or real as in 3D printers. For a carbon polyhedron to be able to efficiently function as a voxel, it should possess the important property of 3D space-filling. That property being satisfied, they could in principle perhaps function as solid controllable building blocks that could be used to assemble any arbitrary 3D structures by juxtaposition.</p><p>Of all nuggets that we studied, in only three of them, the carbon atoms define space-filling polyhedra that could function as chemical voxels: nugget 8 (cubane), nugget 12 (hexaprismane or [6]-prismane) and nugget 24a (a truncated octahedron hydrocarbon).</p><p>Let us first consider nugget 8 (cubane), of point group O h . Cubane's chemical stability with respect to selfdecomposition in the absence of any other reagents is something that can be inferred from its corresponding calculated energy change of reaction. Accordingly, let us consider the possibility of a nugget 8 , cubane, molecule dissociating into either 2 molecules of cyclobutadiene (C 8 H 8 → 2C 4 H 4 ), or into 4 molecules of ethyne (C 8 H 8 → 4C 2 H 2 ), Fig. 6.</p><p>The ΔE ωB97XD/6-31G* values for these reactions are equal to + 368.8 kJ mol −1 and 551.2 kJ mol −1 ; large values that prevent such dissociation from occurring despite cubane's highly tensioned cubic structure. These ΔE ωB97XD/6-31G* values indicate that these entropy-favored self-decompositions, are unlikely to occur spontaneously. These www.nature.com/scientificreports/ findings are consistent with the fact that, as previously mentioned, cubane (nugget 8 ) has already been prepared 6 .</p><p>Further, cubane growth in three dimensions is predicted to be a stable allotrope of carbon. Actually, a carbon allotrope with this 3D-structure could be very well used as an energy storage compound and would probably exhibit a larger mass density when compared with all other allotropes of carbon, including diamond.</p><p>Let us now examine the case of nugget 12 , the hexaprismane, which has the structure of a prism with two parallel hexagonal faces linked through six square faces (Fig. 4). Hexaprismane can be thought of as a face-to-face dimer of benzene. The calculated energy of dissociation of nugget 12 into two benzene molecules (C 12 H 12 → 2C 6 H 6 ) Fig. 7a, yields a ΔE ωB97XD/6-31G* = − 389.8 kJ mol −1 , indicating that, in this case, the spontaneous chemical selfdecomposition of hexaprismane is predicted to be highly likely to occur. As a reinforcement to this affirmation, the thermal cycloaddition of two benzene molecules [6 + 6] is symmetry forbidden 35 . Indeed, so far and despite many attempts, nugget 12 , C 12 H 12 , the hexaprismane, has never been synthesized. These facts point further in the direction that the growth of nugget 12 to three dimensions would quickly spontaneously transform such a hypothetical solid into superimposed layers of graphene, such as graphite. Recently, a vertical stacking of graphene has been evolved into materials with highly tunable electronic properties and unique functionalities: the van der Waals heterostructures (vdWHs) 36 . So, for all practical purposes, it is very unlikely that the hexaprismane hydrocarbon nugget 12 could ever be of practical use as a chemical voxel. Nevertheless, the geometric concept of an hexaprismane polyhedron as a chemical voxel has recently been realized by the synthesis of isoreticular pillar layered metal organic frameworks exhibiting properties such as catalytic activity 37 . Two other self-dissociation reactions that could be thought of for the hexaprismane nugget 12 would be: (i) self-dissociation 7b,c, respectively. These two large positive calculated values reveal, as expected, that the self-decomposition of hexaprismane nugget 12 into two benzene molecules is the one most likely to occur spontaneously. The third and last carbon voxel is nugget 24a , which has the geometric form of a truncated octahedron: a space-filling Archimedean solid displaying many geometric properties, nugget 24a is a hydrocarbon, not the C24 fullerene which presents the same carbon structure 38 , which is geometrically equivalent to both the B 12 N 12 Fullerene reported by Matxain et al. 39 as well as to ZIF-8, a very stable and largely researched metal-organic framework, MOF 40 .</p><p>Due to its high symmetry, and much less strained chemical bonds than either cubane or hexaprismane, nugget 24a is a possibility to be considered as a carbon voxel. Let us now proceed by first examining its three possible forms of self-decomposition of nugget 24a : (a) into 4 benzene molecules, with a ΔE ωB97XD/6-31G* value of − 154.3 kJ; (b) into 6 cyclobutadiene molecules, with a ΔE ωB97XD/6-31G* value of + 2311.6 kJ; and (c) into 12 acetylene molecules, with a ΔE ωB97XD/6-31G* value of + 2858.9 kJ, Fig. 8a-c, respectively.</p><p>These results indicate that nugget 24a , although possibly unstable with respect to a self-decomposition into 4 benzene molecules, can be expanded as voxel into a 3D solid that would constitute an allotrope form of carbon. By being constituted by carbon atoms only, and noncoplanar vicinal six-membered rings, it cannot be split into benzene molecules or into graphene layers that would benefit from electron delocalization for stabilization. The geometric arrangement of the carbon-only hexagons in a such a perfectly packed 3D solid, placing each and every carbon atom in a condition of equilibrium of forces, would most certainly prevent its dismantling. Its infinite 3D expansion leads to a carbon-only solid compound which would constitute an allotrope of carbon. So much so that a sample has been found and properly characterized as a natural, super-hard, and transparent crystalline polymorph of carbon from the Popigai impact crater in Russia, formed because of a natural shockwave event 41 , and established to be consistent with such structure 42 .</p><p>Stability of the nuggets. Now, we turn our attention to the structural stabilities of the non-voxel nuggets.</p><p>Due to their molecular formula, their self-dissociation into ring compounds is a bit more complex, necessarily being at least into a mixture of benzene and cyclobutadiene, according to where n = 6p + 4q, with n, p, and q being integers. Further, there can be multiple combinations of p and q integer numbers that solve this expression for a given integer value of n. However, due to their geometric shapes, it is not always possible for these nuggets to be disassembled into combinations of benzene and cyclobutadiene molecules according to any stoichiometrically possible pair of values of p and q. Indeed, some of these disconnections could www.nature.com/scientificreports/ be shape forbidden. Finally, self-dissociations could also happen into ethyne molecules according to C n H n → (n/2) C 2 H 2 , a reaction that would always be possible since n is necessarily an even number and there are no geometric restrictions for any edges to be detached from the polyhedra. Table 1 shows ωB97XD/6-31G* calculated energies of reaction for all possible shape-allowed self-dissociations of all studied nuggets. From Table 1, complete dissociations into ethyne molecules are unlikely to happen for all nuggets, the same happening for self-dissociations producing any number of cyclobutadiene molecules. Thus, we can divide the nuggets into two groups, according to their energies of self-dissociation reaction ΔE ωB97XD/6-31G* .</p><p>The first group of nuggets is comprised by the ones with at least one of the calculated ΔE values being negative: nugget 12 (hexaprismane), nugget 18 , and all nuggets 24 (including the truncated octahedron, nugget 24a ). These are the nuggets that may perhaps be less stable.</p><p>The second group of potentially more stable nuggets comprises nuggets 8, 14, 16, 20 (a,b,c), 22, 26 (a,b,c) and 28 (a,b,c). This group includes nugget 28b which exhibits polyhedral chirality. As far as we know, so far, none of them have been reported in the literature, not even as a theoretical possibility. These results reveal that most of the designed nuggets are seemingly energetically stable and, probably, not easily capable of self-dissociation into simpler organic compounds.</p><p>On the other hand, the nuggets of formula C 20 H 20 , C 24 H 24 , C 26 H 26 , and C 28 H 28 possess structural isomers. Table 2 shows the energy of isomerization for all energetically favorable possibilities between these isomers. From Table 2, the most stable isomers for each of the molecular formulas are nugget 20c , nugget 24b , nugget 26a , and nugget 28a . However, transformation of one of the isomers into the other, involves fracturing a relatively rigid polyhedron through rearrangements of chemical bonds, thus rendering this type of transformation not likely.</p><p>Vibrational frequencies. We now turn to examine the rigidity of the carbon scaffolds of the nuggets, that is, how they would vary from being hard and inflexible to soft and malleable as the number of vertices (carbon atoms) increases. We regard rigidity as a desirable property in a constrained geometry polyhedral compound, contributing to its structural stability and to other properties such as less susceptibility to thermal relaxation of excited states. Accordingly, in this work, we use the lowest calculated vibrational frequency of each nugget as a measure of its rigidity, the larger this frequency, the more rigid the compound. Indeed, the lowest frequency vibration, generally corresponds to a collective movement of all atoms of the molecule, fluttering in a synchronized manner along the corresponding normal coordinate.</p><p>Table 3 shows frequency values for the lowest vibrational modes for each of the 18 nuggets, after geometry optimization, from ωB97XD/6-31G* density functional theory, DFT, calculations.</p><p>For comparison purposes, Table 3 also shows the lowest vibrational frequency of other compounds, where one can see that, as expected, cyclic compounds are generally more rigid than linear ones. Further, the presence of double bonds certainly increases rigidity in otherwise similar compounds.</p><p>Let us first consider the case of nugget 8 (cubane, C 8 H 8 ), which can be regarded as having been formed by two piled up cyclobutadienes. Cubane (ν ωB97XD/6-31G* = 628 cm −1 ) is more rigid than a cyclobutadiene (ν ωB97XD/6-31G* = 547 cm −1 ), indicating a sturdier structure. On the contrary, nugget 12 (ν ωB97XD/6-31G* = 394 cm −1 ), the [6]-prismane, which can be regarded as having been formed by two piled up benzene molecules, is actually more flexible than benzene, which has a ν ωB97XD/6-31G* value of 414 cm −1 . In general, it can be argued that the sturdier the structure, the more difficult it is for it to get disassembled. Accordingly, as previously discussed, nugget 12 would probably easily self-dismantle into two benzene molecules.</p><p>If we consider all other nuggets, from nugget 14 to nugget 28c , one of them, nugget 24a stands out as being the most rigid, having a very large lowest ν ωB97XD/6-31G* of 372 cm −1 . Nugget 24a is certainly special, displaying a very symmetric structure. This points to a molecular structure with much more balanced forces in each atom than those of the other nuggets. This reinforces the possibility of its 3D expansion, as discussed above, as likely being a very stable carbon allotrope that will probably be found to exhibit unique physical properties.</p><p>All other nuggets display rigidities that are seemingly large enough to guarantee their structural stabilities. As one would expect, the more prolate ones (the "c" ones) are less rigid than the more spherical ones (the "a" ones).</p><p>Naturally, as the number of carbon atoms in their structures increases, the nuggets tend to become less and less rigid. Nevertheless, their rigidities are, of course, still larger by a large difference than those displayed by the n-alkanes, and even by the cyclic alkanes with the same number of carbon atoms. All of this points to the direction that they could all be synthesized, as the synthetically challenging cubane indeed has been 6 .</p><p>As rigid as they are, the nuggets can then be fused together to form even larger structures, generating an assortment of shapes and forms that can bring about regular and irregular solids, porous structures, etc., with many potential applications to materials science. To examine such possibilities, let us now turn to their energetic properties of fusing.</p><!><p>To be able to design novel 1D, 2D, and 3D-scaffolds from the set of nuggets considered in this article, let us now study the ability of these systems to perform face-fusion reactions. Because the nuggets present both square and hexagonal faces, their growths must occur via the fusion reactions of either two square or two hexagonal faces. However, not all these face-fusions may take place because some of the faces of these nuggets, mostly the hexagonal faces, are not exactly flat surfaces, but slightly skew polygons, whose vertices are not all coplanar. In such cases, for a fusion to occur, a requirement of spatial complementarity may not always be possible because the hexagonal faces tend to be all concave. On the other hand, square faces in these polyhedra are almost all invariably planar. Therefore, face-fusion reactions are generally predicted to occur more frequently through square faces, rather than via the usually more skewed hexagonal faces. www.nature.com/scientificreports/ all leading to a huge number of possibilities. Table 4 shows the energies of reactions, one for each type of fusion (whenever possible) that displayed the least ωB97XD/6-31G* energy values of reaction for each pair of identical nuggets. Results on Table 4 indicate that while there are 18 square face fusions, the number of hexagonal face fusions possible is only 5. The values of energy of hexagonal face-fusion reactions range from − 185.5 kJ for nugget 24a to 638.8 kJ to nugget 12 , with the same numbers for square face fusion reactions ranging from − 80.2 kJ, for nugget 26b , to + 427.4 kJ for nugget 8 , cubane. Although the larger the nugget, the more likely it is to display negative face-fusion energies of reaction, we notice an exception to this rule: among the 18 nuggets designed in this article, two identical molecules of the carbon voxel nugget 24a are predicted to perform hexagonal face-fusion reactions with the largest negative value of ΔE ωB97XD/6-31G* = − 185.0 kJ. Therefore, of all nuggets studied, nugget 24a www.nature.com/scientificreports/ is predicted to exhibit the largest aptitude to be applied to growth as 1D, 2D, and 3D-scaffolds, especially when one considers its voxel characteristics.</p><!><p>Upon face-fusion reactions, nuggets can grow into either regular or irregular structures. Let us first consider possible fused compounds displaying structures with regular patterns. The simplest of these patterns are tessellations: covering of the space with nuggets, without overlaps or gaps. Tessellations can occur in one, two or three dimensions, and are the result of face-fusion reactions of a nugget, or of a combination of nuggets, made up by their translations, rotations or reflections. The carbon voxels, nugget 8 , nugget 12 and nugget 24a would be natural candidates. However, as explained above, only nugget 24a would make such a chemically feasible tile for this purpose. Let us therefore turn to consider the growth of nugget 24a in 1 dimension. The idealized self-fusion reaction of two of them via one of its all-equivalent hexagonal faces, 2C 24 H 24 → C 42 H 36 + C 6 H 12 , ΔE ωB97XD/6-31G* is − 185.0 kJ, where C 6 H 12 refers to cyclohexane leads to a generator of the simplest 1D scaffold extension. Figure 9 shows its optimized geometry together with the released cyclohexane for easier visualization.</p><p>Next, to evaluate the ability of nugget 24a in generating 2D-scaffolds, the following idealized fusion reaction was now considered: C 24 H 24 + C 42 H 36 → C 58 H 46 + C 8 H 14 , see Fig. 10 (left), where C 8 H 14 is (1R,6S)-bicyclo[4.2.0] octane, Fig. 10 (right) and whose predicted energy of reaction is − 85.7 kJ. Due to its 2D-structural arrangement its stability is substantially more accentuated when compared with the formation of the essentially linear C 60 H 48 1D compound obtained by fusing together the 1d-generator compound in Fig. 9 with another nugget 24a . This is because now a larger quantity of viable fusion reactions was carried out.</p><p>Finally, let us evaluate the ability of nugget 24a in generating 3D-scaffolds. The following idealized fusion reaction was considered: C 58 H 46 + C 24 H 24 → C 71 H 52 + C 11 H 18 , see Fig. 11, where C 11 H 18 stands for (1s,1aS,4ar,7aR)nonahydro-1H-cyclobuta[de]naphthalene.</p><p>The infinite 3D expansion of this polyhedron will lead to a carbon-only compound that would constitute an allotrope of carbon 42 . A solid model image of a piece of this allotrope can be seen in Fig. 12 below. It is noteworthy that, by acting as a space filling carbon voxel in this manner, at least in principle, nugget 24a could be employed to generate any 3D sculpture with itself as its finest granularity level.</p><p>Another seemingly rigid allotrope of carbon can also be made from nugget 24a in the form of a regular skew apeirohedron. Similarly, but not exactly like the one advanced by Zhou et al. 43 , this will be formed by joining the carbon voxels nugget 24a through hexagonal pyramidal bridges linking hexagonal faces of one to square faces of others, in a manner so that each external square face of the hexagonal prismatic bridge shares an edge with a square face of one of the polyhedra while its opposite edge is shared with a hexagonal face of the other. Figure 13 exemplifies such a hexagonal prismatic bridge between two nuggets 24a . In this case, the idealized chemical 4), these bridged connections of hexagonal faces are more energetically favorable than connections via square faces. Therefore, the regular skew apeirohedron can then be formed by linking together, in this manner, each nugget 24a by 4 of its 8 hexagonal faces according to Fig. 14 below 44 . This putative allotrope of carbon, adding to previous exotic carbon allotropes 45 , would be very stable and rigid. Its density, however, would be evidently smaller than that of the space filling allotrope shown in Fig. 12. The presence of zeolite-like nanoporous cavities inside its structure could be a singular feature, that could perhaps prove to be the origin of many emerging and interesting properties.</p><p>Other types of polyhedral solids, with larger cavities, can also be conceptualized, such as the one made, this time by nugget 16 , via square face-fusions, and whose projection in one plane reveals a semiregular or Archimedean tessellation, that can be grown indefinitely Fig. 15. Such a compound, if ever obtained, would also likely behave as a load resisting skeleton due to its symmetric nature. Furthermore, this structure could also be grown www.nature.com/scientificreports/ in 3D leading to lengthy tubular cavities that could prove eventually useful. Structures such as these, with large cavities in the middle, suggest applications to materials science as catalysts, porous powders, etc. Many more combinations can be conceptualized by connecting the nuggets. Figure 16 shows a helix compound made by fusion of nugget 28b via two of its quasi-planar hexagonal faces. Such a compound, whose form resembles a twisted rope, would exhibit helicity, a form of chirality.</p><p>Besides, these regular and aesthetically appealing structures, several other large structures can be conceived by binding together several of the nuggets, leading to a myriad of hydrocarbon structures that would extend far beyond what is being here presented. The geometric possibilities of molecular structures that could in principle be formed based on these nuggets are truly vast: "symmetries, spirals, trees, waves, foams, tessellations, meanders, cracks, and stripes with fractal dimensions" 46 .</p><!><p>Euler's theorem and topological strategies were employed in order to theoretically design a set of 18 hydrocarbon nuggets of general formula C n H n containing four-and six-membered rings, that exist up to 28 vertexes. From Euler's theorem we demonstrated that all such polyhedra must contain exactly six four-membered rings, for an arbitrary number of six-membered rings equal or greater than two. Among these 18 nuggets, 13 are novel systems, with 3 of them exhibiting polyhedral chirality.</p><p>We also showed that, with the exception of hexaprismane, which is predicted to easily self-dissociate into two benzene molecules, and therefore unlikely to be synthesizable; and also with the exception of nugget 18 , which is presumably expected to dissociate into three benzene molecules, all other nuggets are likely to be relatively stable and not self-dissociate or degrade.</p><p>Subsequently, vibrational properties revealed that the designed nuggets are sufficiently rigid. In this sense, the nuggets with 28 carbons are predicted to exhibit a structural rigidity, in average about 100 times greater than that of the linear alkane n-octacosane C 28 H 58 .</p><p>We also explored the expansions of these nuggets into larger structures by face-fusion reactions involving mainly hexagonal and sometimes square faces.</p><p>Nugget 24a , the carbon voxel, resembles the most a fullerene (6 and 5-membered rings, however) in terms of the spherical shape, and possesses a chemical structure similar to the MOF ZIF-8. Due to its energetically favorable face-fusion reactions, Nugget 24a is deemed to be the most suitable one to have a large potential to be applied to growth as 1D, 2D, and 3D-scaffolds. Accordingly, any 3D sculpture could be generated with nugget 24a www.nature.com/scientificreports/ at its finest granularity level if sufficient synthetic control is one day discovered; or perhaps by carving from the innovative carbon allotrope presented in Fig. 11.</p><p>In conclusion, as mentioned in the previous section, the nuggets could be in principle expanded into all sorts of forms: "symmetries, spirals, trees, waves, foams, tessellations, meanders, cracks, and stripes of fractal dimensions" 46 . Their scaffolds may be decorated with strategically placed substituents as quantized perturbations, to promote attractive forces between them for a potential use in molecular tectonics. Perhaps they can form designer hyperstructures made layer by layer in a precisely chosen sequence where electronic or even exotic phenomena, typically requiring exceptionally low temperatures, can be explored. In summary, these are structures that should www.nature.com/scientificreports/ be considered as possibilities and of interest to researchers from all areas of carbonaceous nanomaterials (e.g., fullerene, nanotube, graphene, etc.). Finally, we also present the perspective of novel carbon allotropes, both space filled, as well as with cavities, hinting at interesting properties if synthesized or found as it appears to be the case with the natural, super-hard, and transparent crystalline polymorph of carbon from the Popigai impact crater in Russia, formed because of a natural shockwave event 41,42 .</p><p>Received: 14 December 2020; Accepted: 12 February 2021</p>

Scientific Reports - Nature

Chemical synthesis and immunological evaluation of new generation multivalent anticancer vaccines based on a Tn antigen analogue

Tumor associated carbohydrate antigens (TACAs), such as the Tn antigen, have emerged as key targets for the development of synthetic anticancer vaccines. However, the induction of potent and functional immune responses has been challenging and, in most cases, unsuccessful. Herein, we report the design, synthesis and immunological evaluation in mice of Tn-based vaccine candidates with multivalent presentation of the Tn antigen (up to 16 copies), both in its native serine-linked display (Tn-Ser) and as an oxime-linked Tn analogue (Tn-oxime). The high valent vaccine prototypes were synthesized through a late-stage convergent assembly (Tn-Ser construct) and a versatile divergent strategy (Tn-oxime analogue), using chemoselective click-type chemistry. The hexadecavalent Tn-oxime construct induced robust, Tn-specific humoral and CD4 + /CD8 + cellular responses, with antibodies able to bind the Tn antigen on the MCF7 cancer cell surface. The superior synthetic accessibility and immunological properties of this fully-synthetic vaccine prototype makes it a compelling candidate for further advancement towards safe and effective synthetic anticancer vaccines.

chemical_synthesis_and_immunological_evaluation_of_new_generation_multivalent_anticancer_vaccines_ba

Introduction<!>Design and synthesis<!>Immunological evaluation<!>Conclusions<!>Ethical statement<!>Conflicts of interest

<p>Cancer immunotherapy approaches based on synthetic vaccines containing tumor-associated carbohydrate antigens (TACAs) represent a topic of high interest. [1][2][3] Anticancer vaccines have been conceived as innovative therapeutic agents for inclusion in multi-therapy settings in order to elicit antitumor immunity in patients with prior surgical resection of a primary tumor. This approach has the potential of preventing, or at least prolonging the time to recurrence at the metastatic sites, while exhibiting reduced toxicity compared to cytotoxic treatments such as radio-and chemotherapy. 4 Active immunotherapy educates the immune system of a patient to recognize tumor-associated antigens displayed on the vaccine construct and to trigger an immune response selectively directed towards malignant cells. 5,6 Extraordinary advances in Immunology and Synthetic Chemistry have led to the development of subunit vaccines based on tumor-associated glycans for the generation of more effective carbohydrate-directed immune responses. [7][8][9][10] The administration of carbohydrate antigens alone in a vaccine formulation can only weakly activate B-cells, resulting in production of low-affinity IgM antibodies and short-living plasma cells. This is because, apart from a few exceptions, 11 carbohydrates are T-independent antigens that are not able to associate with MHC molecules and activate helper T-cells (T H ) to provide the required signals for B-cell stimulation. This, in turn, results in impaired antibody isotype-switching (to highaffinity IgG antibodies) and decient production of both longliving plasma cells and memory B-cells. 12,13 As a consequence, conjugate-vaccine design relies on the hapten-carrier effect, whereby helper T-cell epitopes are included in the form of a protein carrier (semi-synthetic vaccines) [14][15][16] or as discrete T Hepitopes (fully-synthetic vaccines), either directly connected to B-cell epitopes through linker moieties or graed onto nonimmunogenic scaffolds or nanoparticles. [17][18][19] Aberrantly glycosylated versions of the protein mucin-1 (MUC1) are overexpressed in most human epithelial cancers, 20,21 making this glycoprotein a target of high interest for both diagnostic and immunotherapeutic applications. 22 Cancer-related MUC1 tandem repeats display truncated carbohydrate moieties at O-glycosylation sites, exposing glycan epitopes such as the Tn antigen that are normally hidden in mucins expressed in non-transformed cells. The Tn antigen consists of an N-acetyl-D-galactosamine (GalNAc) unit a-O-linked to the serine (Ser) or threonine (Thr) of a peptide backbone, 23,24 and is overexpressed in approximately 90% of breast carcinomas, 25 and in 70-90% of colon, bladder, cervix, ovary, stomach and prostate cancers. [26][27][28] As such, it represents an excellent target for vaccine development, and we and other research groups have focused efforts on the design of anticancer vaccine candidates based on the Tn antigen. [29][30][31][32][33][34][35][36][37][38] The inherent low immunogenicity of carbohydrate antigens is even more critical in TACAs, as they are self-derived antigens and therefore not prone to being recognized as foreign molecules by the immune system. 39,40 Thus, a fundamental challenge for TACAbased vaccines involves the ability to produce functional, isotype-switched, IgG antibodies that are able to recognize native antigens expressed on cancer cells and selectively promote their clearance. Advances in the chemical-immunology eld have provided evidence that through an accurate structural design, it is possible to develop effective TACA-based anticancer vaccines with promising preclinical outcomes. 10,[41][42][43][44][45][46][47][48] To overcome important limitations associated with protein-hapten conjugates, [49][50][51] fully synthetic vaccine approaches are being developed that enable the assembly of structurally dened and easily characterizable constructs via modular chemical strategies. 15,[52][53][54][55] In this context, we report herein the design, synthesis and immunological evaluation of unprecedented fully synthetic vaccines with high Tn-antigen valency that are able to elicit robust and functional immune responses in mice.</p><!><p>Since 2005, some of us have pioneered the use of cyclic Regioselectively Addressable Functionalized Templates (RAFTs) as designed scaffolds for the presentation of B-and T-cell epitopes in synthetic vaccine prototypes. 18,56,57 The RAFT core consists of a cyclic decapeptide featuring two Pro-Gly residues as b-turn inducers, which stabilize its conformation in solution and provide a relatively rigid structure where lysines' side chains point towards two spatially opposite directions, thus dening an upper and a lower domain. 58 In these early prototypes, four units of the Tn antigen moiety were graed onto these scaffolds by using oxime ligation, a versatile chemoselective reaction that enabled the straightforward preparation of tetravalent structures by coupling deprotected aminooxy-glycan moieties without the need for activating agents. 59,60 While tetravalent vaccine constructs were immunogenic, 56,59,[61][62][63] we reasoned that higher carbohydrate valency would enhance recognition properties due to the multivalent effect, leading to increased B-cell receptor (BCR) clustering, [64][65][66] which represents an early and key step that impacts downstream immune signaling, including B-T cell communication. 67,68 Therefore, to go one step further and improve the immunological properties and potency of early prototypes, we developed a new set of fully synthetic Tn-based vaccine candidates displaying a 4-fold increase in B-cell epitope ratio as well as additional T cell epitopes for more efficient immune responses (Fig. 1a).</p><p>Thus, multivalency-driven recognition of carbohydrate antigens by BCRs followed by receptor-mediated internalization of the vaccine construct incorporating a CD4 + epitope would allow antigen-presenting B cells to load the T H epitope onto MHC-II molecules on the cell surface. Direct B-T cell contact via T-cell receptor (TCR) on CD4 + T lymphocytes would provide bidirectional signaling, ultimately leading to B-cell proliferation and differentiation (Fig. 1b). [69][70][71] Initially, we generated a small series of multivalent glycodendrimers as B-cell epitope carriers (Scheme 1, compounds 2, 5-8) to investigate whether a higher order multivalency could be benecial to antibody binding in an anti-Tn mAb interaction assay (Fig. 2). 74 We rst synthesized Tn-based glycodendrimers 2 and 5, in which GalNAc units are a-O-linked to the Ser hydroxyl groups (referred to as "Tn-Ser" along the text), as in the native Tn antigen. Starting from intermediate 1 (see the ESI, Scheme S1 †), bearing four protected Tn-Ser residues, global Oacetyl and Fmoc removal gave tetravalent Tn-Ser glycodendrimer 2 (Scheme 1a). Meanwhile, functionalization of the free amino group of the lower lysine side chain in 1 with Bocaminooxyacetic acid N-hydroxysuccinimide ester (Boc-Aoa-NHS), 75 followed by base-mediated deprotection of the four GalNAc-Ser residues afforded intermediate 3.</p><p>Deprotection of the Boc-aminooxy group on 3 and subsequent oxime ligation with core scaffold 4, 72 which displays four a-oxo-aldehyde groups, 76 provided hexadecavalent Tn-Ser glycodendrimer 5 in four steps from intermediate 1 following a convergent strategy involving unprotected moieties (see the ESI, Scheme S4 †). Conversely, the Tn-analogue glycodendrimer 8, in which the GalNAc units are attached via oxime linkages (referred to as "Tn-oxime" along the text), was synthesized in a divergent fashion by graing the aminooxy-GalNAc moiety onto a hexadecavalent, a-oxo-aldehyde functionalized scaffold as the last step in the route. 73 With our set of compounds in hand featuring hexadecavalent Tn-Ser/oxime structures (Scheme 1, compounds 5 and 8, respectively) and their tetravalent analogues (Scheme 1, compounds 2 and 6 (ref. 61), respectively), we then performed direct interaction assays to evaluate their ability to be recognized by anti-Tn antibodies (anti-Tn mAb clone 9A7). 74 Tetravalent construct bearing GlcNAc-oxime residues (Scheme 1, compound 7 (ref. 73)) was used as a negative control (Fig. 2).</p><p>While hexadecavalent Tn-Ser compound 5 exhibited the strongest binding, the interaction curve of the Tn-oxime glycodendrimer 8 indicated that this multivalent system can serve as an effective analogue of the native Tn antigen. In contrast, tetravalent Tn-containing compounds 2 and 6 showed absorbance values close to the baseline, comparable to those of negative control 7. These initial results prompted us to pursuit the synthesis of the complete vaccine structures based on selected glycodendrimers 5 and 8 for immunogenicity studies in mice. In addition to the hexadecavalent vaccine prototypes, the corresponding tetravalent vaccine constructs derived from 2 and 6 were also synthesized to evaluate the impact of antigen valency in the elicited immune response.</p><p>To complete the design of our synthetic vaccine prototypes, we incorporated T helper CD4 + and CD8 + epitopes from ovalbumin [77][78][79][80] (OVA) into the previous glycodendrimers to generate potent immune responses with strong and long-lasting production of IgG antibodies against the T-cell independent Tn carbohydrate antigen. OVA 323-339 CD4 + T helper and OVA 257-264 CD8 + T cell epitopes were synthesized "in-line" incorporating a cysteine residue at the C-terminus for further chemoselective conjugation to the core cyclopeptide scaffold (Scheme 2). The synthesis started with protected peptide sequence 9, which was obtained using standard Fmoc-based automated solid phase peptide synthesis (SPPS) on a Rink amide resin. Treatment with a TFA/TIS/H 2 O (96 : 2 : 2) cocktail resulted in the removal of all acid-labile side-chain protecting groups, with concomitant cleavage from the resin, affording peptide 10 in a 41% overall yield.</p><p>We rst focused our efforts on the synthesis of the "nativelike" Tn-Ser vaccine prototypes based on glycodendrimers 2 and 5. Starting from building block 1 (see Scheme 1), which displays protected Tn-Ser peripheral residues and a free amino group of the lysine of the scaffold, coupling with the NHS ester of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine (Boc-Cys(NPys)-NHS) in DMF provided intermediate 11 (Scheme 3a). 81,82 Taking advantage of the thiol activating nature of the NPys group, Boc removal (1 : 1 TFA/CH 2 Cl 2 ) from the cysteine residue, followed by disulde bridge formation with cysteine-containing peptide protected Tn-Ser peripheral glycodendrimer S5 to the a-oxoaldehyde-bearing central scaffold 4 through oxime linkages (see the ESI, Scheme S6 †), was functionalized at its lower domain with OVA peptide 10 via disulde bridge formation by using the three-step procedure described above (Scheme 3b). The resulting Tn-Ser hexadecavalent construct 15, however, was found to be unstable to the various deprotection conditions used to remove the O-acetyl and Fmoc moieties (see the ESI, Scheme S8 †), which presumably affected the internal oxime linkages, failing to afford the fully-deprotected vaccine candidate 16.</p><p>To address this problem, we designed an alternative strategy towards a modied vaccine construct (19) in which the internal oxime linkages were replaced with 1,4-triazoles (Scheme 4). Since this modication involves the "internal" part of the nal glycosylated structure and not the external B-cell epitope display, the binding ability of the construct should not be affected. In contrast to the synthetic route towards 16, the new strategy allows for a more convergent assembly via late-stage CuAAC and also enables O-acetyl and Fmoc removal from the Tn-Ser moiety at an earlier stage in the synthesis. In the event, CuAAC reaction between a slight excess of fully-deprotected peripheral glycodendrimer 17 (see the ESI, Scheme S9 †) equipped with an alkyne handle on the lower domain and OVAfunctionalized, azide-bearing core scaffold 18 (see the ESI, Scheme S10 †) was carried out in a degassed DMF/PBS mixture using our previously reported protocol in the presence of With Tn-Ser vaccines 13 and 19 in hand, we next directed our efforts towards the synthesis of the Tn-oxime vaccine candidates. Unlike for the Tn-Ser constructs, the presence of a single free amino acid (Lys on the lower domain of the scaffold) in glycodendrimers 6 and 8 enabled the use of a versatile divergent strategy involving late-stage chemoselective functionalization at this position followed by installation of the T cell OVA epitopes in the last step of the route. Thus, starting from glycodendrimer 6 (see Scheme 1b), vaccine construct 20 was synthesized in 53% yield over three steps via Boc-Cys(Npys) installation, and disulde bridge formation with OVA peptide 10 (Scheme 5). Following this three-step sequence, vaccine candidate 21 was obtained analogously from hexadecavalent glycodendrimer 8, although its poor solubility in DMF required the use of a DMF/ PBS mixture (1 : 1, pH 7.4) to achieve the coupling of the Boc-Cys(NPys) residue with a satisfying 41% yield aer RP-HPLC purication (Scheme 5).</p><!><p>With the synthetic vaccine candidates in hand, we next evaluated their ability to elicit immune responses. Groups of ve C57BL/6 mice were immunized subcutaneously three times every two weeks with each vaccine construct (50 mg dose) (Group A: hexadecavalent Tn-oxime 21; Group B: hexadecavalent Tn-Ser 19; Group C: tetravalent Tn-oxime 20; Group D: tetravalent Tn-Ser 13) in combination with the saponin QS-21 as an adjuvant 84 (20 mg). In addition, a group of control mice (Group E) were immunized with compound 21 without adjuvant (Fig. 3).</p><p>Three weeks aer the last immunization, blood was collected for serological analysis and the mice were sacriced to assess cellular immunity. Notably, no toxic side effects (e.g. local inammation, systemic reactions, mouse weight loss or death) were observed over the course of the immunizations (data not shown), indicating the non-toxicity of the synthetic vaccine constructs. First, we evaluated the ability of the constructs to generate antibody responses and of the antisera to bind the Tnantigen in different presentation modes [native Tn-Ser residues (5, 2) and unnatural Tn-oxime analogues (8, 6), as well as in higher (5, 8) and lower (2, 6) valency, see Scheme 1]. Microtiter plates were coated with the corresponding Tn glycodendrimers lacking the OVA peptide and the total anti-Tn IgG levels in blood sera were detected by ELISA. Group A mice, immunized with hexadecavalent oxime-linked Tn construct 21 in combination with QS-21, exhibited the highest IgG levels against its glycodendrimer counterpart 8 (Fig. 4a). Moreover, this group was the only one in which all ve mice were able to generate humoral responses. In contrast, Group B (hexadecavalent Tn-Ser 19 + QS-21) showed variable but lower IgG levels, while Groups C (20 + QS-21) and D (13 + QS-21) in which mice were immunized with the corresponding tetravalent constructs, exhibited IgG levels similar to the no adjuvant control (Group E). Interestingly, IgG antibodies produced by Group A mice (hexadecavalent Tnoxime 21 + QS-21) were also able to recognize hexadecavalent Tn-Ser glycodendrimer 5 (Fig. 4b), showing therefore no clear preference for the native or unnatural Tn presentation on the scaffold. However, antisera from these mice (Group A) were less efficient in binding the Tn antigen presented through lowvalency glycodendrimers Tn-oxime 6 and Tn-Ser 2 (Fig. 4c and d). Therefore, increased Tn antigen valency was found to be crucial for high-affinity binding of the IgG antibodies to the Tn antigen construct. On the other hand, while Group B mice showed antisera (IgG antibodies) able to bind both high-valency glycodendrimers 8 and 5 (Fig. 4a and b), their levels were considerably lower than those exhibited by Group A, and were not able to bind tetravalent glycodendrimers 6 and 2 (Fig. 4c and d). In these assays, tetravalent compounds 20 and 13 (Groups C and D, respectively) elicited IgG antibody levels similar to the no adjuvant Group E, highlighting the importance of the increased multivalency of vaccine candidates 21 and 19 to generate potent humoral responses. In addition to the presence of the OVA CD4 + T helper epitope, co-administration of QS-21 as an adjuvant was found to be essential for antibody classswitching and elicitation of IgG antibodies, with IgM antibody levels being negligible at the last time point (see the ESI, Fig. S45 †). 85 Antibody titration using oxime-linked glycodendrimer 8 for coating was carried out for the two groups showing the highest OD values, i.e. Group A (mice immunized with hexadecavalent Tn-oxime 21 + QS-21) and B (mice vaccinated with hexadecavalent Tn-Ser 19 + QS-21) (Fig. 5).</p><p>Antibody subtyping of the anti-8 IgG isotypes revealed that Group A mice showed not only high total IgG titers but also elevated levels of the IgG1, IgG2b and IgG2c antibody subtypes. Interestingly, the IgG2c antibody titers in this group were as high as those observed for IgG1 antibodies, suggesting that 21 in combination with QS-21 elicits a balanced Th1/Th2 immune response (Fig. 5e). The IgG2c subtype is of particular interest since it is associated with potent antitumor effect such as complement-and antibody-dependent cell toxicity in mice. 86 In contrast, IgG antibody titers from Group B mice were not signicantly higher than those of the no adjuvant control Group E, and subtyping of these antibodies revealed a bias towards the IgG2b subclass. These results indicate that hexadecavalent vaccine construct 21 bearing oxime-linked Tn antigen analogue is more efficient than its native Tn-Ser counterpart 19 in generating potent humoral responses, and was therefore selected for further immunological studies.</p><p>We then evaluated the ability of the antisera to recognize the Tn antigen in a native context and analyzed the binding of the vaccine-induced serum antibodies to a human cancer cell line (MCF7) expressing the natural Tn antigen by using uorescence microscopy. Notably, IgG antibodies elicited by immunization with compound 21 plus QS-21 (Group A mice) were able to specically bind Tn-expressing MCF7 cells. Moreover, indirect immunouorescence images showed high signal coming from sera from Group A mice, whereas antisera from mice immunized with 21 without QS-21 (Group E) showed no Tn-specic IgG binding (Fig. 6a). Antibody binding of sera from mice Group A showed broad membrane surface localization (Fig. 6b). In contrast, although compound 21 alone (Group E) was able to elicit IgG antibodies to a small extent (Fig. 5e), these antibodies were not able to specically recognize natural Tn antigen expressed in this tumor cell. These results highlight that antibodies elicited by the Tn-oxime analogue vaccine construct 21 coadministered with QS-21 are functional and able to recognize the native Tn antigen expressed on the cancer cell surface, conrming the tumor specicity of the generated antibody responses.</p><p>Next, we also investigated the cellular immune responses elicited by Tn-oxime construct 21 by testing its ability to specically activate T cells. At the time of sacrice (three weeks aer the last immunization, day 49), whole splenocytes were harvested and assayed for T cell restimulation in the presence of full-length OVA protein. Aer 48 h stimulation, splenocytes were analyzed for activation markers using ow cytometry by assessing the percentage of CD4 + CD44 high (ref. 87 and 88) and CD8 + CD107a + (ref. 89) in the restimulated splenocyte pools. Notably, vaccine construct 21 in combination with QS-21 activated CD4 + as well as CD8 + T cells aer stimulation with the specic antigen, conrming the immunogenicity of compound 21 for both B-and T-cells (Fig. 7). Therefore, construct 21 stands out as a potent and safe synthetic Tn analogue vaccine candidate that, coadministered with QS-21, is able to induce robust humoral and cellular immune responses without toxic side effects.</p><!><p>In summary, we report the design, synthesis and immunological evaluation of fully synthetic hexadecavalent Tn-based vaccine candidates with a 4-fold increased carbohydrate epitope ratio compared to earlier generation vaccines. These Tn-based constructs were designed as higher-valency B celltargeting modules to address the need for an effective BCR clustering to boost B cell activation and antibody production. The synthesis of the hexadecavalent Tn-Ser construct resulted especially challenging due to late-stage global deprotection issues that resulted in cleavage of the internal oxime linkages and product decomposition. Instead, synthetic access to an alternative Tn-Ser construct (19, Scheme 4) was achieved by replacing the four internal oxime linkers with triazole groups, enabling a convergent assembly of the modules using CuAAC click chemistry. To circumvent the challenges associated to the native Tn-Ser vaccine candidates, as a more practical chemical solution we generated more synthetically accessible vaccine constructs based on Tn analogues displaying the Tn antigen via oxime linkages. Thus, we developed a divergent and streamlined strategy whereby convenient functionalization of the hexadecavalent Tn-oxime glycodendrimer 8 was carried out in a three-step late-stage sequence to give the nal vaccine structure 21 (Scheme 5). In mouse immunization studies with QS-21 as an adjuvant, hexadecavalent Tn-oxime and Tn-Ser constructs 21 and 19 elicited higher IgG antibody responses than their tetravalent analogues 20 and 13, conrming the high-valency benet of our new-generation vaccines. Between the hexadecavalent constructs, Tn-oxime vaccine candidate 21 elicited the highest IgG antibody titers, consistently higher than those generated by Tn-Ser construct 19, and were also the most efficient in binding the Tn antigen in both presentation modes, i.e. oxime-linked Tn (8) and Tn-Ser (5). Moreover, IgG subtyping of the vaccine-induced antibodies showed different patterns for Group A and Group B sera. Immunization with 21 (plus QS-21, Group A) elicited high IgG1 and IgG2c isotype titers, whereas vaccination with 19 (plus QS-21, Group B) revealed a bias towards the IgG2b subtype (Fig. 5). Notably, antibodies generated by Group A mice were also able to bind Tn-expressing MCF7 cancer cells as assessed by uorescence microscopy (Fig. 6), conrming the tumoral specicity of the produced antibodies. Furthermore, vaccine construct 21 was also able to activate CD4 + and CD8 + cellular responses, as assessed by ow cytometry analysis of OVA-restimulated splenocytes (Fig. 7). In conclusion, we have developed a synthetically accessible newgeneration vaccine candidate (21) based on a multivalent oxime-linked Tn-antigen analogue that showed no toxicity and was able to generate potent and functional humoral and cellular immune responses. The superiority of this fully synthetic Tnbased vaccine construct 21, in terms of both its synthetic accessibility and immunological properties, makes it a promising candidate for further development towards cancer immunotherapy applications.</p><!><p>Animals were cared for and handled in compliance with the Guidelines for Accommodation and Care of Animals (European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientic Purposes) and internal guidelines. Mice were housed in standard cages and fed on a standard diet ad libitum. All the experimental procedures were approved by the appropriate local authorities. The CIC bioGUNE animal facility is fully accredited by AAALAC International.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Aging Enhances Production of Reactive Oxygen Species and Bactericidal Activity in Peritoneal Macrophages by Up-Regulating Classical Activation Pathways

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection is central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3\xe2\x80\x934 mo) and aged (14\xe2\x80\x9315 mo) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in macrophage recruitment into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to LPS. Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in proteins linked to immune cell pathways under both basal conditions and following LPS activation. Immune pathways up-regulated in macrophages isolated from aged mice include proteins critical to formation of the immunoproteasome. Detection of these latter proteins are dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases many proteins involved in immune cell function in aged Balb/c mice. Collectively these results indicate that macrophages isolated from old mice are in a pre-activated state that enhances their sensitivities of LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection to general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the formation of reactive oxygen species is likely to contribute to observed age-dependent increases in oxidative damage that underlie many diseases of the elderly.

aging_enhances_production_of_reactive_oxygen_species_and_bactericidal_activity_in_peritoneal_macroph

<!>Materials<!>Macrophage Elicitation<!>Macrophage Isolation<!>Nitric Oxide Measurements<!>Bactericidal Activity<!>Macrophage Lysis<!>Cysteine Alkylation and Trypsin Digestion<!>LC-MS Analysis and Peptide Identification<!>Quantitative Measurements of Protein Abundance Changes<!>Macrophage Isolation and Characterization<!>In vivo Functional Measurements of Macrophage Recruitment<!>Ex vivo ROS Generation and Bactericidal Activity and<!>Protein Identification and Pathway Interrogation<!>Identification of Protein Abundance Changes<!>DISCUSSION

<p>Within the animal kingdom, the innate immune response is highly conserved. Specific classes of immune cells recognize characteristic pathogen-associated biomolecules (e.g., conserved cell wall components like lipopolysaccharides (LPS1)) to orchestrate their rapid clearance at localized sites. For example, macrophages rapidly engulf and kill entrapped pathogens through a coordinated oxidative burst, simultaneously releasing inflammatory cytokines (e.g., TNFα) to recruit additional immune cells to the site of infection. In vertebrates, activated macrophages also act as an interface with the adaptive immune system through the presentation of antigenic determinants derived from engulfed pathogens that act to activate and maintain T cell activation following their recruitment to sites of infection. Microbial infections, in turn, reprogram cytotoxic lymphocytes and T helper cell responses as part of the adaptive immune system to induce their proliferation and promote the release of IFNγ to sensitize macrophages at distant sites. IFNγ exposure acts to promote macrophage activation upon bacterial recognition to up-regulate antigen presentation, the release of inflammatory cytokines, and the production of reactive oxygen species (ROS) that act to kill microorganisms. Control of macrophage activation is critical as the oxidative burst, resulting from their activation, inflicts collateral damage to host macromolecules and tissues, which contribute to a range of different age-related diseases (1–2).</p><p>Common models of macrophage aging emphasize the accumulation of oxidative DNA damage that results in their functional dysregulation, reported to impair the ability to respond to LPS, generate ROS, and present antigens through class-I and class-II major histocompatibilty complex (i.e., MHC-I and MHC-II) pathways necessary for the activation of cytotoxic lymphocytes and T-helper cells (3). Alternatively, changes in levels of circulating cytokines may induce differentiation to alter macrophage polarity, acting to modify the repertoire and magnitude of available functional responses. Complicating an understanding of age-dependent declines in immune function is the coupling between the innate and adaptive immune systems, which act together to coordinate cellular responses against pathogen exposures. While it is understood that adaptive immunity declines with age (4–5), discrepant results have been reported regarding age-dependent changes in macrophage function and their importance in downgrading immune defenses, which predispose aged animals to infection and contribute to the development of many of the diseases of the elderly (6–8). For example, isolated resident peritoneal macrophages from middle-aged (12 mo) and old (21 mo) mice have been reported to show an enhanced ability to kill herpes simplex virus in comparison to young (2 mo) controls, despite reported decreases in their ability to generate nitric oxide and other reactive oxygen species (ROS) known to mediate bacterial killing (9). Similar increases in pathogen clearance are observed for aged Balb/c mice (18 mo) when animals were challenged with a bacterial infection involving Leishmania major, despite the absence of any significant differences in rates of phagocytosis, bacterial killing, or nitric oxide production following isolation and uniform sensitization of peritoneal macrophages by IFNγ (10). An important clue to these apparently contradictory results comes from the observation that enhanced rates of bacterial killing observed in aged mice depend on their prior exposure to normal circulating pathogens, since housing under clean room conditions abrogate age-dependent increases in bacterial resistance (10). These latter results suggest that age-dependent alterations in macrophage function result from an enhanced sensitivity to environmental exposures, which may occur due to age-dependent reductions in physical barriers that limit pathogen entry and act to sensitize macrophages to promote their rapid activation in response to infection.</p><p>As environmentally induced changes in macrophage function should result in characteristic alterations in the proteome that are indicative of shifts in activation pathways, we have examined possible linkages between protein abundance changes and molecular pathways involving immune function, and their relationship to phenotypic changes in macrophage function. Peritoneal macrophages were isolated from young (3–4 mo) and aged (14–15 mo) Balb/c mice, which represents a normal model for use in aging measurements (9–10). The aged mice (14–15 mo) used in our measurements are near the average lifespan of this mouse strain, which is 485 ± 9 days (about 16 months) (11), permitting an investigation of age-dependent cellular changes prior to the onset of late-life pathologies that can complicate mechanistic interpretations. Macrophage functions were assayed using simple in vivo measurements that indicate age-dependent increases in the abundance of macrophages in the peritoneum in response to elicitation by an irritant injected into the peritoneum that is indicative of increased motility and macrophage activation. Mechanistic linkages between observed phenotypic changes in macrophage function and molecular pathways involving immune function were identified using mass spectrometry based measurements of protein abundance changes, which involved a shotgun proteomic analysis in which tandem mass spectrometry was used to identify 1,847 proteins with annotated functions for subsequent quantitation of protein abundance changes using the accurate mass and elution time (AMT) tag proteomic strategy. Consistent with observed functional changes and hypotheses developed using tandem MS data, we observe an age-dependent up-regulation of specific immune cell pathways (e.g., antigen presentation) and abundance changes of 77 proteins linked to macrophage activation. There is no indication of any impairment of normal cellular pathways involving macrophage activation in aged animals; rather, these results are indicative of an age-dependent up-regulation of normal classical activation pathways.</p><!><p>Male Balb/c mice (3–4 mo. and 14–15 mo.) were from Charles River Laboratories International Inc. (Wilmington, MA). Bio-Gel P-100 polyacrylamide beads (45–90 µm) were from Bio-Rad (Richmond, CA). Bacterial LPS from E. coli strain O127:B8 was from Sigma Chemical (St. Louis, MO). Interferon was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Ampicillin, RPMI 1640 medium (#0030078DJ), fetal bovine serum (FBS), penicillin, and streptomycin were from Gibco (Carlsbad, CA). FITC-labeled anti-mouse F4/80 macrophage specific antibodies were from eBioscience, Inc. (San Diego, CA). Calcein acetoxymethyl (AM) ester and 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI) were from Invitrogen (Carlsbad, CA).</p><!><p>When indicated, macrophages were elicited by injection (1mL) of a 2% (v/v) sterile solution of washed and hydrated polyacrylamide beads in phosphate buffered saline (PBS) into the peritoneal cavity five days prior to their isolation, as previously described (12). Our experimental design is in accordance with all prior reports that address age-dependent changes in macrophage function, which commonly isolate peritoneal macrophages following elicitation at a single time point 3–5 days following the introduction of an irritant (in our case Bio-Gel polyacrylamide beads) (8, 13–20). As described by Melnicoff and coworkers (21), homogeneous populations of peritoneal macrophages are isolated within this time window to avoid contamination by neutrophils and resident macrophages that becomes problematic when macrophages are collected at longer times following elicitation.</p><!><p>Resident or elicited peritoneal macrophages were isolated using standard peritoneal lavage procedures following asphyxiation using CO2 (12). Briefly, phosphate buffered saline (PBS) (10 mL) was injected into the caudal half of the peritoneal cavity using a 25-gauge needle, whole mice were gently rocked for 10 seconds, and peritoneal cells were slowly withdrawn using a 19-gauge needle and collected into a conical tube on ice. Isolated cells were plated in RPMI 1640 medium supplemented with heat-inactivated fetal bovine serum (10% v/v), penicillin (1% v/v), and ampicillin (1% v/v) at a constant cell density (1 × 106 cells per p100 plate) and incubated for 1 hr at 37 °C. Nonadherent cells were removed by washing plates five times in warm PBS (500 µL), and remaining adherent macrophages were maintained in a humidified atmosphere of 5% CO2 and 95% air at 37 °C overnight prior to treatment and harvest. Purity and viability of isolated cells were analyzed using an Agilent 2100 Bioanalyzer Microfluidics platform for flow cytometry with 2-color analysis of fluorescently stained cells and standard immunohistochemistry methods using a Nikon Eclipse TE200 epifluorescence microscope equipped with Metamorph imaging software to quantify the correspondence between multiple fluorescence signatures. Macrophage purity was determined to be greater that 95% of isolated cells through a comparison of FITC-labeled anti-mouse F4/80 macrophage specific antibodies to identify cells in comparison to total cells measured using DAPI labeling of double stranded DNA. Viability was measured by comparing fluorescence signals associated with cell esterase activity visible upon cleavage of calcein AM in comparison to FITC-labeled anti-mouse F4/80 macrophage specific antibodies.</p><!><p>Time-dependent increases in nitric oxide were measured by following the accumulation of the stable nitrite end product using the Griess reagent (Pierce Inc., Rockford, IL). Prior to measurement, all nitrate was enzymatically converted to nitrite using nitrate reductase. Nitrite concentrations in conditioned media were determined on the basis of standard curves calibrated using sodium nitrite as a standard, as we have previously described (22).</p><!><p>Primary peritoneal or RAW 264.7 macrophages were challenged with S. typhimurium at a multiplicity of infection of 100, essentially as previously described (23–25). Briefly, S. typhimurium 14028 cells were cultured the day before the assay in LB broth at 37 °C overnight, and were harvested by centrifugation (12,000 rpm for 1 minute) before resuspension at a final density of 5 × 105 cells/ml in 24-well cell-culture plates (i.e., 500,000 macrophages/well). Prior to bacterial challenge, macrophages were treated with 100 U/mL of interferon gamma (IFNγ), rinsed, and the prepared S. typhimurium-laden media were directly added to the plate of macrophages (26). The infection proceeded as plates were placed back into the incubator under standard conditions (37 °C in 5% CO2). Following incubation for 30 minutes to permit macrophages to phagocytize the S. typhimurium, extracellular bacteria were rinsed off with PBS. Fresh media (12.5 µg G418 per mL) replaced the bacteria-laden media to eliminate extracellular bacteria and prevent the extracellular replication and invasion of any S. typhimurium remaining on the plate. Macrophages infected with S. typhimurium were then further incubated, and at indicated times cells were washed and lysed (PBS, 1% triton X-100, and 0.1% sodium dodecyl sulfate [SDS] for 5 minutes at room temperature). Remaining viable bacteria were detected as colonies following the serial dilution of the cellular lysates onto LB agarose plates that were incubated overnight at 37°C.</p><!><p>Following removal of media, cells were rinsed once with chilled D-PBS (Invitrogen, Carlsbad, CA) and incubated in chilled 20 mM Tris (pH 8.0), 1% Nonidet P-40, 0.15 M NaCl, 1 mM Na2PO4, and 1 mM EGTA. Cell lifters were used to manually scrape and transfer cells into a chilled glass homogenizer, and following cell disruption (a ten-stroke homogenization), lysates were immediately centrifuged for 30 minutes at 9,300 × g at 4°C. Supernatant was removed, and stored at −80°C. Cellular disruption in the presence of detergent permits quantitative measurements of total lysates to be evaluated as membrane disruption and protein solubilization enhances overall proteomic coverage. In the absence of protein solubilization, membrane associated proteins are lost from the sample during sample processing. As many of the key proteins associated with macrophage activity are linked to membrane processes, it is critical to include a solubilization step for accurate assessments of proteomic changes. Lysates were subjected to low-speed centrifugation (9,300 × g on a table top centrifuge) prior to analysis to prevent any large particles from clogging the LC equipment, and there was no detectable loss of protein following this procedure.</p><!><p>Lysates were dialyzed against ammonium bicarbonate prior to addition of 8 M urea to denature proteins. Cysteine residues in lysates were reduced using tris(2-carboxyethyl)phosphine (TCEP) (Bond Breaker TCEP solution) (5 mM) (Thermo Scientific, Rockford IL), alkylated using iodoacetamide (25 mM), and sonicated in a UTR200 sonoractor (Hischler, Teltow,Germany) at 50% intensity for 3 min, as described before (27). Following a 10-fold dilution of samples into freshly prepared 50 mM ammonium bicarbonate solution (pH 7.8) and 1 mM CaCl2, samples were digested using trypsin (1:50, wt/wt ratio of trypsin to sample protein)for 1 min in a Barocycler™ NEP-3229 instrument, as previously described (28). Each digest was desalted using Supelco (St. Louis, MO) Supelclean C-18 tubes, as described elsewhere (29), and concentrated (1 mg/mL) using vacuum centrifugation.</p><!><p>A quantitative analysis of changes in the cellular proteome involves of two-step process. First, in stage 1 tandem MS (MS/MS) measurements are made following proteolytic digestion and strong cation exchange (SCX) fractionation of proteins from peritoneal macrophage lysates obtained from young and aged animals into 30 liquid chromatography (LC) fractions for analysis using a linear trap quadrupole (LTQ) iontrap mass spectrometer (ThermoElectron Corp., San Jose, CA), as described previously (30). The MS/MS spectra were analyzed using the SEQUEST Bioworks 3 release version (31) against an IPI Mus musculus database downloaded from NCBI (i.e., M_Musculus_2006-07-25_IPI which contains 94,146 entries). The mass tolerance for the precursor and fragment ions were respectively 2.5 Da and 0.8 Da. Database search parameters included dynamic modification of +16 Da for Met oxidation and fixed +57 for Cys carbamylation. Only peptides with +2 and +3 charges were analyzed for fragmentation and SEQUEST analysis. Confident peptide identification with a false discovery rate of 1% (i.e., q < 0.01) involved fitting the data to a sum of Gaussian distributions following correction of Xcorr values to take into account peptide length, where corrected Xcorr = ln(Xcorr)/ln(peptide length) and corrected ΔCn = (ΔCn)½ (27, 32). Sequences, charge states, masses, and peptide identification scores obtained from the SEQUEST algorithm for all identified peptides are included as an excel file in Supporting Online Information. No upper limit was prescribed for the number of missed or non-specific peptide cleavages. However, as tabulated in Supporting Online Material, the vast majority (>92%) of identified peptides contain two tryptic cleavage sites – providing enhanced support that the SEQUEST algorithm combined with the mass resolution of our instrumentation provides accurate peptide assignments. We find that 6% of the identified peptides contain a single tryptic cleavage site. Peptides identified from these tandem MS measurements were used to build an accurate mass and elution time (AMT) database that matches the masses of individual peptide sequences and normalized elution times to permit unique peptide identifications, as previously described (33). The AMT database is necessary for all quantitative changes in the abundances of individual proteins.</p><!><p>Using the AMT database, in stage 2 we use a high-resolution reversed phase capillary chromatography coupled to a high-resolution LTQ-Orbitrap XL MS to quantify changes in the abundance of individual peptides from a consideration of the ratio of ion currents for individual AMT tags, as previously described (34–35). LC-MS spectra are first processed to detect charge and isotopic masses in individual mass spectra using the program Decon2LS (http://ncrr.pnl.gov/software/) and identified spectra are further processed using the VIPER program to calibrate elution times, refine the mass calculation, and match the LC-MS features to AMT tags in the reference database (36). In these latter measurements, the grouped features for each identified peptide are represented by the median value obtained across three LC-MS runs. These data were loaded into the software tool DAnTE for quantitative data analysis, which allows a direct comparison of identified peptides across data sets to analyze and visualize abundance differences (37). Peptide abundances were first log base 2 transformed, and an outlier check was applied by observing the Pearson correlations between datasets. Any prominent outlier dataset that had weak correlations (< 0.7) is excluded from further analysis, as previously described (38). A linear regression based normalization method (available in the program DAnTE) was applied next, within each replicate category. The central tendency adjusted peptide abundances were used to infer the corresponding protein abundances via the 'Rrollup' algorithm in DAnTE (37). This tool permitted a determination of protein abundance changes, where the most abundant peptide across all data sets is used as a reference to calculate the ratios of protein abundances. During the Rrollup step the Grubbs outlier test was applied with a p-value cutoff of 0.05 to further remove any outlying peptides. Finally, a t-test identified significant abundance differences, using a p-value< 0.05. Protein abundances are the median of the resulting peptide abundances, where statistical significance is calculated from the ratio of the protein abundances using a cut-off at 5% FDR. IPI protein identifiers obtained from the individual proteins were used for data mining and retrieval using internal cross identifier mappings (i.e., mapping form protein to gene identifiers) and further pathway and functional enrichment data retrieval in conjunction with the Bioinformatics Resource Manager (BRM) software (39). Estimates of total proteome coverage utilized David Bioinformatics Resources (http://david.abcc.ncifcrf.gov/content.jsp?file=citation.htm) to analyze 28 major cellular pathways expected to be present in macrophages (40–41).</p><!><p>Homogenous populations of viable macrophages were isolated from Balb/c mice. Macrophage purity was determined to be greater that 95% of isolated cells through a comparison of FITC-labeled anti-mouse F4/80 macrophage specific antibodies to identify macrophages in comparison to total cells measured using DAPI labeling of double stranded DNA (Figure S1 in Supporting Information). Viability was measured by comparing fluorescence signals associated with cell esterase activity visible upon cleavage of calcein AM in comparison to FITC-labeled anti-mouse F4/80 macrophage specific antibodies, and represents greater than 90% of isolated macrophages following correction for signals associated with uncomplexed FITC-labeled antibody visible even in the absence of added cells.</p><!><p>To assess age-dependent alterations in the inflammatory response of macrophages, in vivo macrophage recruitment was measured following injection of an irritant (i.e., sterile Bio-Gel P-100 polyacrylamide beads) into the peritoneal cavity of Balb/c mice. The large 45–90 micron size of the Bio-Gel P-100 polyacrylamide beads avoids possible artifacts associated with phagocytosis and results in the isolation of a population of macrophages that remain largely quiescent in comparison with other elicitation protocols using, for example, thioglycollate (TG) (42–43). The number of macrophages isolated from young (3–4 mo) mice were compared with those isolated from older animals near the average mouse lifespan (14–15 mo) (44). Prior to elicitation, we respectively isolate an average of 1.3 ± 0.2 × 106 and 0.90 ± 0.02 × 106 resident macrophages from each young and old mouse (Figure 1A). The observation that young mice yield more resident macrophages than aged mice is consistent with earlier observations (13). Following elicitation, there are substantial increases in the number of isolated macrophages, equaling an average of 9 ± 2 × 106 and 12.6 ± 0.8 × 106 macrophages respectively isolated from each young and old mouse (Figure 1B); overall yields are not statistically different, which is in agreement with prior reports that indicate age-dependent differences in the yield of resident macrophages disappear following elicitation (13). These results indicate an age-dependent enhancement in the recruitment of macrophages into the peritoneal cavity of aged animals (14 ± 1 fold) in comparison to young control (7 ± 2 fold) to yield equivalent numbers of macrophages. Age-dependent increases in macrophage recruitment may be relevant to understanding previous measurements that have identified age-dependent increases in cellular inflammatory responses and an enhanced resistance of infected mice to introduced bacterial pathogens (10).</p><!><p>To assess possible age-dependent alterations in macrophage function, we measured nitric oxide production, measured as total nitrite (Figure 1C). We observe increased levels of nitric oxide production during the first two hours following exposure to bacterial endotoxin LPS. These latter results are in agreement with suggestions that increases in the generation of reactive oxygen species by macrophages contribute to age-dependent increases in the oxidative damage to cellular proteins (45). At longer times, comparable levels of nitric oxide are detected irrespective of the age of the donor animals. These observations indicate that the age-dependent increase in nitric oxide production does not represent an intrinsic limitation in the capacity of macrophages isolated from the younger animal to produce nitric oxide. Rather, macrophages isolated from aged animals are highly responsive to activation upon exposure to the bacterial endotoxin LPS.</p><p>Additional clarification of functional differences involved measurements of bacterial killing following their phagocytosis, which involves increases in nitric oxide production as part the oxidative burst. These measurements involved assays of the bactericidal response of isolated macrophages following phagocytosis of live bacteria (i.e. Salmonella typhimurium). Macrophages were plated to the same density in all cases (i.e., 500,000 macrophages/well). At various times following phagocytosis macrophages were lysed and colonies that arise from surviving intracellular bacteria were measured using a plate assay, as previously described (25). In comparison to a commonly used macrophage-like cell line derived from Abelson leukemic virus-induced tumors in Balb/c mice (i.e., RAW 264.7 cells), all isolated peritoneal macrophages have a substantially increased bactericidal activity that is indicative of highly coordinated metabolic pathways that are retained in aged animals (Figure 1D). However, macrophages isolated from old mice effectively kill 99.9% of phagocytosed bacteria during the first two hours following infection (Figure 1E). In comparison, macrophages isolated from young control animals kill only 90% of the phagocytosed bacteria. Increased bacterial killing is consistent with observations that there is an enhanced basal level of nitric oxide production and a more rapid response following exposure to LPS. These results, in total, suggest that measurements of protein abundance changes following LPS exposure provide a realistic indication of age-dependent changes in macrophage response pathways.</p><!><p>To access mechanisms associated with increased recruitment and pathogen killing in macrophages isolated from aged mice, we have identified expressed proteins using tandem mass spectrometry. To achieve in-depth proteome coverage and to build an AMT database, proteolytically digested macrophage lysates were fractionated using strong anion exchange (SCX) chromatography, permitting the facile identification of 19,055 peptides using a 1% false-discovery threshold (q < 0.01) (Table 1; see Figure S2A in Supporting Information)(27, 32). The 19,055 identified peptides correspond to 6,578 unique peptides that were used to build an accurate mass and elution time (AMT) database useful for quantitative measurements of protein abundance changes (see below), that matches the masses of individual peptide sequences and normalized elution times to permit unique peptide identifications (33). Identified proteins demonstrate, as expected, that pathways involving macrophage function are substantially enriched (Table S1 in Supporting Information).</p><p>Using gene ontology software to link individual proteins to cellular function, we have identified 1,847 macrophage proteins (see Table S5 in Supporting Materials). Irrespective of aging or macrophage activation, the distribution of proteins within gene ontology categories are very similar (Figure 2A,B). Nevertheless, there are substantial gaps in the proteome coverage, as we identify only 36% of the proteins in highly conserved central metabolic pathways (see Figure S3 in Supporting Information). These results indicate that MS/MS proteomic measurements selectively identify the most abundant proteins, and that increases in the identification of proteins from specific pathways are indicative of increases in the proteins within these pathways. As a result, the substantial (greater than 2-fold) increase in the number of identified proteins linked to immune response pathways for macrophages isolated from aged Balb/c mice, in comparison to young controls, suggests that there is an age-dependent up-regulation of inflammatory pathways in macrophages that is broadly consistent with observed age-dependent increases in inflammatory damage reported for a range of animal models (Figure 2C,D).</p><p>This is apparent from a consideration of identified proteins linked to antigen presentation as part of the MHC-1 pathway (see Figure S4 in Supporting Information), where substantially more of the proteins linked to the formation of the immunoproteasome necessary for peptide degradation and antigen presentation are detected in macrophages isolated from aged animals (see Table S2 in Supporting Information). Further, there are substantial increases in the number of detected proteasomal subunits following LPS exposure for macrophages isolated from aged mice; in comparison, LPS exposure has essentially no effect with respect to the number of identified proteosomal proteins in macrophages isolated from young animals. As antigen presentation is highly sensitive to macrophage activation (46), these latter results are consistent with enhanced levels of macrophage activation in aged animals. Likewise, there are age-dependent increases in the number of identified proteins associated with immune response pathways within a range of macrophage response pathways, including those linked to cytoskeleton/motility, phagocytosis/signaling, antigen presentation/differentiation, and activation/stress response (Figure 2E,F). Within the list of identified proteins, there are also substantial age-dependent increases in the number of proteins assigned to play a role in classical activation pathway (Figure 2G,H). However, for the majority of detected proteins LPS exposure results in only modest (24–28%) increases in the fraction of identified proteins linked to these immune response pathways that is very similar for macrophages isolated from either young and aged mice, suggesting the involvement of a subset of pathways (e.g., proteasome function) in age-dependent alterations in macrophage function. A similar insensitivity to LPS activation is apparent from a consideration of a range of proteins linked to antigen presentation pathways not involving the proteasome, which appear to be constitutively present irrespective of activation (Table S3). These latter results are consistent with known regulatory control mechanisms in which pathway control typically involves the first committed step of a pathway (in this case antigen presentation as part of the MHC-1 immune response).</p><!><p>Age-dependent differences in the abundance of specific proteins identified in both young and aged animals were resolved using quantitative proteomics using an accurate mass and time tag (AMT) database to compare ion currents of individual peptides (33). In all reported proteins, abundance differences represent averages and standard deviations for more than two unique peptides in each protein, as previously described. For example, in the case of arginase 1, abundance changes during aging involve pair-wise comparisons of ion current for seven different peptides, as fully described in Experimental Procedures (see section entitled Quantitative Measurements of Protein Abundance Changes)(Figure 3). Observed decreases in the peptide abundances for the seven resolved peptides vary from a 99.5% decrease (i.e., REGLYITEEIYK) to a maximal decrease in abundance of > 99.9% (i.e., LKETEYDVRDHGDL). Collectively, the mean decrease in arginase I abundance is 99.7 ± 0.1%.</p><p>Following LPS exposure, there are significant changes in the abundance of 54 proteins, where 90% of affected proteins increase in their abundance in macrophages isolated from aged mice (Figure 4; see Table S4 in Supporting Information). Of these proteins, we observe substantial increases in the abundance of diagnostic proteins linked to nitric oxide generation (e.g., annexin I, aldehyde dehydrogenase 2, and cystatin B) as well as co-regulated antioxidant proteins that are part of the classical activation pathway of macrophage activation (47–51). Further, of the 54 sensitive proteins, 26 have previously been co-localized within intracellular vacuoles associated with the compartmentalization and killing of bacterial pathogens (52). The other 28 proteins that undergo age-dependent changes in abundance are all linked to known pathways associated with the macrophage immune response involving cytoskeletal motility, phagocytosis and signaling, antigen presentation and macrophage differentiation, and activation pathways involving reactive oxygen species and stress responses (see Table S4 in Supporting Information). Thus, age-dependent alterations in macrophage function involving enhanced mobility, increases in the formation of nitric oxide, and higher rates of bacterial killing are all the result of a coordinated up-regulation of normal pathways involving immune cell activation. There is no indication of any dysregulation of normal cellular pathways linked to macrophage activation.</p><p>Prior to macrophage activation by bacterial endotoxin LPS a smaller number of proteins change their abundance in macrophages isolated from aged mice, involving the down-regulation of 11 proteins and the up-regulation of 12 proteins (Figure 4). Of particular interest is the large decrease in the abundance of arginase I in macrophages isolated from aged animals (Figure 3), whose expression is under the control of cytosolic interleukins (such as IL-4) that act to induce a phenotypic switch that favors an alternative activation pathway (3). This latter result is consistent with earlier observations that have demonstrated age-dependent declines in the ability of T helper (CD4) cells to mount antigen-specific Th2 responses involving the release of IL-4 and other cytokines that promote this alternative pathway of macrophage activation (10). Rather, T helper cells in aged animals primarily exhibit a Th1 phenotype involving the release of inflammatory cytokines IFNγ, IL-2, and TNFα that promote classical pathways of macrophage activation that enhance generalized antimicrobial responses involving the up-regulation of iNOS. These latter results are consistent with the functional data demonstrating higher levels of nitric oxide production in the resting state of aged macrophages as well as a hypersensitivity of macrophages isolated form aged animals to activation by LPS (Figure 1C).</p><!><p>We report an age-dependent up-regulation of coordinated pathways normally associated with macrophage activation, which provides a strong indication that increases in macrophage sensitivity to activation is fundamental to observed age-dependent changes involving innate immunity. These results do not support models involving genetically programmed cellular changes during aging that might be causal in the induction of a pathological state that involves a loss of coordination between normal pathways. Rather, our results support prior suggestions that "many of the aberrant responses seem to be dependent on the host environment, the milieu in which the cells reside, and might not be entirely dependent on the innate immune cells themselves" (53). Consistent with this model, our data suggests that environmental effects associated with immune cell exposure act to sensitize macrophage to induce a chronic inflammatory response that is consistent with the vast majority of data concerning age-dependent phenomena. Apparent contradictory results in the literature can be understood in terms of differences in environmental exposures.</p><p>The data presented here represents the first global assessment of protein abundance changes of the aging process in macrophages, demonstrating an up-regulation of immune pathways in macrophages isolated from aged mice. Prior to the identification of age-dependent changes in the macrophage proteome, we first confirmed prior observations of in vivo and ex vivo functional differences between peritoneal macrophages isolated from young (3–4 mo) and aged (14–15 mo) Balb/c mice (Figure 1). Observed age-dependent functional changes involving increases in: i) macrophage recruitment following elicitation, ii) basal rates of nitric oxide generation that result in a reduction in the time needed for full activation of senescent macrophages following LPS exposure, and iii) bactericidal activity following phagocytosis of Salmonella are all in agreement with prior observations, as well as suggestions that age-dependent increases in the generation of reactive oxygen species by macrophages contribute to age-dependent increases in oxidative damage to a range of biomolecules (9–10, 13, 45). Using these characteristic macrophage samples, it is therefore possible to employ proteomic measurements that identify specific proteins and related pathways to investigate fundamental mechanisms responsible for age-dependent changes in macrophage function.</p><p>To quantitatively address possible changes in immune specific pathways, protein abundance changes were measured using the quantitative AMT-tag approach. We observe age-dependent abundance changes of 77 proteins known to play central roles in promoting macrophage activation (Figure 4; Table S4 in Supporting Information). Virtually all identified proteins that undergo age-dependent abundance changes map onto normal processes associated with macrophage activation in terms of processes (Figure 4) or cellular locations (i.e., 26 of the 54 proteins whose abundance is altered during aging following LPS exposure co-localize with intracellular vacuoles associated with bacterial killing, a process linked to macrophage activation) (52). As reflected in the heat maps in Figure 4, observed abundance differences are highly reproducible (Figure 3; see tabulated errors in Table S4). Of particular interest is the large 99% decrease in the abundance of arginase I in macrophages isolated from aged animals, whose expression is under the control of cytosolic interleukins (such as IL-4) that act to induce a phenotypic switch that favors an alternative activation (Th2-like) pathway. These latter results are consistent with substantial increases in the abundance of diagnostic proteins linked to nitric oxide generation (e.g., annexin I, aldehyde dehydrogenase 2, and cystatin B) as well as co-regulated antioxidant proteins that are part of the classical (Th1-like) macrophage activation pathway. The vast majority of the other proteins that undergo age-dependent changes in abundance are linked to known pathways associated with the macrophage immune response involving cytoskeletal motility, phagocytosis and signaling, antigen presentation and macrophage differentiation, and activation pathways involving reactive oxygen species and stress responses (Figure 4) that are consistent with observed age-dependent changes in macrophage function (Figure 1).</p><p>Specific examples of the mechanistic relationship between the quantitative AMT data and macrophage function include the identification of increases in the abundance of central proteins linked to normal pathways involving macrophage activation (see Table S4 in Supporting Information). Consistent with age-dependent increases in macrophage elicitation and trafficking, we observe abundance increases in major structural proteins necessary for cytoskeletal motility that include actin, specific myosin isoforms linked to motility, tubulin, and macrophage capping protein (known to be critical to resist infection). Likewise, age-dependent increases in bactericidal activity are consistent with quantitative increases in the abundance of key proteins that have previously been co-localized within intracellular vacuoles associated with bacterial killing (52). These proteins include moesin (linked to TNF production), coronin (actin binding protein linked to phagosome formation), and transgelin (actin binding protein that suppresses MMP-9 and whose expression is triggered by TNF). Key proteins indicative of the up-regulation of activation pathways associated with antigen presentation and macrophage polarization resulting from classical activation pathways include filamin alpha (initiates actin polymerization to reorganize cytoskeletal complexes that promotes MAPK-dependent signaling and ERK phosphorylation), cathepsin D (endopeptidase in lysosomes that promotes apoptosis through activation of caspase 8), histocompatibility-2 (surface glycoprotein linked to antigen processing and presentation via MHC-1 immune response), and heat shock protein 8 (involved in protein transport to endoplasmic reticulum critical for antigen presentation). Observed age-dependent increases in nitric oxide generation are consistent with abundance changes for proteins linked to the respiratory burst and adaptive macrophage functions involving chaperones and antioxidants that are critical to macrophage activation, which include arginase I (decreases in abundance are diagnostic of classical activation pathway, as arginase depletes arginine to inhibit nitric oxide production by NOS), cathepsin B (increases in cysteine protease linked to antigen degradation and inflammatory processes associated with trafficking TNF containing vesicles to membrane), arachidonate-5-lipoxygenase activating protein (downstream of Toll-receptors this protein plays a key in forming leukotrienes linked to inflammatory responses), annexin A2 (promotes tyrosine kinase activation), lymphocyte cytosolic protein (actin binding protein critical to adhesion-dependent respiratory burst), and aldehyde dehydrogenase (antioxidant protein linked to nitric oxide production). Collectively, these results indicate a coordinated increase in normal pathways involving macrophage activation, and do not support models that emphasize a dysregulation of normal cellular pathways linked to macrophage activation (3).</p><p>Observed age-dependent alterations in macrophage function are consistent with known age-dependent changes in the adaptive immune system, which involve shifts in T-helper cellular responses that favor the production of the inflammatory cytokines IFNγ, IL-2, and TNFα (i.e., Th1 response) (10). Such soluble factors induce the activation of resting macrophages to enhance nitric oxide production, antigen presentation, enhanced cytokine biosynthesis, and phagocytosis (54). Thus age-dependent increases in the resistance of macrophages to pathogens following their phagocytosis (Figure 1D,E)(9), as well as the increased resistance of old Balb/c mice themselves to introduced infections (10), are consistent with well-documented age-dependent changes involving population shifts in T-lymphocytes to favor Th1 inflammatory responses. Apparent contradictory results in the literature, which detect both increases and decreases in macrophage function (9, 14–15, 55–57), can be understood in terms of environmental factors and assay conditions that uncover age-dependent differences in macrophage sensitization to activating signals (e.g., LPS). For example, increases in nitric oxide levels, routinely observed upon challenge of elicited peritoneal macrophages by bacterial endotoxin (14) (Figure 1C), are abolished if macrophages are first uniformly sensitized by IFNγ exposure prior to endotoxin challenge (8). Likewise, increases in the bacterial resistance of aged mice are dependent on environmental exposures to normal pathogens, and are lost when mice are housed in sterile (clean room) conditions (10). These observations are consistent with the considerable phenotypic heterogeneity present within macrophage populations, which is modulated in response to environmental conditions, which include responses to the generation of inflammatory cytokines (e.g., INF-γ, IL-2, and TNFα) and other soluble factors from T-lymphocytes that act to induce changes in the macrophage proteome and differentiation (58–59).</p><p>What is currently unclear in the etiology of immune dysregulation during aging is the role that macrophages may play in promoting long-term shifts in T-lymphocyte population heterogeneity. Understanding the causal relationships that lead to age-related changes in the immune system requires an understanding of the functional coupling between the innate and adaptive immune systems. Specifically, antigen presenting cells (i.e., macrophages, dendritic cells, and B cells) inform and amplify adaptive immune system responses and have the potential to induce shifts in innate and adaptive immunity characteristic of that seen in aged animals. For example, maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection is central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. In aged animals macrophages exhibit a hyper-responsive and coordinated initiation of classical macrophage activation pathways, which enhance nitric oxide production and bactericidal activity to minimize infection from general microbial threats (9–10), which are likely to be exacerbated by age-dependent decreases in the efficacy of physical barriers that limit pathogen entry to enhance environmental exposures. Upon pathogen recognition, macrophages release cytokines (e.g., IL-12) to induce T-helper cell differentiation to favor a Th1 response and release of inflammatory cytokines by T-helper cells that is associated with macrophage activation and T-helper cell proliferation in response to pathogen entry (Figure 5). Cross-talk with T-helper cells thereby amplify inflammatory signals that promote macrophage activation in response to age-dependent decreases in physical barriers to pathogen entry that enhance exposures and further modify adaptive immunity through responses that originate in the innate system. These results suggest that therapies aimed to alleviate immune system dysregulation should include strategies aimed at neutralizing these amplification cascades between macrophage and T-lymphocytes, involving key targets of macrophage function that include not only inflammatory cytokines but also key signaling pathways involving macrophage activation, and pathways associated with antigen presentation.</p>

PubMed Author Manuscript

Anomalous p-backbonding in Complexes between B(SiR3)3 and N2: Catalytic Activation and the Breaking of Scaling Relations

Chemical transformations of molecular nitrogen (N2), including the nitrogen reduction reaction (NRR), are difficult to catalyze because of the weak Lewis basicity of N2. In this study, it was found that Lewis acids of the types B(SiR3)3 and B(GeR3)3 bind N2 and CO with anomalously short and strong B-N or B-C bonds. B(SiH3)3•N2 has a B-N bond length of 1.48 Å and a complexation enthalpy of -15.9 kcal/mol at the M06-2X/jun-cc-pVTZ level. The selective binding enhancement of N2 and CO is due to p-backbonding from Lewis acid to Lewis base, as demonstrated by orbital analysis and density difference plots. The p-backbonding is found to be a consequence of constructive orbital interactions between the diffuse and highly polarizable B-Si and B-Ge bond regions and the p-regions of N2. This interaction is strengthened by electron donating substituents on Si or Ge. The p-backbonding interaction is predicted to activate N2 for chemical transformation and reduction, as it decreases the electron density and increases the N-N bond length. The binding of N2 and CO by the B(SiR3)3 and B(GeR3)3 types of Lewis acids also has a strong s-bond contribution. The relatively high sbond strength is connected to the high positive surface electrostatic potential [VS(r)] above the B atom at the pyramidal binding conformation. Electron withdrawing substituents increase the potential and the s-bond strength, but favor the binding of regular Lewis acids, such as NH3 and F -, more strongly than binding of N2 and CO. Molecules of the types B(SiR3)3 and B(GeR3)3 are chemically labile and difficult to synthesize. Heterogenous catalysts with the wanted B(Si-)3 or B(Ge-)3 bonding motif may be prepared by B-doping of nanostructured silicon or germanium compounds. B-doped silicene show promising properties as catalyst for the electrochemical NRR.

anomalous_p-backbonding_in_complexes_between_b(sir3)3_and_n2:_catalytic_activation_and_the_breaking_

Introduction<!>Methods and theoretical procedures<!>+01<!>Lewis acidities<!>Orbital analysis of p-backbonding<!>Density difference maps<!>Electrostatic contribution to s-bonding<!>Covalent character of s-bonding<!>Realizing the B(Si-)3 and B(Ge-)3 bonding motifs<!>Conclusions<!>N2 reduction reaction (NRR)

<p>Nitrogen in its elemental formal is highly inert due to the very strong chemical bond of the nitrogen molecule. The high bond energy of the nitrogen triple bond in connection with the relatively modest bond energies of single and double bonds involving nitrogen transforms to high kinetic barriers for utilizing molecular nitrogen in chemical transformations. An important example is the Haber-Bosch process for converting nitrogen and hydrogen gas to ammonia, which relies on high temperatures and pressures in combination with transition-metal catalysis. Nature also depend on transition-metal catalysis for nitrogen reduction, but the nitrogenase enzymes manage the task at ambient conditions due to a more advanced catalytic machinery. 1,2 The development of efficient catalysts for nitrogen reduction has been hindered by the weak Lewis basicity of N2. Traditional Lewis acids, such as the boron trihalides, do not form donor-acceptor complexes with N2. Very strong Lewis acids, such as B(CF3)3, bind N2 but are too reactive to be useful. To avoid catalyst inhibition, chemical activation of N2 requires Lewis acids that preferentially binds N2 with binding energies that are stronger or at least on par with the binding energies for other Lewis bases that may be present. In the terminology of theoretical catalysis, N2 binding needs to break scaling relations for binding energies. In particular for electrochemical nitrogen reduction, the binding of N2 has to be competitive with the binding and reduction of the proton to avoid inhibition of the catalyst. 3 A number of transition metal compounds have been developed that can bind and catalytically activate N2. [4][5][6][7][8][9][10] Their function has largely been attributed to the presence of low lying d-orbitals that allow for the concurrent acceptance of electron density from the N2 sorbital and p-backdonation towards the N2 p* orbital. The CO molecule is isoelectronic to N2 but a stronger Lewis acid and its catalytical activation follows a similar protocol.</p><p>Main group chemistry has been less successful in nitrogen activation, but recent studies have demonstrated fixation and reduction of N2 by some novel hypo-valent borylene compounds. 11,12 These are argued to work by a similar mechanism as the transition metal catalysts, with N2 s-donation into an empty sp 2 -hybrid orbital and pi-backdonation from a fully occupied p-orbital on boron. However, it should be noted that N2 is bound to one borylene unit B at each end and thereby a delocalized p-system similar to that of a conjugated hydrocarbon is formed.</p><p>In an attempt to characterize the N2 binding properties of trivalent boron Lewis acids, we observed an unexpected behavior upon replacing carbon for silicon as the atom bonded to boron. Whereas B(CH3)3 does not bind N2, B(SiH3)3 forms a complex with a very short and strong B-N bond. In Fig. 1 , we show the geometries and complexation enthalpies of the complexes of B(SiH3)3 with N2, NH3 and CO, together with the same properties for the corresponding complexes of B(CF3)3. All values have been obtained at the DFT M06-2X/juncc-pVTZ level of theory, and for the B(SiH3)3 complexes we also compare with coupled cluster calculations with values in italics, i.e. CCSD/6-31G+(d´,p) for geometries and CCSD(T)/juncc-pVTZ for energies. Beginning with the B(SiH3)•N2 complex, we find a very short B-N bond of 1.48 Å (1.53 Å). This is even shorter than the sum of the covalent single bond radii, which amounts to 1.56 Å. 13 The formation of the complex results in slight increases in the B-Si and N-N bond lengths by 0.014 (0.003) and 0.012 (0.015) Å, respectively. The complexation enthalpy is -15.9 (-12.4) kcal/mol, which is congruent with a strong B-N bond, but a somewhat higher value than could have been anticipated based upon the bond length.</p><p>Comparing the B(SiH3)3•N2 complex to the B(SiH3)3•NH3 complex, we find a considerably longer B-N bond of 1.64 Å (1.65 Å) but a stronger interaction with a complexation enthalpy of -35.2 (-34.1) kcal/mol in the latter complex. Considering that NH3 is a much stronger Lewis base than N2, a much larger difference in the complexation enthalpy could have been anticipated. The formation of the B(SiH3)•CO complex results in similar changes to the geometry as the formation of the B(SiH3)3•N2 complex, but the B-N bond of the former is slightly longer than the B-C bond of 1.46 Å in the latter. The complexation enthalpy of -45.4 kcal/mol is much lower than for the N2 complex, and despite CO being a significantly weaker Lewis base than NH3 the CO-complex is stronger than the B(SiH3)3•NH3 complex.</p><p>We continue with comparing the binding geometries and complexation enthalpies of the B(SiH3)3 complexes with those of the B(CF3)3 complexes. It should first be noted that B(CF3)3 is a very strong and chemically labile Lewis acid that only has been detected as a transient intermediate from thermal dissociation of B(CF3)3•CO. 14 The complexation enthalpy of the B(CF3)3•N2 complex is only slightly higher compared to the B(SiH3)•N2 complex, i.e. -14.7 vs ΔH Cmpl -15.9 kcal/mol, but the B-N bond is much longer in the former, i.e. 1.62 vs 1.48 Å. However, B(CF3)3 forms a very strong complex with NH3 with a complexation enthalpy of -61.2 kcal/mol, which is almost twice the strength of the interaction in the B(SiH3)•NH3 complex. On the other hand, the difference in B-N bond length compared to B(SiH3)3•NH3 is relatively small with a bond length of 1.60 Å in B(CF3)3•NH3. In contrast to B(SiH3)3, B(CF3)3 also forms a much stronger complex with NH3 than with CO, and the bonding in B(CF3)3•CO is significantly weaker than in the B(SiH3)•CO complex, although the B(CF3)3•CO bond is strong with a complexation enthalpy of -30.1 kcal/mol.</p><p>Summarizing the geometrical and energetics data of Fig. 1, it is indicated that B(CF3)3 binds all three Lewis bases with a similar mechanism and the variation in complexation enthalpy agrees with their relative Lewis basicities. B(SiH3)3 seems to bind NH3 following a related mechanism, whereas the binding of N2 and CO invokes an additional component to the binding that results in much shorter intramolecular bond lengths (B-N or B-C) and enhanced binding strengths.</p><p>The physical origin of the enhanced binding of N2 and CO is nontrivial to deduce, but it is reasonable to anticipate a connection to the p-backbonding mechanism prevalent in transition metal compounds that activates N2 and CO, and which has been indicated in the N2 activating hypo-valent borylene compounds. In this study we attempt to investigate this hypothesis in greater detail but we also take a comprehensive perspective in analyzing the Lewis acid-base interactions for this type of compounds. Furthermore, we investigate the potential for optimizing the selectivity for N2 binding and activation by means of chemical derivatization. Finally, we seek to identify nanostructured materials that are synthetically accessible and invoke the necessary chemical functionalities for use as heterogenous catalysts.</p><!><p>Structures of molecules and molecular complexes have been optimized at the M06-2X/jun-cc-pVTZ level of Kohn-Sham density functional theory. The M06-2X density functional is highly accurate for main-group chemistry, including non-covalent interactions, and it is parameterized to include short and mid-range London dispersion interactions. 15 This functional has been shown produce highly accurate energetics for classical Lewis adducts, as well as frustrated Lewis pairs, with an average deviation of 0.6 kcal/mol relative complete basis set extrapolated DLPNO-CCSD(T) energies. 16 The jun-cc-pVTZ basis set is the cc-pVTZ basis set augmented with diffuse s, p, d functions on non-hydrogen atoms. 17 The geometries and energies obtained with M06-2X have been compared with coupled cluster calculations at the CCSD(T)/jun-cc-pVTZ//CCSD/6-31G+(d´,p) level of theory for selected systems.</p><p>To characterize the capacity of the Lewis acids to supply electrons for p-backdonation, the average local ionization energy [Ī(r)] was computed on molecular surfaces defined by the 0.001 au electron density contour. The Ī(r) is rigorously defined by eq. 1 within generalized Kohn-Sham density functional theory. 18,19 𝐼 ̅ (𝐫) = − ) 𝜀 + 𝜌 + (𝐫) 𝜌(𝐫) -./.</p><!><p>where ei is the energy of orbital i, ri(r) is the density of the orbital, and r(r) is the total electron density. According to Janak's theorem, 20 the negative values of the orbital energies can be considered approximations to the ionization energies, and Ī(r) can be interpreted as the average energy needed to ionize an electron at a point r in the space of a molecule or atom. Surface Ī(r) [ĪS(r)] has been demonstrated to be an effective tool for predicting local reactivity for electrophilic processes, such as electrophilic aromatic substitution reactions. 19 Minima in ĪS(r) [ĪS,min] reflect the positions most likely to donate electrons and thus most susceptible for electrophilic attack.</p><p>To characterize the p-holes of the Lewis acids and their capacities to participate in electrostatic interactions with Lewis bases, the surface electrostatic potential was computed at the same isodensity contour as in the ĪS(r) computations. The V(r) is defined by,</p><p>where ZA is the charge on nucleus A located at RA, and ρ(r) is the electron density function.</p><p>V(r) is a physical observable, and qV(r) corresponds to the electrostatic interaction energy for a positive or negative point charge (q) at different positions (r) in space. Surface maxima in V(r) [VS,max] has been demonstrated to be an efficient indicator of the most active positions for nucleophilic noncovalent interactions, such as halogen and hydrogen bond donating sites, 19 but also for characterizing interaction sites at Lewis acids, such as BCl3 and BH3. 21 All DFT and ab initio computations have been performed using the Gaussian16 suite of programs. 22 The computations of ĪS(r) and VS(r) have been performed using the Hs95 program 19 of Tore Brinck and the ELF computations using the topchem2 program. 23</p><!><p>To improve the understanding of the chemical mechanism and chemical requirements for selective binding of N2 and CO, we have listed computed properties for a series of boron-based Lewis acids and their complexes with N2, CO, NH3 and Fin Table 1. The complexation enthalpy of Fis included as a fluoride ion affinity has been used a general descriptor for assessing Lewis acidity. 24 For comparison, we have also included some related Lewis acids with Al or Ga instead of B as the coordinating atom.</p><p>First of all, we note that the suspected p-backbonding behavior that we found in complexes of B(SiH3)3 with N2 and CO seems to be confined to compounds where a central boron atom is bonded to Si or Ge. Only in the B(SiR3)3 and the B(GeR3)3 compounds do we observe the short intramolecular B-N or B-C bonds and the enhanced binding strengths for N2 and CO when compared to binding strengths for NH3 and F -. Substituting B for Al, or Si for C, H or Cl increases the bond length and complexation enthalpies. In particular, B(CH3)3 or BCl3 do not form a stable complex with N2. .8 a Surface electrostatic potential maximum (VS,max) above the B or A atom. b VS,max of the Lewis acid in the geometry of the complex with N2. c Surface minimum of the average local ionization energy (ĪS,min) in the region above the B-Si bond. Geometry of Lewis acid from its complex with N2. d Intermolecular B-N (or Al-N) bond distance for the complex with N2. e Forms no stable complex with N2. f VS,max or ĪS,min for the geometry of the Lewis acid in its complex with NH3. g No ĪS,min above the B-H bond. ĪS(r) value above the B-H bond.</p><!><p>It is not obvious why a compound of the type B(SiR3)3 or B(GeR3)3 should participate in pbackbonding as there are no occupied lone-pair p-orbitals on B in these compounds that can interact with the p*-orbitals of N2 or CO. However, an analysis of the occupied orbitals of B(SiH3) in a distorted pyramidal geometry corresponding to that in B(SiH3)3•N2, shows that the two degenerate orbitals 18 and 19 as well as the degenerate HOMOs have the proper symmetry for the orbitals to interact with the p and p* orbitals of N2 or CO. The first type of orbitals (18,19) has very little density on the B and thus by itself will overlap only weakly with the ptype orbitals. On the other the orbital energy of 18 and 19 (-16.5 eV) is similar to the p orbitals (-14.8 eV) and should favor a constructive interaction.</p><p>The second type of orbitals, the HOMOs (27,28), has a shape and extension that should enable a good overlap with the p and p* orbitals. On the other hand, the HOMO energy (-8.8 eV) is intermediate between the p energy (-14.8 eV) and the p* energy (0.8 eV), and thus we do not expect a simple interaction with either of these. Overall, the interaction of the two sets of degenerate orbitals of B(SiH3)3 with the p and p* orbitals of N2 will generate four set of degenerate orbitals, eight in total, where the six lowest energy orbitals are likely to be occupied.</p><p>Investigating the occupied orbitals of B(SiH3)3•N2, we indeed find three sets of degenerate orbitals that are occupied and consistent with such an interaction, as shown in Fig. 2 The first two of these orbitals, i.e. number 22 and 23 with an energy of -16.9 eV, are strongly p bonding between B and N. These orbitals have large contributions from 18 or 19 on B(SiH3)3 and the p orbitals on N2, but also a smaller contribution from one of the HOMOs that enhances the B-N p bonding. The orbital energy (-16.9 eV) is slightly lower than orbital energy (-16.5 eV) of 18 and 19. The second type of orbital (25, 26) have contributions from the same orbitals as in 22 and 23, but is clearly non-bonding between B and N and the orbital energy (-15.9 eV) is slightly higher. Thus, orbitals 22,23 together with 25,26 will result in a significant p bonding contribution to the B-N interaction.</p><p>Fig. 2 The top row to the left shows the orbitals of B(SiH3)3 that have the correct symmetry for interaction with the p and p* orbitals of N2. The bottom row shows the orbitals of B(SiH3)3•N2 that are formed due to these interactions. Note that orbitals 22,23 contribute strongly and 34,35 weakly to the p-bonding of the complex. Orbitals 25,26 are non-bonding in this respect.</p><p>The third set of orbitals is the HOMOs, i.e. number 34 and 35 with an energy of -8.8 eV. These orbitals have a very similar shape and energy as the HOMOs of bare B(SiH3)3, but the orbitals of the complex has an additional contribution that can be expressed as a linear combination of the N2 p and p* orbitals, or more exactly as a very small in phase contribution from p(p) on N(1) in the B-N(1)-N(2) sequence and a somewhat larger out of phase p(p) on N(2). At first glance, it may seem the HOMOs of B(SiH3)3•N2 are nonbonding with respect to B-N but a closer inspection shows that they have a slight bonding character due to the shape of the contributing HOMO of B(SiH3)3, which extends over the B-N bond region, and the sign (in phase) of the p(p) on N(1). The change in sign of the wavefunction between N atoms also means that the orbital weakens the N-N p bond and the larger contribution from p(p) on N(2) shifts electron density towards N(2). The fourth set of degenerate orbitals is the LUMOs of B(SiH3)•N2, they are essentially identical in shape and energy to the p* orbitals of N2 and only have a minor contribution from the B(SiH3) orbitals. These orbitals may be important for photochemical activation of B(SiH3)•N2 or could become partly occupied upon reduction of the complex.</p><p>Summarizing our findings we note that the B-N p-bonding characters of orbitals 22,23,34 and 35 are strengthened by a reduction of the B-N distance thereby explaining the very short B-N distance in B(SiH3)•N2. For comparison, we have analyzed the occupied orbitals of the B(CF3)3•N2 complex, and in this complex there is no orbital that has a significant p-bonding character between the B and N.</p><p>As indicated by the orbitals of Fig. 2 the p-bonding interaction in B(SiH3)3•N2 is different from the classical picture of p-backbonding as a donation of electrons from occupied d-orbitals or p-orbitals into the antibonding p*-orbitals of N2. However, as discussed by Pettersson and Nilsson, the classical picture is simplified and it is not representative for the pbackbonding of transition metal surfaces with N2 or CO. 25 Instead the orbitals responsible for the p bonding to the ligand on those surfaces resemble the orbitals of Fig. 2 that provides the p bonding in B(SiH3)3•N2, i.e. it is orbitals similar to 22 and 23 of B(SiH3)3•N2. It is important to remember that all orbitals have to be orthogonal; this restricts their potential shapes and symmetries, and the classical picture of p-backbonding is not consistent with the orthogonality requirement. Caution should be taken when interpreting the bonding between certain atoms in a molecule based on a few orbitals, as the canonical orbitals typically are complex and delocalized; it is the combination of all occupied orbitals that gives the total electron density and determines the bonding in the molecule. To estimate the capacity for p-bonding interaction with the N2 porbitals, we argue that the surface average local ionization energy [ĪS(r)] is a better descriptor than the energy of any individual orbital of the Lewis acid, e.g. the HOMO, as ĪS(r) can be defined as a functional of the total electron density and is invariant to orbital rotation. The positions with the lowest ĪS(r), the ĪS,min, are the position from which electrons are most easily removed or donated, and the values of the ĪS,min are indicative of the average electron binding energy at those positions.</p><p>As shown in Fig. 3, there is a ring shaped region of low ĪS(r) above the B-Si bond region in B(Si(CH3)3)3 where there is a p-bonding interaction with N2 in B(Si(CH3)3)3•N2. Similar ĪS,min regions are found in all the compounds of the types B(SiR3)3 and B(GeR3)3 that form short and strong p-type bonds with N2 and CO, as well as in B(CH3)3, which forms a p-type bond with CO but not N2. The ĪS,min value increases with the substituent on B in the order, Ge(CH3)3 > Si(CH3)3 > GeH3 > Si(OH)3 > SiH3 ≈ Si(SiH3)3 ≫SiF3 which seems to reflect the p-bond donating capacity, as the B-N bond length increases in approximately the same order, with B(Ge(CH3)3)3•N2 having the shortest bond (1.46 Å) and the B(SiF3)3•N2 the longest bond (1.51 Å). Inductive donors, such as CH3, are expected to donate electrons into the B-Si (or B-Ge) bond and strengthen the p-bond with N2, whereas inductive acceptors, particularly F, withdraw electron from the B-Si or B-Ge bond and weaken the B-N p-bond.</p><p>It is important to remember that there is both a s and p contribution to the bonding of N2 and CO, and that inductive acceptors strengthens the s-bond. Thus, there is no simple correlation between the strength of the p-bond, as indicated by the B-N bond length or ĪS,min, and the complexation enthalpy. When it comes to B(CH3)3, it does not bind N2 and binds CO only weakly; this is partly a consequence of a weaker s-bond than in B(SiH3)3, but primarily due to a much weaker p-bond as indicated by a higher ĪS,min, 12.89 eV as compared to 11.53 eV for B(SiH3)3. BH3 binds N2, but weakly, due to a much stronger s-bond, as indicated by highly positive VS,max (vide infra), and a relatively low ĪS(r) (13.0 eV) in the p-bonding region of the Lewis acid.</p><!><p>To better understand the bonding interactions of B(SiH3)3, we have computed the density difference (DD) maps for the complexes with N2, NH3 and F -, see Fig. 4. For comparison, we have also included the DD for the B(CF3)3•N2 complex. The overall shapes of the DD are all rather similar, with a buildup of electron density above and below the B nucleus with a shape that is intermediate between a p and a sp 3 orbital. Thus, in all the complexes there is an accumulation of electron density in the B-N bond region; for the DD of B(SiH3)3•N2, the upper part is wider than in the other complexes consistent with a partial B-N p-bond. On the other hand, the donut shaped depletion of electron density above the B-Si bond region should not be seen as the result of donation of electron density into the p-bond, as the corresponding depletion is slightly bigger for B(SiH3)3•NH3 and much bigger for B(SiH3)3•F -. Instead we interpret this depletion as the results of a polarization of electron density from the B-Si bond region towards the region below the B resulting from the interaction with the lone pair of N2. The depletion in B(SiH3)3•Fis bigger and more diffuse, as Fcarries a full negative charge and the distribution of negative charge is not as localized as in the lone pairs of N2 and NH3.</p><p>An important observation from the DD of B(SiH3)3•N2 is that there is a density depletion in the N-N bond region; this indicates that the B(SiR3)3 compounds not only binds N2 strongly, but also weakens the N-N bond. The weakening of the N-N bond together with the buildup of p density at the outer nitrogen (N(2)) activates the molecule for chemical transformation.</p><p>Comparing the DDs of the B(SiH3)3•N2 and B(CF3)3•N2 and reveals interesting information about the difference in bonding and reductive activation between these complexes. The overall pictures are rather similar, but there is much smaller depletion in the B-C bond region of the latter compared to that in the B-Si region of B(SiH3)3•N2; this difference is consistent with the B-Si bond density being more diffuse and polarizable and with the electron withdrawing effect of the CF3 group. There is also a considerable difference between the N2 pregions of the two complexes. In both complexes there is a buildup of p-density at N(1) ,the N closest to the B, which can be viewed as the result of a polarization of N2 p-density due to the high positive electrostatic potential on B. In B(SiH3)3•N2, there is additionally a buildup of electron density at the N(2), and we interpret it as the result of the p-bonding character of the interaction and the contribution to the density from the HOMOs, which have a significant p(p) contribution at N(2) (see Fig 2). As already indicated, this build up may be important for the catalytic activation of N2.</p><!><p>Recent studies have shown that even strong donor-acceptor interactions, such as halogen and hydrogen bonds involving charged soft Lewis bases (e.g. Br -) , that traditionally are considered to have a significant charge transfer contribution often can be characterized and quantified by only considering electrostatics and polarization. 26,27 Here we will analyze the variation in sbond strength among the different Lewis acids and bases and argue that an electrostatic model can provide at least a semi-quantitative agreement.</p><p>Beginning with the B-Si compounds it can be noted that their high Lewis acidities to a significant extent can be traced to a high surface electrostatic potential [VS(r)] at the B, i.e. a high VS,max value, see Fig. 3. The high VS,max is not surprising considering that the B-Si Lewis bases are electron deficient with an empty p-orbital as the LUMO. The value of the VS,max becomes further increased when the Lewis acid is distorted to the pyramidal geometry present in the complexes. In fact, the VS,max value in many cases exceed 50 kcal/mol, which is much higher than the values typically found in neutral molecules except for the acidic hydrogens of strong hydrogen bond donors.</p><p>The Lewis acidity does not follow the VS,max value strictly; as an example N2 binds stronger to B(Si(CH3)3)3 than to B(SiH3)3 despite B(Si(CH3)3)3 having a lower VS,max. This behavior can be traced to the combination of a stronger p-bond and a stronger polarization in the N2 interaction with B(Si(CH3)3)3, i.e. the importance of polarization in B(Si(CH3)3)3•N2 is enhanced because of the very short B-N distance due to the p-bond interaction and the higher polarizability of CH3 compared to H. The introduction of strongly electron-withdrawing substituents, such as CF3, SiF3 and CN, substantially increases the VS,max value, and thus enhances the Lewis acidity. However, this effect is more important for the interactions with NH3 and Fthan for interactions with N2 and CO, as the electron-withdrawing substituents weaken the p-bond interaction. This is particularly evident for B(CN)3, which is one of the strongest binders of NH3 and F -, but does not form stable complexes with N2 and CO. The effect may be enhanced by the resonance withdrawing capacity of CN as CF3 and SiF3 are inductive electron acceptors.</p><p>To obtain a Lewis acid that preferentially binds N2 and CO, substituents that donates electron density into the B-Si or B-Ge bond are preferred as these promote the formation of a p-bond with N2 and CO. Somewhat surprisingly, we find that the detrimental effect of electron withdrawing substituents is bigger for CO binding compared to N2 binding. Intuitively, this is surprising considering that CO by all measures have a stronger lone pair and thus should have a stronger electrostatic interaction with B. However, the p-bonding interaction seems to be more important for CO compared to N2 and since the electron withdrawing substituents reduce pbonding, this effect takes precedence in the complexes with CO.</p><p>It is also interesting to note that there are enhanced binding strengths of NH3 to B(SiR3)3 and B(GeR3)3 when compared to traditional boron based Lewis acids, e.g. B(CH3)3, BCl3 and BH3. This is consistent with previous reports that also NH3 can participate in p-backbonding interactions. 28 In comparison, the fluoride affinity follows the VS,max of the Lewis acid more closely indicating an interaction dominated by electrostatics. However, due to small size and negative charge of F -, polarization plays an integral role and explains why B(CH3)3 and BH3 binds Fmore weakly compared to the other Lewis acids. Because of the anomalously large polarization effect and the generally very high binding strength, it can be argued that fluoride affinity is not a representative scale of general Lewis acidity.</p><p>We have also compared the boron based Lewis acids to some aluminum and gallium based Lewis acids with similar structures. Despite featuring very high VS,max at Al or Ga, these Lewis acids bind N2 and CO only weakly while being relatively intermediate binders of NH3 and strong binders of F -. The strongest Lewis acid of the Al-compounds is Al(SiF3)3, and it has the highest VS,max of all the non-charged Lewis acids that are investigated in this study. Accordingly it has the lowest Fcomplexation enthalpy of the neutral Lewis acids, whereas the N2 complexation enthalpy is relatively modest at -13.0 kcal/mol. We note that AlCl3, in contrast to BCl3, binds N2 with a negative complexation enthalpy, but that the binding strength is reduced going to GaCl3. In this context, it should be noted that all the Al and Ga based Lewis acids remain nearly planar around the central coordinating Al or Ga atom after coordination to N2 and that the bonding distance is larger than the sum of the covalent radii.</p><!><p>In contrast to the p-interactions, which has been analyzed in terms of orbital interactions, the s-bond contribution to the interactions of the boron based Lewis acids bases has so far been rationalized only in terms of electrostatics and polarization. This analysis has provided a mean for explaining the variations in the complexation enthalpy with respect to Lewis bases and the substituents on the Lewis acid. In this context, it is interesting to note that ELF-analysis, which has been shown to be a stringent tool for distinguishing between physical and covalent bonding, shows the B-N bond to be non-covalent. 29 However, as pointed out by several researchers, it is more appropriate to consider a gradual scale between covalent and non-covalent bonding. 21,[30][31][32][33] Politzer et al. argue that the bonding in BCl3•NH3 is of significant coordinative covalent character based on the strength of the interaction, the relatively short B-N bond length, and the pyramidal structure of the BCl3 in the complex. Following similar arguments we suggest that all the stable BR3•N2 and BR3•CO complexes have some covalent character to the intermolecular s-bonding, but that the covalent character in many cases is relatively weak considering the significant p-contribution to the binding and the relatively low binding strength. In contrast, the Al and Ga based Lewis acids form complexes with N2 that have relatively long Al-N or Ga-N bonds and tetragonal structure. This indicates non-significant p-bonding and a covalent character of the s-bonding that is much lower than for the B compounds. Interestingly, we find that even the carbocation C(CH3)3 + binds N2 weekly with a complexation enthalpy of -2.2 kcal/mol and with a B-N distance as long as 2.97 Å. This observation confirms that the N2 binding of the boron based Lewis acids cannot be rationalized by electrostatic considerations alone.</p><!><p>The B(SiR3)3 and B(GeR3)3 compounds are promising candidates for N2 and CO activation because of their strong and selective binding of these Lewis bases. In addition, the p bonding mechanism is likely to provide catalytic activation for chemical transformation, including reduction. However, none of the Lewis acids in this category in Table 1 has yet been synthesized. To our knowledge, B(SiPh3)3 is the only molecule of this type that has been prepared. 34 We have made some preliminary calculations on this molecule and found it to bind N2 relatively weakly with a complexation enthalpy of around -9 kcal/mol. The poor binding seems to be the consequence of a combination of electronic and steric crowding, and additionally there may be a kinetic barrier for binding due to a steric shielding of the B atom by the phenyl groups in the free Lewis acid.</p><p>We hypothesize that it may be easier to prepare heterogenous catalysts with the favorable B(Si-)3 or B(Ge-)3 bonding motif. Solid silicon and germanium have been prepared in the forms of crystals, 2-D materials and nanoparticles. This type of materials is commonly doped with boron to obtain semi-conductors. In particular for nanoparticles, it has been shown that the boron atoms accumulate at the surface, and a similar behavior is expected for larger particles and crystals. 35,36 B-doped silicon nanoparticles have also been shown to be resistant against oxidation in air, which is an import property if they are to be used as electrocatalyst. In Fig. 5 we show the DFT-PBE optimized structure of boron substituted and hydrogenated silicene, which is the silicon analog of graphene. As seen from the figure, this material has the advantage that the B(Si-)3 unit has a pyramidal geometry already before binding N2, and thus is preorganized to bind N2. The B-doped H-silicene is a relatively strong N2 binder and has a similar B-N bond length and N2 binding energy as B(SiH3)3 when computed using the PBE functional. Assuming that the error due to the functional is similar for both compounds, we predict a N2 binding enthalpy close to -16 kcal/mol for the B-doped H-silicene. Preliminary calculations of the distal pathway for electroreduction to ammonia, indicate that the first reductive step is rate-determining with a limiting potential close to 1.5 V. This is an encouraging result, but shows that further structural and chemical optimization will be needed to afford selective and efficient catalysts.</p><!><p>Lewis acids of the types B(SiR3)3 and B(GeR3)3 are found to bind N2 and CO with anomalously short and strong B-N or B-C bonds. The very short B-N bond in the complexes with N2 is particularly remarkable considering that N2 is a very weak Lewis acid. This selective binding enhancement is attributed to p-backbonding based on an analysis of the occupied orbitals in the complexes with N2, and an analysis of the density differences associated with the formation the complexes. However, the classical picture of p-backbonding as a donation into the unoccupied p*-orbitals of N2 is found to be a simplification. The p-bonding is a consequence of constructive orbital interactions between the diffuse and highly polarizable B-Si and B-Ge bond regions and the p-regions of N2. The B-Si and B-Ge bond regions are characterized by a ring shaped region of low ĪS(r). The value of the ĪS,min in these regions reflects the p-bond strength in the complexes, as ĪS,min follows the order of the B-N bond length, i.e. the shorter the bond, the lower the ĪS,min. The p-backbonding interaction is expected to activate the N2 unit for chemical transformation and reduction, as it decreases the electron density and increases the length of the N-N bond. The binding of N2 and CO by the B(SiR3)3 and B(GeR3)3 Lewis acids also has a strong s-bond contribution. The relatively high s-bond strength is connected to the high positive surface electrostatic potential [VS(r)] above the B atom, the boron VS,max. The magnitude of the VS,max is further increased when the B-Si coordination becomes pyramidal upon interaction. Introduction of electron withdrawing R-substituents increases the VS,max value and thereby the s-bond strength, but also leads to a higher ĪS,min and reduced p-backbonding strength. Thus, such substituents increase the general Lewis basicity, but will favor the binding of regular Lewis acid such as NH3 and Fmore strongly than binding N2 and O2. Another observation is that the boron based Lewis acids in contrast to Al-based Lewis acids generally have a significant covalent contribution to the s-bonding, which is indicated by intermolecular B-X bonds that are significantly shorter than the sum of the van der Waals radii and pyramidal geometries around the central B atom in the complexes.</p><!><p>Our computational results for the B(SiR3)3 and B(GeR3)3 Lewis acids indicate that these types of molecules have the potential to catalyze nitrogen reduction reactions. Unfortunately, they are highly reactive and difficult to synthesize. It may be easier to prepare heterogenous catalysts with the wanted B(Si-)3 or B(Ge-)3 bonding motif. Boron doped crystals, 2-D materials and nanoparticles may be prepared by regular synthesis techniques used for preparation of semiconductor materials. We have shown that such materials will have the B(Si-)3 unit in a favorable bonding geometry for N2 ligation. Preliminary calculations of electrochemical reduction of boron-substituted and hydrogenated silicene indicate potential for efficient catalysis but shows that further studies and optimization of chemical composition and nanostructure are needed.</p>

ChemRxiv

Metal-Binding Foldamers

Sequence-defined oligomeric molecules with discrete folding propensities, termed foldamers, are a versatile source of agents with tailored structure and function. An inspiration for the development of the foldamer paradigm are natural biomacromolecules, the sequence-encoded folding of which is the basis of life. Metal ions and clusters are common features in proteins, where the role of metal varies from supporting structure to enabling function. The ubiquity of metals in natural systems suggests promise for metals in the context of folded artificial backbones. In this minireview, we highlight efforts to realize this potential through a survey of published work on the design, synthesis, and characterization of metal-binding foldamers.

metal-binding_foldamers

Introduction<!>Nucleic Acid Mimetic Backbones<!>Aromatic Backbones<!>Peptoids<!>\xce\xb2-Peptides and Heterogeneous Backbones<!>Conclusions

<p>Sequence-encoded folding of biomacromolecules and the functions of the precisely structured architectures that result are the basis of life. The versatility of proteins and nucleic acids has driven chemists to seek to broaden the scope of backbones capable of such sequence-encoded folding. The agents arising from these efforts are termed "foldamers"—sequence-defined artificial oligomers that adopt discrete folded conformations.[1] Foldamers vary considerably in chemical composition, from chains highly reminiscent of proteins or nucleic acids to others that lack close analogy in nature.[2] Since the emergence of the field in the 1990s, foldamers have shown remarkable scope in terms of the structures and functions possible in designed synthetic backbones.</p><p>Metals play vital roles in natural proteins and have proved a powerful basis for protein design and engineering.[3] Introduction of unnatural amino acids and/or metal cofactors has further broadened the scope of functions possible in these systems.[4] Both rational design and computational approaches have been used to create de novo metal-dependent folds, including some capable of catalysis and electron transfer.[5] Complementary to efforts to make use of metals in protein design and engineering, metals have also found wide use in exerting control over peptide and protein self-assembly.[6]</p><p>The above two bodies of precedent on folded artificial backbones and metalloproteins suggest significant potential for metals in the context of foldamers. Progress on a number of fronts over the last two decades has helped to demonstrate this potential. In this review, we provide a survey of published work on metal-binding foldamers. As with their metalloprotein counterparts, the roles played by the metals in these systems are diverse (e.g., controlling folded structure, imparting redox-activity, enabling catalysis). Further diversity is found in the covalent connectivity of the folded artificial backbones as well as the chemical details of how these oligomers engage with metal. Collectively, these efforts show significant potential for metals as a means to broaden the scope of structure and function possible in foldamers.</p><!><p>While most work toward metal-binding foldamers has centered on chains with connectivity inspired by proteins or lacking close analogy in nature, discussed in subsequent sections, significant early progress was made with artificial backbones that mimic nucleic acids. The foundation for these efforts was the introduction of artificial metal-binding motifs into DNA. As an anionic biopolymer, DNA has long been known to bind metal cations.[7] Seeking to overcome limitations in information storage possible with a four-base genetic code, researchers have explored the utility of engineered metal-binding sites in DNA. The essential strategy was to replace natural hydrogen-bonded base pairs in the DNA double helix with a metal complex involving ligand moieties on neighboring sites in the duplex.[8] After demonstration of the idea in DNA, this concept was extended to artificial DNA-like oligomers.</p><p>Peptide nucleic acid (PNA) is a structural mimic of DNA where nucleobase moieties are displayed on an amide-based backbone.[9] PNA of suitable sequence can form duplex or triplex assemblies with complementary PNA, DNA, or RNA strands.[10] In 2003, Achim demonstrated incorporation of metal-binding moieties to replace canonical hydrogen-bonded base pairs in PNA.[11] Introducing a pair of bipyridine ligands led to a high-affinity binding site for Ni2+ in a PNA-PNA duplex. Metal binding did not disrupt the structure of the duplex; however, the stability was reduced relative to a corresponding assembly bearing a canonical T-A base pair at the same site. Based on the hypothesis that the lower stability was a consequence of a non-coplanar arrangement of bipyridine ligands, later efforts showed that a pair of 8-hydroxyquinoline moieties yielded a dramatically more stable duplex (Figure 1A).[12] The above technology was later advanced to create metal-directed PNA triplexes.[13]</p><p>Complementary to efforts to incorporate metal binding sites in the core of PNA duplex and triplex assemblies, Metzler-Nolte has explored the introduction of redox-active metals at PNA termini. One motivation in such efforts is the use of the resulting agents in electrochemical biosensors for DNA.[14] In one example demonstrating the power of this approach, copper-catalyzed azide-alkyne cycloaddition was used to modify PNA with a ferrocene moiety;[15] the resulting labeled chain was used to construct a PNA-based sensor (Figure 1B) able to detect DNA with high sensitivity and discrimination of single nucleotide polymorphism in the target.[16]</p><p>Exploring metal-dependent folding in another DNA-inspired artificial backbone, Meggers introduced metal-binding sites in place of hydrogen-bonded base pairs in glycol nucleic acid (GNA), a minimalist analogue of DNA.[17] Hydroxypyridone nucleobases were incorporated at two sites in a self-complementary GNA sequence; the resulting oligomer formed a duplex in the presence of Cu2+ (Figure 1C).[18] The motivation for creating sites for metals in the assembly was to facilitate high resolution structure determination. It was envisioned that the metal would aid in crystallization by stabilizing the duplex and provide a means for experimental phasing of the X-ray diffraction data. In the event, both goals were realized, and the resulting X-ray structure provided an atomic-level picture of information storage in a bio-inspired polymer with potential prebiotic relevance.</p><!><p>While some foldamers show a close relationship to biomacromolecules (proteins or DNA) in their chemical composition, others manifest the paradigm of sequence-encoded folding in backbones that lack clear analogy in nature.[2b] Owing to their rigidity and capacity to support noncovalent intrachain interactions, aromatic moieties have proved a privileged functionality in such work. Despite the challenge of designing folding patterns without a natural fold as a template for mimicry, aromatic oligomers have been shown capable of many complex structures and functions. In some examples, surveyed below, metal binding plays a key role.</p><p>Building on pioneering work toward the development of meta-phenylacetylenes as non-biological oligomers that can fold to form hollow helices,[19] Moore prepared derivatives with metal-binding nitrile groups lining the lumen of the molecular channel.[20] The resulting molecule was shown to fold in the presence of Ag+ to form a helix with coordinated metal ions in the cavity. Folding was dependent on metal in solvent systems unable to support folding of the parent chain. Lehn demonstrated that an aromatic oligomer based on alternating naphthyridine / pyrimidine moieties folds and assembles in the presence of K+ to form extended helical fibers.[21] This approach was later extended to chains based on alternating pyridine / hydrazone motifs that fold to form a helix in the presence of Pb2+ (Figure 2A).[22]</p><p>A class of foldamer related to the aromatic oligomers described above has aryl groups in the chain linked by amide bonds.[23] These scaffolds benefit from the functional versatility and predictable conformational behavior inherent to aromatic backbones as well as the synthetic accessibility afforded by amide-based couplings to construct sequence-defined chains. Fox reported metal-dependent folding in aromatic oligoamides through incorporation of a core salen or salenophen moiety as a metal-binding motif in the backbone.[24] The resulting chains fold in the presence of Ni2+ or Cu2+ to form a single-stranded helix. The stability of the fold depends on the presence of metal as well as the structural details of the coordination geometry. Electrochemical reduction in the case of the Cu2+ complex leads to structural reorganization and disruption of the fold. Subsequent related work later showed that absolute helicity in an otherwise achiral oligomer could be controlled through a stereocenter remote from the central metal binding site in the helix.[25] This system found use as a host for the comparison of helix-directing ability of different chiral motifs.[26]</p><p>Huc has demonstrated metal-dependent folding in aromatic oligoamides based on a designed helical capsule previously discovered and developed in their group.[27] One example involved construction of a derivative of the capsule with a central pyridazine-pyridine-pyridazine moiety (Figure 2B).[28] This monomer undergoes a conformational change in the presence of Cu+ or Ag+ from a backbone arrangement incompatible with the helical fold to one that promotes it through encapsulation of the metal in the interior cavity formed in the folded state. A high-resolution crystal structure of one of the helical capsules provided key support for the design hypotheses and the role of the metal in the structure. Later exploration of a broader panel of alkali and alkaline earth metals showed affinity of the oligomer for a variety of ionic guests with high selectivity for Mg2+.[29]</p><!><p>The most common class of metal-binding foldamers are those that mimic natural proteins in chemical composition and/or folded structure. In some of such systems, the backbone connectivity is a subtle modification of natural α-peptide, but the fold adopted by the artificial chain is completely distinct from a biological counterpart. One example of this class are peptoids, glycine-based oligomers with side chains displayed on the amide nitrogen rather than the Cα position in natural peptides.[30] The tertiary amides along the backbone are subject to cis/trans isomerization, impacting the folded states accessible. Further, due to the lack of hydrogen-bond donors in the main chain, folding is driven by local stereoelectronic effects and short-range hydrophobic interactions involving side chains. Despite these complexities, peptoids have emerged as a valuable foldamer class in terms of both structures and functions possible.[31]</p><p>In the first example of a metal-dependent peptoid fold, Zuckermann reported a helix-turn-helix with a high affinity metal coordination site.[32] The approach was guided by the propensity of peptoid chains bearing a preponderance of hydrophobic monomers with bulky chiral side chains to fold into a polyproline-I (PP-I) helical secondary structure. Two peptoid PP-I helices were linked by a flexible α-peptide loop and fluorescent donor and quencher incorporated at the termini as a probe for helix-helix association. Using this scaffold as a host sequence, metal-binding imidazole and thiol side chains were systematically introduced at different sites on the opposing helices. Optimal placement of two thiols and two imidazoles near the helix termini led to an oligomer that binds Zn2+ with a dissociation constant in the low nM range (Figure 3A).</p><p>Kirshenbaum reported a metallopeptoid by a different approach.[33] Rather than targeting a tertiary fold, the design paradigm was to introduce metal-binding 8-hyroxyquinoline side chains at adjacent turns of a PP-I helical secondary structure (Figure 3B). The resulting peptoid was able to form complexes with Cu2+ and Co2+. The high affinity observed was attributed to a pre-organized metal binding site based on molecular modeling, which showed a similar helical conformation adopted by the peptoid in the metal-free and metal-bound states.</p><p>Maayan, who carried out the work on metal-binding peptoids with Kirshenbaum, went on to make contributions that have collectively expanded the structural and functional scope of metal-binding peptoids. In a follow up to the above study, exploration of sequence effects in the hydroxyquinoline-functionalized helix revealed an interplay between conformational behavior inherent to the peptoid backbone and distortions imposed by metal coordination geometry.[34] Engineering the system further yielded peptoids with multiple metal-binding sites in a single chain.[35] Thus, incorporation of four hydroxyquinoline moieties in consecutive turns of a PP-I helix led to an oligomer that can simultaneously bind two Cu2+ or Co2+ ions. A noteworthy aspect of this system was the use of monomers with hydrophilic chiral side chains, which rendered the metallopeptoid complex soluble in water. Providing new routes to enable synthetic access to metal-binding peptoids, it was shown that copper-catalyzed azide alkyne cycloaddition could be used to generate functionalized peptoids where the newly introduced triazole moieties also serve as metal binding ligands.[36]</p><p>In all the examples discussed to this point, the metallofoldamer complex involves a single type of metal ion. Many proteins in nature bind to multiple different metals at distinct sites, inspiring researchers to seek foldamers able to do the same. In one effort to this end, Maayan developed a helical peptoid able to switch its fold in response to the composition of metal ions present in solution.[37] The design was based on an oligomer bearing 8-hydroxyquinoline and 2,2ʹ:6ʹ,2ʹʹ-terpyridine side chains at adjacent turns in a PP-I helix. In the presence of Cu2+, this peptoid forms a 1:1 complex in which the two chelating side chains bind in a square pyramidal geometry. When a mixture of metal ions composed of Cu2+ in combination with another species Mn+ (Mn+ = Zn2+, Fe3+, or Co2+) is present, a hetero-bimetallic complex forms with two peptoid chains binding simultaneously to two different metals. In related later work, a peptoid was constructed with two distinct metal binding sites in a single chain.[38] Positive allosteric cooperativity was observed in the system, a hallmark in natural proteins.</p><p>As mentioned in the introduction, metal coordination can be a powerful means of directing peptide and protein self-assembly. Exploring this idea in the context of peptoids, Maayan has shown that suitably designed sequences bearing 2,2ʹ-bipyridine moieties can fold and associate in the presence of metal ions including Ru2+.[39] Chiral induction from the peptoid chain to the metal center was observed, and later work demonstrated alterations in the peptoid sequence could be used to tune photoluminescent properties of the complex.[40] Related bipyridine-functionalized peptoids were shown to form complexes with other metals, including Cu2+, Co2+, and Ni2+.[41]</p><p>While most metalloproteins involve a single metal ion at each binding site, multimetallic clusters are found in some systems. Seeking to expand the scope of peptoids as metal-binding scaffolds, Zuckermann developed sequences able to fold and bind such clusters (Figure 3C).[42] The design was based on a short chain with appropriately positioned carboxylate side chains as ligands. A combination of rational design to develop a core sequence motif and combinatorial screening to optimize and extend the oligomer led to a peptoid able to replace three of the four acetate ligands in the cobalt oxo cluster Co4O4(OAc)4(py)4 [OAc = acetate, py = pyridine].</p><p>Maayan advanced metal-binding peptoids into the realm of catalysis through the creation of sequences able to promote aerobic oxidation of alcohols.[43] A series of peptoids were designed in which TEMPO and a Cu+-binding 1,10-phenanthroline ligand were introduced as side chains. The resulting peptoids were shown to catalyze the oxidation of benzyl alcohol to benzaldehyde with turnover numbers >10-fold improved relative to a simple mixture of TEMPO and phenanthroline catalysts under identical conditions. Comparison of sequence-function relationships revealed a minor role for folded structure in determining catalytic efficiency; however, control experiments showed the peptoid backbone was essential. This system was later extended to develop catalysts for the oxidative synthesis of imines[44] and electrochemical water oxidation.[45]</p><p>Inspired by the observation of azole (thiazole, oxazole) moieties as common metal-coordinating groups in peptide-derived natural products, Fuller recently described a new class of metal-binding foldamer backbone based on oligomers that combine peptoid and azole monomer units in a 1:1 alternating pattern.[46] One such chain with a terpyridine moiety at its terminus was shown to form a high affinity 2:1 complex with Zn2+.</p><p>All the above examples of metal-binding foldamers involve linear chains as ligands; however, cyclic species also have significant potential. De Riccardis prepared a series of different sized macrocyclic peptoids in an effort to identify receptors able to bind metals.[47] The six-residue macrocycle was shown to bind a variety of alkali metals, and an X-ray crystal structure of the complex with Sr2+ showed the peptoid adopts a symmetrical conformation with metal ion bound in the core via backbone carbonyl groups (Figure 3D). In a follow-up study, the series of peptoid macrocycles was extended to include eight- and ten-residue homologues.[48] The larger oligomers were also able to bind a range of alkali metals with comparable affinity, and some were shown able to transport ions across a membrane bilayer. Similar macrocyclic peptoids have also found application in metal-directed self-association to form supramolecular materials.[49]</p><p>A useful practical aspect of peptoids as a foldamer class is their facile synthesis via the sub-monomer approach,[50] which is highly amenable to combinatorial methods. Francis has reported combinatorial synthesis and screening to identify peptoid ligands able to bind toxic heavy metals. In one example, a library consisting of ~2400 four-residue peptoid sequences was synthesized in a one-bead one-compound format and screened to find oligomers that bind hexavalent chromium species in aqueous solution. A polymer resin functionalized with the peptoid oligomer depleted toxic chromium salts from contaminated samples of creek and ocean water.[51] This platform was later extended to identify peptoid ligands able to extract Cd2+ from human serum.[52]</p><!><p>As with peptoids, oligomers composed of β-amino acid residues (β-peptides) have a rich history in foldamer research.[2a] That said, among the many structures and functions elicited from β-peptides, systems involving metals are rare. In one early example, Seebach showed metal-dependent stabilization of isolated β-peptide helix and hairpin secondary structures.[50] Their goal in this effort was to construct a β-peptide analogue of the ββα zinc finger domain, a natural protein tertiary fold where helix and hairpin simultaneously engage a Zn2+ ion (Figure 4A). Thus, β3-residue homologues of the natural metal-binding amino acids histidine and cysteine were incorporated into two separate β-peptides—at adjacent turns in one helix-forming sequence and at cross-strand positions in another hairpin-forming sequence. NMR structures in the presence of Zn2+ showed that both the helix and hairpin were able to accommodate the bound metal ion. Combining the two sequence motifs into a single β-peptide chain yielded evidence of Zn2+ binding; however, structural details on the nature of the complex remained elusive.</p><p>In efforts to advance the structural complexity possible in foldamers beyond isolated secondary structure, Schepartz reported an amphiphilic β-peptide that self-assembles to form an octameric helix bundle quaternary structure.[53] In later work, this system was used to develop an assembly with binding sites for metal ions.[54] Addition of the β3-residue homologue of the natural amino acid cysteine to the C-terminal position of the parent bundle-forming β-peptide sequence led to an analogue that forms a similar octameric bundle but with new potential metal coordination sites. This helix bundle was screened for the ability to bind various divalent metal ions, leading to the identification of Cd2+ as uniquely able to form a defined complex. Additional experiments revealed a preferred stoichiometry of two bound Cd2+ ions per eight-chain helix bundle and a degree of positive allostery between the two metal binding sites.</p><p>An advantage of foldamers based on oligoamides is the modularity of design and synthesis inherent to a backbone accessible by iterative coupling of amino acid subunits. This modularity has been exploited to produce a variety of foldamers that blend multiple monomer types in a single chain. A number of these mixed or "heterogeneous" backbones have been applied in the creation of metal-binding systems.</p><p>Shionoya, a forerunner in the development of metal-mediated base pairing in DNA, later explored metal-binding in foldamer backbones through the incorporation of oximes as ligand groups into an amide-based oligomer chain.[55] The resulting heterogeneous backbone was shown to adopt a range of different secondary structures (helix, hairpin, double-hairpin) in the presence of Pd2+, depending on the chain length and other supporting ligand(s) present.</p><p>Zhao has explored metal binding foldamers containing amino acid analogues of the steroid cholic acid.[56] Noteworthy in these oligocholate scaffolds is that folding does not rely on strong intrachain interactions and is thus highly sensitive to environmental effects. This characteristic makes the system well suited to operate as a switch responsive to external stimuli. Using metal binding as the trigger for conformational interconversion, Zhao developed an oligocholate sensor for Hg2+.[57] The design was based on a heterogeneous oligomer chain with a 1:1 alternation of cholate monomer and the amino acid methionine. The former serves as a structural element, and the latter provides a metal-binding side chain moiety. Introduction of fluorescent donor and acceptor groups at the termini allowed a FRET-based readout of folding. The oligocholate probe was shown to be a sensitive and selective sensor for mercury. Incorporation of the scaffold into surfactant micelles allowed the sensor to function effectively in aqueous solutions.[58]</p><p>Schafmeister has developed and applied a foldamer class based on spiro-ladder oligomers referred to as "bis-peptides."[59] These oligomeric chains lack freely rotatable bonds in the backbone and can thus be considered as modular molecular rods with shape and functional group display predictably determined by covalent connectivity. Seeking to construct a macromolecule that undergoes a defined conformational change upon interaction with metal, two rigid bis-peptide chains with metal-binding 8-hydroxyquinoline moieties at their termini were connected by a flexible amino acid linker.[60] In the presence of Cu2+, the heterogeneous chain was shown to undergo a conformational change from an extended "open" state to a "closed" state in which the ends of the rod are brought in proximity.</p><p>Our lab has a longstanding interest in the development of foldamers based on engineering backbone connectivity in natural amino acid sequences.[61] An advantage of this approach is that it provides facile access to heterogeneous-backbone mimics of a variety of complex tertiary folding patterns. Among systems explored in this work is the ββα zinc finger domain. We created mimics of zinc finger 3 from the human transcription factor Sp1 (Sp1–3) in which β3-residues or N-Me-α-residues bearing the side chain of the replaced α-residue were incorporated in the helix and hairpin regions, respectively.[62] The resulting heterogeneous-backbone oligomer showed native-like metal coordination and folded stability, and an NMR structure indistinguishable from the prototype domain. An unexpected observation in this work was that modification in the metal-binding turn led to a variant unable to fold or bind metal. In an effort to gain insight into this finding, we examined the impact of backbone alteration in a related zinc finger domain from the same protein (Sp1–2), which has a slightly elongated loop relative to Sp1–3.[63] The Sp1–2 metal-binding turn was shown to be more permissive to backbone alteration, and comparison of several artificial turn inducers led to identification of an Aib-Gly motif as most native-like in structure and stability. This finding was leveraged to generate a foldamer mimic of the domain where the helix, hairpin, and turn were simultaneously modified (Figure 4B).</p><p>The above strategy toward heterogeneous-backbone mimics of the zinc finger domain involves backbone modifications interspersed throughout a sequence. An alternate approach is prosthetic modification, where a contiguous segment corresponding to a single secondary structure element is replaced by a foldamer analogue. Building on their pioneering foundational work to develop aliphatic oligoureas as a foldamer class,[64] Guichard demonstrated the prosthetic replacement of the α-helix in a ββα zinc finger from the transcription factor Egr1 (Figure 4C).[65] An analogue was synthesized in which the helical region in the chain was replaced with a helix-forming oligourea bearing a similar side chain set. The chimeric variant was shown to bind Zn2+ with high affinity, and an NMR structure confirmed that the foldamer helix was compatible with the metal-dependent tertiary fold of the native domain. Importantly, the chimeric zinc finger was shown to bind to duplex DNA, an essential function of the prototype domain.</p><!><p>In summary, we have reviewed here published work on the range of structures and functions possible in artificial oligomers with discrete folding behavior and defined metal binding characteristics. As with natural metalloproteins, these metallofoldamers are remarkably diverse in terms of the metals involved and the roles they play in determining structure and function. Further, reflecting a central tenet of the foldamer concept, the chemical details of the backbones and the folded architectures they adopt also vary widely among these systems. Already in work to date, functions have been documented including metal-dependent folding, allosteric cooperativity, molecular recognition, catalysis, and ion transport. Collectively, these efforts suggest tremendous promise for metals to further advance the scope of structures and functions possible in foldamers.</p><p>Among frontiers for the metallofoldamer field are expanding structural complexity and expanding functional scope in this molecular class. In terms of structure, the intricate folds shown possible to date, while impressive, pale in comparison to the complexity and diversity of folds seen in natural metalloproteins. While nature has enjoyed the benefit of evolution to establish countless folds from a common set of building blocks, chemists have an arsenal of backbone connectivities available to use in the rational design of artificial metal-dependent folds. In the realm of function, the vast scope of behaviors seen in natural metalloprotein systems suggests unrealized potential for metallofoldamers in contexts including macromolecular recognition, catalysis, and energy transduction. Moreover, artificial metal-dependent folds have the potential to be used in eliciting functions that lack analogy in nature. These novel functions, in turn, enable new applications (e.g., sensors, materials, devices). Ongoing research on metal-binding foldamers now and in the years ahead will help to further realize their potential.</p>

PubMed Author Manuscript

Boronic acids for functionalisation of commercial multi-layer graphitic material as an alternative to diazonium salts

A novel radical-based functionalisation strategy for the synthesis of functionalised commercially obtained plasma-synthesised multi-layer graphitic material (MLG) is presented herein. 4-(trifluoromethyl)phenyl boronic acid was utilised as a source of 4-(trifluoromethyl)phenyl radicals to covalently graft upon the graphitic surface of MLG. Such a methodology provides a convenient and safer route towards aryl radical generation, serving as a potential alternative to hazardous diazonium salt precusors. The structure and morphology of the functionalised MLG ( Ar f-MLG) has been characterised using XPS, Raman, TGA, XRD, SEM, TEM and BET techniques. The XPS quantitative data and Raman spectra provide evidence of successful covalent attachment of 4-(trifluoromethyl)phenyl groups to MLG.

boronic_acids_for_functionalisation_of_commercial_multi-layer_graphitic_material_as_an_alternative_t

Introduction<!>Synthesis of Ar f-MLG<!>Analysis and characterisation of Ar f-MLG<!>X-ray photoelectron spectroscopy<!>Determining the decomposition characteristics by TGA<!>Examining the graphitic structure by Raman spectroscopy<!>Analysis of interlayer spacing using X-ray diffraction data<!>Analysis of surface morphology using SEM and TEM<!>Surface area and porosity analysis<!>General remarks<!>Characterisation methods<!>Conclusions<!>Conflicts of interest

<p>Graphene describes an allotropic form of carbon consisting of a single layer hexagonal array of sp 2 hybridised carbon atoms. The combination of stacked graphene planes makes up the well-known structure of graphite. Whilst graphite has been known for several hundred years, graphene itself was not isolated until 2004. 1 Graphene possesses excellent mechanical, electrical, optical, thermal and biocompatible properties. A whole host of potential applications for these materials has been touted by both academia and industry. 2,3 Although research on this material is still in its early stages of development, plenty of applications have been proposed which harness and exploit the aforementioned properties.</p><p>Notwithstanding all of the aforementioned properties of these materials, there are some significant challenges which hinder the practical application of graphene materials on any large or industrial scale. 4 Graphene is a challenging material to work with due to its limited dispersibility within most solvents. Additionally, characterisation challenges are very common. 5 Pristine graphene is only available in small laboratory scale quantities whilst larger scale industrially derived graphene contains many defects, oxygen functionalisation and are typically obtained as multi-layer graphitic material (MLG). 6 Furthermore it is typically obtained with a broad distribution of sizes and morphologies.</p><p>Issues such as processability can be addressed via functionalisation. Functionalisation can also alter the properties of the material in a number of ways. For example, addition of new functional groups can enhance solubility and/or generate new composite materials with specifically designed properties. 7,8 The incorporation of additional organic or inorganic moieties to the surface of such materials also creates steric repulsion between the sheets and stacks, thus providing an energy barrier against undesired clumping. 9 Furthermore, covalent functionalisation can also alter the graphitic structure by introducing a band gap, thereby allowing the material to switch to lower conductance states. 10 It is thus imperative to obtain new, safe routes to introduce covalent functionality to new materials. 7,8 Over the 16 years since graphene was first isolated, a vast amount of research has been devoted to the development of methods towards covalently functionalised graphene. [11][12][13][14][15][16][17][18] Many of these methodologies utilise hazardous conditions, such as diazonium salts. From a synthetic point of view, diazonium salts provide an excellent and straightforward source of radicals from which the only by-product is nitrogen gas. 19 This offers a clean methodology for attaching functional groups to the material. Such radicals combine with radicals present on the surface to form new covalent bonds as demonstrated by a number of groups. [20][21][22][23][24][25][26][27] Their application, however, does have some undesirable synthetic conditions which makes it challenging for large scale industrial production. Many diazonium salts decompose violently and can be sensitive to friction and shock. 28 As a result, access to radicals through alternative precursors is desirable for the purpose of large-scale functionalisation of graphene and graphitic material.</p><p>Through an industrial collaboration, we embarked on an investigation to explore alternative starting materials which allow access to aryl radicals required for covalent functionalisation. From an industrial perspective, plasma-synthesised multilayer graphitic material 29,30 is more accessible and as such there is significant value in finding synthetic methodologies for its derivatisation. As such we aimed to explore these more accessible materials for functionalisation as opposed to graphene. Herein, we wish to report a preliminary account of these investigations. We show that 4-(trifluoromethyl)phenyl radicals, accessed via oxidative conditions of 4-(trifluoromethyl)phenyl boronic acid, can be used to covalently functionalise the outer surfaces of plasma-synthesised MLG, thus forming 4-(trifluoromethyl)phenyl functionalised MLG ( Ar f-MLG). We also explore the impact of carrying out this functionalisation by examining the changes in relation to the original material. The functionalised material was characterised using XPS, Raman, TGA, XRD, SEM, TEM, and BET spectroscopic and analytical methods.</p><!><p>As a means of exploring alternatives to generate aryl radicals in situ for the purposes of covalently functionalising MLG, we looked at aryl boronic acids as potential sources. Aryl boronic acids are well known for their applications in cross coupling reactions, leading to the formation of C-C bonds. It has previously been demonstrated that aryl radicals can be generated from aryl boronic acids under various oxidative conditions, as an alternative strategy for forming C-C bonds. For example, oxidants and combinations of oxidants such as Mn(OAc) 3 , 31 AgNO 3 / K 2 S 2 O 8 , 32,33 have all been employed for these purposes.</p><p>Baran recently reported the application of a mixture of silver nitrate and potassium persulfate for the formation of C-C bonds. 32,33 It has been postulated that the persulfate is reduced by the silver to form both [SO 4 ] 2À and [SO 4 ] À , which in turn react with the aryl boronic acid to generate aryl radicals (Scheme 1). 32 The silver(II) is then reduced back to silver(I) as shown.</p><p>The transformations proceed within a biphasic aqueous/DCM solvent system.</p><p>We utilised the Baran protocol for generating aryl radicals in the presence of MLG as a means of functionalising this material. For the purposes of aiding the subsequent characterisation, we chose 4-(trifluoromethyl)phenyl boronic acid as the aryl radical source. This precursor was chosen since it was reported to be most the efficient of those tested (due to the electron withdrawing nature of the CF 3 group) 32 and it contained fluorine which could be readily observed by X-ray photoelectron spectroscopy (XPS). Prior to the reaction with the graphene material, a control reaction was undertaken. In this control reaction, 4-(trifluoromethyl)phenyl boronic acid was reacted with silver nitrate and potassium persulfate in a 1 : 1 mixture of water and DCM (Scheme 2). We found that 44 h stirring at room temperature was sufficient for complete conversion of the starting material. Two products were separated from the DCM layer following work up. These products were identified as the homo-coupled product 4,4 0 -bis(trifluoromethyl)-1,1 0 -biphenyl (A) and 4,4 0 -bis(trifluoromethyl)-1,1 0 -biphenyl ether (B) by NMR spectroscopy and mass spectrometry, in an approximate 1 : 1 ratio. The former product (A) was expected, whilst the oxygen-bridged compound (B) was not initially anticipated. We believe that this product originates from the radical species reacting with water to form phenol, which then undergoes a C-O coupling reaction to form the ether product. It was important to confirm the identify of these products since they can also form intermolecular interactions with the surface of the material, which can confuse later interpretation and its characterisation.</p><p>With evidence of radical generation, we proceeded to repeat the same conditions in the presence of MLG with the aim of functionalising this material with the aryl groups (to form Ar f-MLG) with the expectation that both 4,4 0 -bis(trifluoromethyl)-1,1 0 -biphenyl and 4,4 0 -bis(trifluoromethyl)-1,1 0 -biphenyl ether organic side products would also be present within the reaction mixture (Scheme 3). After 44 h, the newly formed Ar f-MLG was isolated from the reaction mixture. This involved purification steps to ensure that organic and inorganic by-products were mostly removed from aryl functionalised MLG ( Ar f-MLG). The reaction mixture was centrifuged and the resulting powder was subjected to repeated dispersion/centrifugation cycles using large volumes of water, acetonitrile and DCM. The remaining Scheme 1 Reported mechanism for the generation of aryl radicals using the approach by Baran and co-workers. 32 Scheme 2 Reaction of 4-(trifluoromethyl)phenyl boronic acid with silver nitrate and potassium persulfate in a biphasic water and DCM reaction.</p><p>solid material was then placed under high vacuum (10 À6 bar) for 168 h.</p><!><p>The new functionalised material, Ar f-MLG, was analysed and characterised via a range of techniques in comparison with the original MLG sample. This allowed us to examine the degree of functionalisation and its impact on the graphitic structure. The results acquired from these techniques are outlined below.</p><!><p>Both original and functionalised samples were examined by XPS, the results of which are presented in Fig. 1-4 and Table 1. Additional details and spectra are provided in the ESI, † in Fig. S1.</p><p>We initially characterised MLG and subsequently Ar f-MLG to identify and compare surface characteristics, as well as the changes to its elemental composition. The data confirms changes to the elemental composition of the material upon functionalisation (Table 1 and Fig. 1). Most notable is the incorporation of fluorine into Ar f-MLG with a relative atomic concentration of 3.5% (at%) for the F 1s orbital. Deconvolution of the high-resolution XPS spectrum of C 1s spectrum (Fig. 2a) shows that MLG consists of seven environments for the carbon atoms. These were as follows with the binding energies within brackets: C sp 2 (284.5 eV), p-p* (290.9 and 294.0 eV), CQO (288.3 eV), C-O (286.6 eV), C sp 2 (284.8 eV) and O-CQO (289.6 eV). A breakdown of the elemental compositions of these components is shown in Table 1. Carbons represent 94.9 at% on the total elemental composition at the surface as expected from graphitic material.</p><p>The corresponding C 1s spectra for Ar f-MLG (Fig. 2b), consists of nine components corresponding to C sp 2 (284.5 eV), p-p* (290.9 and 294.0 eV), CQO (287.7 eV), C-O (286.4 eV), C sp 2 (284.8 eV), O-CQO (289.0 eV), C-F x (284.9 eV) and CF 3 (292.7 eV). Whilst both show graphitic character, Ar f-MLG shows additional C-F functionality, originating from the reaction of 4-(trifluoromethyl)phenyl radicals with the graphitic material. Overall, there is a reduction in the percentage carbon in this sample. This is consistent with functionalisation. Furthermore, there is a large increase in the concentration of sp 3 hybridised carbon centres, with respect to sp 2 hybridised carbons, from 8.8 at% up to 15.8 at%. Some of this increase has be attributed to oxidation of the MLG sample as a result of the oxidising conditions of the reaction (vide infra). As outlined below however, the level of increase in the oxygen content does not account for such a large increase in sp 3 hybridised centres. These observations are therefore consistent with some degree of the covalent attachment of the aryl functional groups to the conjugated sp 2 network to form Ar f-MLG.</p><p>The O 1s spectrum for MLG (Fig. 3a) consists of five components CQO (531.7 eV), O 1s C-O-C (533.1 eV) and O 1s satellite structures (535.1 eV, 536.9 and 538.9 eV). This confirms the presence of a significant degree of oxygen functionality already within the starting material which originates from its plasma processing. The presence of these oxygen functionalities in MLG corresponds to 4.8 at%. The O 1s spectra for Ar f-MLG looks similar to MLG (Fig. 3b), displaying the same components corresponding to CQO (531.7 eV), C-O-C (533.1 eV) and O 1s satellite structures (535.1, 536.9 and 538.9 eV), with similar respective ratios. Upon functionalisation however, the oxygen content almost doubled, increasing from 4.8 to 8.5 at%. This increase in oxygen content is attributed to the oxidising reagent which is required for radical generation. In addition to generating the 4-(trifluoromethyl)phenyl radical it also increased the level of oxidation of the graphene surface to some degree. In order to confirm this, we carried out a control reaction under the same conditions with only K 2 S 2 O 8 and a sample of MLG. We refer to this new material as c Ox-MLG. Its surface elemental composition as determined by XPS is also presented in Table 1 for comparison. In this case, a large increase in the oxygen concentration was indeed observed in the sample up to 10.7 at%. Even though the percentage of sp 3 carbon centres in Ar f-MLG is higher than c Ox-MLG, the increase in oxygen content is much lower (cf. 8.5 at% vs. 10.7 at%). This is consistent with the fact that the additional sp 3 centres originate from aryl group functionalisation. Furthermore, an increase in oxygen content may also be explained by the fact that a portion of 4-(trifluoromethyl)phenoxide [OC 6 H 4 (CF 3 )] groups could also be covalently bonded to MLG in addition to C 6 H 4 (CF 3 ). The ether species (B) was of course found in the control reaction in the absence of MLG (where both A and B were formed).</p><p>Fig. 4 highlights the XPS spectra at higher binding energies, indicating the presence of other functional groups. Upon functionalisation, a peak at 687.9 eV (Fig. 4a) confirms the presence of fluorine in the Ar f-MLG material. This is not present within the starting material (see Fig. 1a and Table 1). This binding energy is consistent with the presence of the 4-(trifluoromethyl)phenyl functional group. 36 Alongside this evidence of incorporation of this functional group are changes to the elemental compositions of carbon and oxygen. The carbon content reduced to 86.5 at%, as expected. Furthermore, the scenario of physisorption of the starting material (trifluoromethylphenyl boronic acid) on to the MLG surface can be ruled out due to the absence of any boron content in the XPS data. The material was washed multiple times and the corresponding filtrates were monitored by 19 F and 1 H NMR spectroscopy to ensure that the material was free of any unreacted or residual organic species.</p><p>The presence of silver (3d orbitals) was observed in the spectrum for Ar f-MLG at 367.0 and 372.8 eV. The former Ag 3d 5/2 peak was further deconvoluted revealing signals at 367.9 eV and 366.9 eV (Fig. 4b). These resemble silver salts in the form of AgCl/Ag and Ag 2 SO 4 , respectively. 37,38 This is likely to be due to silver salts retained within the material. These proved rather challenging to remove and they appeared to be trapped within the material despite multiple washings steps. Furthermore, both Ar f-MLG and c Ox-MLG materials revealed a small percentage of sulfur incorporation as a result of the oxidising agent. Again, these proved challenging to remove completely. Some nitrogen moieties are present within both MLG and Ar f-MLG at relatively consistent compositions (see Fig. S4, ESI † and Table 1). The retention of these impurities within Ar f-MLG results from the inherent nature of this plasmasynthesied graphitic material where the silver, for example, can get trapped within pores and defects in the material (vide infra).</p><!><p>Thermogravimetric analysis (TGA) enables us to examine how the materials decompose, which can be very useful in confirming the nature and degree of functionalisation within the material. Accordingly, the thermal stability of both MLG and Ar f-MLG was investigated using TGA measurements at temperatures up to 700 1C. A comparison of the results is depicted in Fig. 5. In both cases, a gradual mass loss is observed with distinct or well-defined mass loss regions. Pristine graphite has been found to show little decomposition until around 600 1C within an O 2 atmosphere and up to 1000 1C within a nitrogen atmosphere. Accordingly, mass loss within both samples at these lower temperatures can be attributed to the decomposition of covalently and non-covalently bonded functionality over a gradual period. 39,40 Initial mass loss (up to 100 1C), is attributed to the removal of water from the surface. Decomposition from this point is then assigned to the removal of covalently bonded oxygen functionality. In the case of graphene oxide, it has been found that the decomposition of covalently bound oxygen functionality takes place above 150 1C. 41 Ar f-MLG shows a lower thermally stability than MLG, decomposing at a faster rate. This is assigned to the increased covalent functionality it possesses, including more oxygen functionality and 4-(trifluoromethyl)phenyl moieties. The presence of silver salts on the surface of Ar f-MLG may also open up other pathways leading to a more rapid mass loss from this material.</p><!><p>Raman spectroscopy was used to examine the graphitic structure and extent of defects within MLG and Ar f-MLG, using a wavelength of 514 nm. Raman spectra for the functionalised and unfunctionalised material are depicted in Fig. 6. The spectra for the control material, c Ox-MLG (K 2 S 2 O 8 only) is presented in Fig. S3 (ESI †). A comparison of the spectra for MLG and Ar f-MLG revealed some changes to the structure of the two materials. Both contained the characteristic peaks for graphitic materials, most notably the G band which appeared at 1576.6 cm À1 for MLG and 1580.3 cm À1 for Ar f-MLG. This doubly degenerate E 2g band confirms the sp 2 carbon network within the materials. 10,42,43 The presence of strong D bands indicates the low crystallinity of the materials and also a high degree of sp 3 carbon centres. This is expected for plasma-synthesised MLG's. The level of defects is revealed by the D bands, which occur at 1344.6 cm À1 (for MLG) and 1350.9 cm À1 (for Ar f-MLG). The D band represents the breathing modes with A 1g symmetry involving phonons near the K zone boundaries. 44 The 2D bands at 2704.0 cm À1 (MLG) and 2708.0 cm À1 ( Ar f-MLG) correspond to the second order symmetry allowed overtone of the D band. Within both spectra, the 2D bands are broad and heavily upshifted in respect to that of single layer graphene, suggesting the presence of multiple layered stacks. This is further confirmed by examining the relative intensity of the G band compared to the 2D band, thus providing I 2D /I G ratios. 45 I 2D /I G ratios lower than one are indicative of multi-layered structures. 46 The ratios for MLG and Ar f-MLG are 0.44 and 0.47, respectively. Additional defect-induced bands, which are more intense in the spectrum for Ar f-MLG, are observed at 1618 cm À1 and 2930 cm À1 . 47,48 The higher level of defects within the MLG material most likely originates from the plasma processing during the synthesis. This can lead to an increased number of covalently attached oxygen functionalities at the outer surfaces of the sheets. Addition of these oxygen groups to the sp 2 3 sites, and thus, this confirms an increase in the covalent functionalisation in comparison to the original MLG material. This increase is likely to originate from the desired 4-(trifluoromethyl)phenyl incorporation and additional oxygen functionality. As highlighted above, the XPS analysis indeed confirmed a significant increase in oxygen functionality which was attributed to the oxidising agent K 2 S 2 O 8 . In order to confirm this, the Raman spectrum of c Ox-MLG was also examined. This exhibited similar changes to those found in Ar f-MLG, however, to a lesser extent with respect to the starting MLG. For example, the I D /I G and I 2D /I G ratios were found to be 0.56 and 0.40 (cf. with the values 0.65 and 0.47 for Ar f-MLG). This is consistent with the incorporation of 4-(trifluoromethyl)phenyl groups onto the MLG material alongside some increase in the oxidation level of the material. It should be noted that the level of functionalisation is estimated to be lower than some diazonium salt methodologies and thus further optimisation of this new strategy is needed. Nevertheless, both the Raman spectroscopic and XPS data are both consistent with functionalisation of MLG with trifluoromethylphenyl groups.</p><!><p>In order to gain more detailed information on the interlayer spacing and orientation of the planes within MLG and Ar f-MLG, an X-ray diffraction (XRD) investigation was carried out. The XRD spectra for both MLG and Ar f-MLG are shown in Fig. 7. The spectra show that both materials contain hexagonal ABAB stacking (2H) and rhombohedral ABCA (3R) stacking. This is consistent with that found in commercially available graphene and graphitic-based materials. 49 Strong diffraction peaks are present within both spectra at 26.61, corresponding to graphitic 2H (002) and 3R (003) planes, with an interlaying spacing of 3.35 Å. Analysis of the line shape of this signal suggests that for the majority of the materials, the number of graphene layers within both MLG and Ar f-MLG are in the region of 58 to 73 layers (see ESI † for further details). As such, the material could therefore also be described as graphite nanostructures. 50 For MLG, two smaller intensity lines are also observed at 42.591 and 44.561, which correspond to the 2H, (100) and (101) stacking planes. Two further small intensity lines are observed at 43.441 and 46.201, which correspond to the 3R, ( 101) and (012) stacking planes. The presence of this four-lined pattern is more clearly seen in the expanded section of Fig. 7. This provides evidence of the 3R and 2H phases within the structure. For these, a ratio of 49 : 51% (3R : 2H) was determined (see ESI † for details). These are consistent with that observed within various related materials. 49,51 Again, for MLG two additional lines at 54.731 and 77.621 are observed which correspond to graphite 2H (004) and ( 110) planes.</p><p>Many additional diffraction lines are present within the spectrum of the Ar f-MLG sample, suggesting other chemical species have been incorporated within the structure. The characteristic four-line pattern also appears to be present within this spectrum albeit the 2H (101) and 3R (012) lines are obscured by some new lines. This confirms that there is no significant change to the interlayer spacing upon functionalisation. As highlighted with asterisks (*), the majority of these correspond to the diffraction of the AgCl, Ag and Ag 2 SO 4 impurities incorporated into the material (PDF card numbers: 00-006-0480, 52 01-073-6977 53 and 01-074-1739 54 ). The presence of these silver compounds is in agreement with XPS data presented above. There are no observable lines at 2y angles lower that 26.61 This suggests that largest interlayer spacing value between planes corresponds to the graphitic planes. Therefore, it is most likely that functionalisation has taken place at the outer surfaces of the material. Functionalisation within the internal MLG structure would, of course, result in an increase in the interlayer spacing. The lines corresponding to the 2H and 3R arrangements in both MLG and Ar f-MLG show only minor differences confirming the functionalisation has only a small impact on the crystallinity and interlayer spacing of the MLG structure. Again, this is consistent with functionalisation at the outer positions only.</p><!><p>Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to investigate the surface morphology and lateral dimensions of the graphitic sheets. Four SEM images were taken of a representative example of each material at low and high magnifications (Fig. 8 and 9). The images reveal that both materials consist of multi particulate material as agglomerates which range in diameter between 10-30 mm for MLG and o10 mm in size for Ar f-MLG. The quantity of agglomerates within MLG exceeds that of Ar f-MLG, suggesting that the functionalisation process has led these loosely bound aggregates to unravel. At lower magnifications MLG and Ar f-MLG display a powder like appearance. At increased magnifications, it can be seen that these agglomerates are irregularly arranged consisting of flakes comprised of mostly multi-layered stacks, physically aggregated by p-p interactions. These stacks adopt wavy topography and curled edges. The non-uniform nature of the morphology of these materials is most evident in the higher resolution images. These defects are likely to result from the plasma ablation process during synthesis. The particle size distribution is due to the method of processing of this material.</p><p>Under the conditions utilised to record the images, it was found that the samples underwent charging. In order to obtain more enhanced images, gold coating was added using a sputter coater. The gold coating can be seen with a scaly appearance, as exemplified in Fig. 10. Additional SEM images of Ar f-MLG are provided in Fig. 11. These highlight some of the features of the materials related to their porosity. As can be seen in the image on the left, there is a macro sized pore (circled) which contains some additional material. We believe that this could be some smaller flakes of Ar f-MLG which become trapped within the in-plane pore, and for this reason are difficult to remove during the purification process. The presence of material which could be trapped silver nanomaterials can also be seen on an image in Fig. S7 (see ESI †). The image on the right of Fig. 11 highlights the slit like channels which are found within the spacing between subsequent stacks. The implications of these features on the surface area and porosity of the MLG and Ar f-MLG materials are discussed in the following section.</p><p>Selected TEM images of MLG and Ar f-MLG are presented in Fig. 12. These also shown that both materials consist of multiple platelets with significant clumping. These multi-layered structures can be observed by differences in the contrast. Darker areas within the TEM images represent more dense areas within increased number of layers, whilst light areas represent those of less layers. It can be observed by TEM images that the stacks of layers exhibit lateral dimensions between 100-500 nm in diameter. As with the SEM images, they also indicate material which contains a broad range of morphologies across the surface of both materials.</p><p>When comparing the SEM and TEM images for Ar f-MLG with those of MLG, it can be concluded that there are no visual changes of major significance in the morphology upon functionalisation. Nevertheless, the orientation of stacks in Ar f-MLG appear to be less agglomerated than those within MLG. This causes the individual stacks to appear much clearer within the SEM images. TEM images also reveal that the flakes remain very similar in size and distribution.</p><!><p>Nitrogen adsorption-desorption measurements were used to determine the surface area and pore size distribution of MLG and Ar f-MLG, utilising Branauer-Emmett-Teller (BET) analysis and Barret-Joyner-Halenda (BJH) analysis. 55 The results of these investigations are presented in Fig. 13. It was found that MLG and Ar f-MLG both exhibited Type IIb N 2 adsorptiondesorption isotherms typical of materials composed of platelike particles (Fig. 13a). 56 A Type H3 hysteresis loop is present in each isotherm, indicating the occurrence of capillary condensation within pores of B4 nm in size. 57 Hysteresis of this type is usually associated with plate-like aggregates or adsorbents containing slit-like pores.</p><p>MLG and Ar f-MLG were found to exhibit BET surface areas corresponding to 380.22 and 192.13 m 2 g À1 , respectively (Table 2). A significant contribution of this area originates from pores between 1.7-300 nm. These pores are mostly present within the space between neighbouring stacks which correspond to 75.5% (MLG) and 87.4% Ar f-MLG of the total surface area.</p><p>The pore size distribution profile was examined by plotting the pore size distribution as a function of incremental pore volume (Fig. 13b) and incremental pore area using the adsorption branch of the isotherm (Fig. 13c). Overall, the curves reveal similar patterns for both MLG and Ar f-MLG in each plot, suggesting a minor impact to the overall structure upon functionalisation. In parallel, the average pore size for MLG and Ar f-MLG were 9.53 nm and 11.70 nm, respectively. The main difference, however, is a decrease in the area and volume of pores affecting mostly pores with diameters of less than B75 nm. This is an indication that functionalisation causes a significant reduction in the quantity of pores within the mesoporous region. This finding coincides with the significant decrease in BET surface area (almost half of MLG). We suggest that the functionalised sheets re-orientate themselves to adopt curled, scroll-like edges due to enhanced interactions between the functionalised stacks.</p><p>With such a situation, many of the slit-like pores and channels between neighbouring stacks would become inaccessible to the nitrogen adsorbent molecules. In addition, upon functionalisation, we also observe blockage of pores with smaller aggregates (as seen in Fig. 11). This is can be clearly seen in the SEM image in Fig. 11a which shows a macropore with a width of 700 nm in addition to Fig. 11b where stacks of sheets can be observed. 58</p><!><p>All solvents and reagents were purchased from commercial suppliers and used with no further purification. The MLG material was synthesised and provided by Perpetuus Carbon Technologies. Synthesis of MLG. Natural flake plasma processing of natural graphite was carried out using a custom-made multi-electrode dielectric barrier discharge (DBD) plasma reactor as described elsewhere. 29 Control reaction: reactivity of radicals generated from 4-(trifluoromethyl)phenyl boronic acid in the absence of MLG. This reaction was carried out using similar conditions to that of previous works. 32 The reagent 4-(trifluoromethyl)phenyl boronic acid (0.092 g, 4.83 Â 10 À4 mol) was dissolved in a solvent mixture (40 mL) consisting of water and DCM (1 : 1), followed by the addition of K 2 S 2 O 8 (0.266 g, 9.85 Â 10 À4 mol) and AgNO 3 (0.027 g, 1.60 Â 10 À4 mol). The mixture was allowed to stir for 44 h at room temperature. The biphasic mixture was filtered and the DCM layer was separated from the filtrate using a separating funnel. The DCM layer was then evaporated to dryness to give a yellow oil consisting of two compounds: 4,4 0 -bis(trifluoromethyl)biphenyl (A) and bis((trifluoromethyl)diphenyl)ether (B). Characterisation of A: Control reaction: MLG treated with potassium persulfate ( c Ox-MLG). MLG (0.160 g) and K 2 S 2 O 8 (0.570 g, 2.120 Â 10 À3 mol) were dispersed in a solvent mixture (20 mL) consisting of water and DCM (1 : 1). The mixture was allowed to stir for 44 h at room temperature. The reaction mixture was then centrifuged and the resultant c Ox-MLG solid was separated and washed repeatedly with water, acetonitrile, and DCM through several dispersion/centrifugation cycles. The resultant material was dried in vacuum for 1 week.</p><!><p>X-Ray photoelectron spectroscopy (XPS) analysis was performed using a Kratos Axis Ultra-DLD photoelectron spectrometer with Thermal gravimetric analysis (TGA) was carried out using a PerkinElmer TGA 4000 instrument. The samples were heated from room temperature up to 900 1C (5 1C min À1 ) under a nitrogen atmosphere (50 mL min À1 ).</p><p>Raman spectroscopy was performed using a Renishaw inVia confocal Raman microscope equipped with an Ar + visible green laser with an emission wavelength of 514 nm. Spectra were collected in a reflective mode by a high sensitive charge couple device (CCD) detector.</p><p>Powder X-ray diffraction (XRD) patterns were collected using a Panalytical X'Pert diffractometer with a Cu anode irradiation (l = 1.541 Å) operating at 40 kV and 40 mA. Phase identification was performed by matching experimental patterns against entries in the ICDD standard database.</p><p>Scanning electron microscopy (SEM) images were obtained using a Zeiss Supra 35VP (FEG) SEM instrument. The samples were gold-coated using a sputtering coater to enhance the resolution of the images.</p><p>Transmission microscopy (TEM) images were obtained using a Jeol 2100 field emission gun (FEG) TEM with a 200 kV power source.</p><p>The surface area and porosity characteristics of the materials were analysed using a Micromeritics ASAP 2020 physisorption analyser. Samples were degassed under 0.667 Pa for 720 minutes at 150 1C with a heating rate of 10 min À1 . The surface area and pore size distribution were measured at 77 K using Brunauer Emmett Teller (BET) and Barrett Joyner Halenda (BJH) cumulative pore volume methods, respectively. 1 H and 19 F NMR spectra were performed on a Bruker 400 MHz Ascendt 400, which operated at 400 MHz for 1 H nuclei and 376.6 MHz for 19 F nuclei. Chemical shifts are reported in parts per million (ppm). NMR spectra were obtained in CD 3 CN solvent (1.93 ppm) and internal reference for 19 F.</p><p>Mass spectrometry (MS) was carried out on compounds A and B using a Thermoscientific ISQ Single quad with direct insertion probe and the identity of the compounds were confirmed for the preinstalled library of compounds.</p><!><p>In this article, we provide a preliminary account outlining the successful covalent functionalisation of MLG with 4-(trifluoromethyl)phenyl radicals. This has been achieved by using 4-(trifluoromethyl)phenyl boronic acid as a radical source utilising Baran's protocol. The newly formed material, Ar f-MLG, was found to be decorated at a number of positions at the outer surface as confirmed by a number of spectroscopic and analytical techniques.</p><p>At this stage of the development, some challenges associated with this functionalisation methodology and the nature of the plasma-synthesised multi-layer graphitic material have been identified. The attachment of 4-(trifluoromethyl)phenyl moieties is accompanied by an increase in oxygen functionality around the outer surfaces of the MLG stacks, as a result of the oxidising conditions. Furthermore, the task of removal of entrapped impurities, particularly silver in this case, will need to be addressed. Nevertheless, early indications suggest that this approach could provide access to aryl radicals in a costeffective and safer alternative to hazardous diazonium salts. As a result, this methodology could offer a novel safer approach to synthesise functionalised MLG materials on a larger scale with potential to be developed industrially. We are currently investigating ways in which this methodology can be optimised for practical application and exploring other derivatives. This may assist further processing and provide enhanced interaction with other materials. The development of new functionalisation strategies on commercially derived graphitic materials, of course, becomes increasingly important applications across materials science.</p><!><p>There are no conflicts to declare.</p>

Royal Society of Chemistry (RSC)

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry and Tandem Mass Spectrometry: IV. 3-Ring and 4-Ring Isomers

Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) is used to characterize methylenedianiline (MDA) 3-ring and 4-ring species. Building on our previous MALDI-MS 2-ring MDA isomer study, here we compare 3-ring and 4-ring electrospray ionization (ESI) and MALDI results. In ESI, 3-ring and 4-ring MDAs each form one single [M+H]+ parent ion. Whereas in MALDI, each MDA multimer forms three unique precursor ions: [M+H]+, [M.]+, and [M-H]+. In this study, 3-ring and 4-ring MDA precursors are characterized to identify the unique fragment ions formed and their respective fragmentation pathways. In addition to the three possible precursors, the 3-ring and 4-ring species are higher-order oligomer precursors in polyurethane (PUR) production and thus provides additional insight into the polymeric behavior of these PUR hard block precursors. The combination of ion mobility-mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS) allow the structural characterization of these larger MDA multimers.

structural_characterization_of_methylenedianiline_regioisomers_by_ion_mobility-mass_spectrometry_and

<!>Materials.<!>Traveling Wave MALDI-IM-TOF/MS.<!>Differences between ESI and MALDI Spectra for [M+H]+<!>Characterization of 3-Ring and 4-Ring MDA by MS/MS<!>Characterization by Ion Mobility-Mass Spectrometry<!>3-Ring MDA<!>3-Ring MDA [M+H]+ Species (304 Da)<!>3-Ring MDA [M.]+ Species (303 Da)<!>3-Ring MDA [M-H]+ Species (302 Da)<!>4-Ring MDA<!>4-Ring MDA [M+H]+ Species (409 Da)<!>4-Ring MDA [M.]+ Species (408 Da)<!>4-Ring MDA [M-H]+ Species (407 Da)<!>CONCLUSIONS

<p>Polyurethane (PUR) copolymers are one of the most versatile polymeric materials, commonly manufactured for use in rigid and flexible foams, coatings, adhesives, sealants, elastomers, membrane materials, laminates, fibers, and composites. Methylenedi-aniline (MDA) is a precursor to methylene bisphenyl diisocyanate (MDI), a major hard block component in the manufacturing of PURs. MDA is formed from the reaction between aniline and formaldehyde, generating the 2-ring MDA regioisomers 2,2'-MDA,2,,4'-MDA, and 4,4'-MDA, as well as larger multimeric species including 3-ring and 4-ring MDAs.1,2</p><p>In our previous work, 2-ring MDAs (2,2'-MDA, 2,4'-MDA, and 4,4'-MDA) were characterized using electrospray ionization-ion mobility-mass spectrometry (ESI-IM-MS)1 and matrix assisted laser desorption/ionization-ion mobility-mass spectrometry (MALDI-IM-MS),3 two MS techniques commonly used to analyze PUR polymers.4–8 In those studies, one unique parent ion ([M+H]+ = 199 Da) was observed for each isomer using ESI-MS; however, when studied by MALDI-MS we observed three distinct precursor ions, [M-H]+ = 197 Da and [M.]+ = 198 Da, in addition to the [M+H]+ = 199 Da species.</p><p>More recently, we used ESI-IM-MS to structurally characterize 3-ring and 4-ring MDAs by studying their preferred sites of protonation, gas phase stability, and fragmentation pathways.2 In this present study, 3-ring and 4-ring MDAs were similarly characterized using MALDI-IM-MS and the results were compared to our previous ESI-IM-MS findings. In Figure 1, 3-ring and 4-ring MDA parent species are illustrated with their respective protonation sites denoted with asterisks. Consistent with our 2-ring MDA study, we observed one unique parent ion formed during ESI for both 3-ring ([M+H]+ = 304 Da) and 4-ring MDA ([M+H]+ = 409 Da). During the MALDI process we observe three parent ions; two of which are unique to the MALDI process for each 3-ring and 4-ring MDA: [M+H]+ = 304, 409 Da, [M.]+ = 303, 408 Da, and [M-H]+ = 302, 407 Da, respectively. The additional rings in these species provide greater flexibility and thus more complex consequences for the fragmentation patterns observed. The novelty of this work compares ESI and MALDI-MS ionization and the unique precursor species that arise from 3-ring and 4-ring MDA species. This work highlights the complexity when investigating fragmentation pathways of larger multimeric MDA species. The combination of IM-MS and MS/MS allow for unique structural characterization of the three precursor ions formed and their respective fragment ion pathways observed during the MALDI process.</p><!><p>Sample preparation and experimental details have been reported previously for 3-ring and 4-ring MDA ESI studies2 and 2-ring MDA ESI and MALDI studies1,3, but a few details will be mentioned here. Methylenedianiline (MDA) 3-ring and 4-ring samples were provided by Dr. Stefan Wershofen (Bayer MaterialScience AG, 47812 Uerdingen, Germany). Optima grade methanol and water with 0.1% formic acid were obtained from Fisher Scientific (Waltham, MA, USA). Tetralkylammonium salts, α-cy-ano-4-hydroxycinnamic acid (CHCA) and alkali salts were obtained from Sigma-Aldrich (St. Louis, MO, USA). Samples were dissolved at a concentration of 1 mg/mL in 9:1 methanol/water containing 0.1% formic acid (v/v). Each MDA isomer was combined in a 10:1 CHCA matrix-to-analyte ratio and two or three layers of 0.5 µL of samples were spotted on a 100-well MALDI plate.</p><!><p>A traveling wave IM-MS (TWIM-MS) Synapt G2-S (Waters Corporation, Milford, MA) was used for acquiring ESI and MALDI MS, MS/MS, and IM-MS data.9 Collision cross section (CCS) values cannot be obtained directly from TWIM experimental drift time values, therefore quaternary ammonium salts were used as CCS calibration standards to obtain CCS data from TWIM.10–12</p><p>The Synapt G2-S mass spectrometer MALDI source has a frequency-tripled Nd:YAG laser which emits 355 nm at 1kHz pulse repetition rate. Laser attenuation ranged from 190–300 (arbitrary units), with 270 selected for all experiments unless noted otherwise. CHCA matrix cluster peaks were used for TOF calibration. Further experimental details can be found from our earlier study2, but briefly the TWIM drift cell settings were as follows: TWIM pressure, 3 mbar nitrogen (2.25 Torr); electrodynamic wave height, 35 V; wave velocity, 700 m/s; TOF resolving power, ca. 18,000 m/Δm at 200 m/z. Collision-induced dissociation (CID) experiments were performed using argon gas. In our previous studies, experiments were conducted to evaluate the influence of the MALDI laser fluence and the resolving power of the MS/MS resolving quadrupole on the resulting precursor ion intensities. From those studies, the m/z resolving quadrupole was set to 18 resolving power for the MS/MS experiments providing the best separation of the three precursor ions, without significant loss of signal.3 To perform MS3 experiments, the ion of interest is selected by the quadrupole and is fragmented in the "Trap" region located prior to the TWIM drift cell. The first generation of product ions are then separated by the TWIM and subjected to a second stage of CID in the "Transfer" region to produce 2nd generation and 1st generation product ions (i.e., MS/IM/MS experiments).</p><!><p>The full ESI-MS and MALDI-MS spectra in Figure 2 were acquired for both 3-ring (Figure 2a-b) and 4-ring MDA (Figure 2c-d), respectively. The ESI-MS and MALDI-MS spectra for the 3-ring and 4-ring MDA species are vastly different. Using ESI, we observed one precursor species [M+H]+ and additional characteristic peaks at 211 and 106 Da for the 3-ring (Figure 2a) and 316, 211, and 106 Da for 4-ring MDA (Figure 2c). In addition, peaks observed at 152.6 m/z and 158.6 m/z correspond to the doubly charged 3-ring and 4-ring MDAs (Figure 2a,c). With MALDI we observe more complex mass spectra for both 3-ring and 4-ring MDA (Figure 2b,d). Each of the three unique precursor ions is observed in the 3-ring and 4-ring MALDI spectra, similar to our previous study on 2-ring MDA regioisomers. In MALDI, we observe a characteristic peak at 211 Da for 3-ring MDA and CHCA matrix cluster peaks at 337, 335, 190, and 172 Da (Figure 2b). In addition, the MALDI spectra for 4-ring MDA show the [M+Na]+ peak at 431 Da, characteristic peaks at 316 and 211 Da, and CHCA matrix cluster peaks at 379, 335, 190, and 172 Da (Figure 2d). Precursor ions unique to 3-ring MDA include: [M+H]+ = 304 Da, [M.]+ = 303 Da, and [M-H]+ = 302 Da (Figure 2b). In addition, precursor ions characteristic to the 4-ring MDA include: [M+H]+ = 409 Da, [M.]+ = 408 Da, and [M-H]+ = 407 Da (Figure 2d). In MALDI, the 3-ring and 4-ring [M+H]+ precursor is the least abundant precursor ion compared to [M.]+ and [M-H]+ species (Figure 2b,d). In ESI, the 3-ring and 4-ring [M+H]+ species and characteristic fragment peaks appear more stable than the [M+H]+ precursor and fragment ions produced by the MALDI process. This difference observed for the [M+H]+ precursor indicates that these MDA multimers behave significantly differently when ionized by MALDI than by ESI.</p><!><p>Figure 3 details the MALDI MS/MS spectra for each precursor ion formed: 3-ring (Figure 3a-c) and 4-ring (Figure 3d-f) MDAs, [M+H]+, [M.]+, and [M-H]+. In the MS/MS spectra, different collision energies were used to generate fragment ions for each precursor due to differing precursor stabilities. Figures S1 and S2 in Supporting Information compare all precursor species fragmented at 15 eV compared to 20 eV (laboratory frame). Due to an increase in the number of fragment ions observed for the [M+H]+ species for both 3-ring and 4-ring MDA, a 15 eV collision energy was used to characterize specific fragment ions derived from the [M+H]+ precursor species, and 20 eV was used to characterize fragment ions derived from the [M.]+ and [M-H]+ species. The MS/MS spectra in Figure 3 show unique fragment ions for each 3-ring and 4-ring precursor species: 15 eV was used for [M+H]+ (Figure 3a,d) and 20 eV was used for [M.]+ and [M-H]+ species (Figure 3b-c,e-f). Additional MS/MS spectra at various collision energies for both 3-ring and 4-ring MDA precursors can be found in Supporting Information (Figures S3-S4). Due to the numerous fragment ions formed uniquely for each precursor, this manuscript will focus on the interpretation of major low energy fragment ions formed by the MALDI process and will be introduced by schemes proposing fragment ion pathways and collision-induced dissociation ion breakdown curves (CID curves). Additional MS3 data found in Supporting Information (Figures S5 and S6) was also acquired and used to support the interpretation of unique fragment ion pathways.</p><p>It should be noted that there is a major difference between interpretation of the MALDI spectra of the 3-ring and 4-ring MDAs relative to the 2-ring MDAs from our previous work. This difference arises from the multiple amine groups in the 3-ring and 4-ring compounds. Not only do the amine groups have somewhat differing basicities, but their steric effects can vary significantly. These effects were observed to some extent in the ESI spectra of these compounds,2 but the complexities in the MALDI spectra are more severe due to the larger MDA species and formation of three unique precursor ions in the MALDI process. The net result indicates that it is generally not possible to identify a unique structure for a given mass loss. Supporting Information Figures S7 and S8 address this problem in greater detail with an illustration provided to highlight the complexity of these specific compounds. There-fore, specific structures described in the manuscript are tentative and may not be the only structures possible for a given fragmention.</p><!><p>The experimental IM spectra provided in Figure 4 are shown for the [M+H]+ (blue), [M.]+ (black), and [M-H]+ (red) ion forms of the 3-ring and 4-ring MDA precursor species. In Figure 4a, the TWIM spectrum obtained for the 3-ring species indicate a single distribution for each precursor, whereas in Figure 4b the 4-ring MDA species each exhibit two partially-resolved structural populations, indicative of multiple gas-phase conformations. In our previous ESI studies, computational modeling was used to study the [M+H]+ 3-ring and 4-ring MDA. Theoretical structures were generated to align with experimental CCS values. In those studies, the [M+H]+ 3-ring MDA was shown to undergo the most favorable protonation on the internal amine, when compared to the external amines. In the ESI [M+H]+ 4-ring MDA studies, two conformations were seen, which is similar to what is experimentally observed in the MALDI studies described here. These two conformational families were attributed to external amine protonation and internal amine protonation, respectively. Computational modeling in the ESI study suggests protonation on the external amines is more favored due to the 4-ring MDA species wrapping around the proton to share between the two external amine groups. As demonstrated in previous literature, IM-MS separations can differentiate between both aniline protomers coexisting in the gas-phase: Nprotonated and ring protonated aniline.13–14 Similar to aniline studies, we can confirm that IM-MS techniques can aide in identifying multiple sites of protonation within 3-ring and 4-ring MDA precursor species. In Figure 4a, the IM-MS results show 304 Da [M+H]+ and 303 Da [M.]+ species to have the same CCS (140.8 Å2). However, the 302 Da [M-H]+ species show a smaller CCS (139.2 Å2; this difference in CCS for the 3-ring MDA can be attributed to the structural differences exhibited by the [M-H]+ bridged fluorene-backbone, as depicted in Figure 1. The 4-ring MDA precursors behave much differently than the 3-ring species. First, we notice two conformations present for each precursor, each conformation having different abundances. Next, the 409 Da [M+H]+ and 408 Da [M.]+ species each share the same CCS both for the larger conformation (158.3 Å2) and smaller conformation (164.1 Å2). The 407 Da [M-H]+ species is uniquely different from all of the 3-ring and 4-ring precursors. The 407 Da IM-MS trace shows two isomeric species; the less abundant isomer exhibits the smaller CCS (156.0 Å2), whereas the more abundant isomer has the larger CCS (164.2 Å2). When comparing the [M-H]+ species between 3-ring and 4-ring MDAs, we notice that the 302 Da species has a smaller CCS, whereas the 407 Da species (more abundant isomer) has a larger CCS value. The differences observed between the precursors and 3-ring and 4-ring MDA can be attributed to either structural differences or multiple sites of protonation. Structural differences between 3-ring and 4-ring MDA include the addition of a bridged methyl-aniline and differences between [M+H]+ and [MH]+ for each MDA multimer include the bridged fluorene backbone. Possible protonation sites on the 3-ring and 4-ring MDAs are illustrated in Figure 1, differences include exterior or interior amine protonation. As seen in our previous 3-ring and 4-ring ESI studies, the MDA species must be oriented such that the additional proton is in proximity to the most basic site on the adjacent aniline ring to represent the most energetically favorable position. Curve resolution analysis for the 4-ring MDA precursors (Supporting Information S9) and additional IM-MS data can be found in Supporting Information on Pages S10-S11</p><!><p>In the MS/MS spectrum for each 3-ring MDA precursor, Figure 3a-c shows major fragment peaks observed at low lab frame collision energies: [M+H]+ = 15 eV, [M.]+ = 20 eV, and [M-H]+ = 20 eV. The conversion from precursor ion to major fragment ion was monitored as a function of applied collision energy as shown in Figure 5. Collision energy was ramped from 0 to 50 eV for all unique precursor species in 5 eV increments and performed in triplicate. Figure 5a,c,e monitors CID of major fragment ions having a percent relative ion current (%RIC) of 20% or more, and Figure 5b,d,f illustrates %RIC of low abundance fragment ions formed in the range of 5–20% along the 0–50 eV ramp. All of the CID curves in Figure 5 represent %RIC values of 5% or more along the 0–50 eV ramp. Additional CID curves for minor fragment ions formed at 35 eV or higher are provided as Supporting Information (Figure S12). Major fragment ions observed for each precursor species will be discussed in further detail.</p><!><p>In Figure 3a, the MS/MS spectrum highlights major fragment ions observed for the 304 precursor species at 15 eV. The MS/MS spectrum shows the 304 Da species dissociating into four major fragment ions 303, 211, 209, and 198 Da, as seen in Scheme 1a and in the total percent ion current table found in Supporting Information Figure S13. These fragment ions are derived directly from the 304 Da species and each forms with an intensity greater than 5%. In Figure 5a-b, the [M+H]+ precursor depletes at 15 eV forming fragment ions 303 ([M-1]+), 211 ([M-93]+), 209 ([M-95]+), and 198 Da ([M-106]+); a dashed line is drawn for visual alignment of major and minor fragment ion formation at 15 eV. Major fragment ion 303 Da corresponds to the loss of one hydrogen, and the transfer of charge to the ring (8%, 15 eV). Major fragment ion 211 Da forms from the loss of aniline (7%, 15 eV), from its parent species 304 Da. Fragment ion 209 Da corresponds to the loss of aniline and two hydrogens; this fragment ion forms at higher collision energy (6%, 30 eV). In addition, mass 198 Da forms in high abundance due to the loss of methylene-aniline (30%, 15 eV). Scheme 1a outlines a proposed fragmentation pathway for each fragment ion (>5%) originating from the [M+H]+ 304 Da species. As seen in Scheme 1a, minor fragment ions such as 210 ([M-93]+), 197 ([M-106]+), 181 ([M-17]+), and 180 Da ([M-17]+) form by depletion of the major fragment ions at higher collision energy. Scheme 1a proposes that mass 303 Da can form two possible fragments ions: 210 Da and 197 Da. Mass 210 Da is formed by the loss of aniline (10%, 25 eV) and ion 197 Da forms from the loss of methylene-aniline. In Figure 5a-b we observe the depletion of mass 198 Da leading to the formation of the 181 Da (25%, 40 eV) species, resulting from the loss of –NH2 and one hydrogen forming the fluorene-bridge. Similarly, minor fragment ion 180 Da (16%, 40 eV) results from the loss of –NH2 and one hydrogen forming the fluorene-bridge, from major fragment ion 197 Da. The MS3 data found in Supporting Information Figure S5 is consistent with the pro-posed Scheme 1a outline. Additional possible structures of the proposed fragment ions derived from the 304 Da [M+H]+ are outlined in Supporting Information (Figure S14). Proposed fragmention pathways, CID curves, and intensity values for additional ions formed at 35 eV or higher are also described in Supporting Information (Figures S12).</p><!><p>When comparing the Figure 3b MS/MS spectrum of the [M.]+ species to Figure 3a [M+H]+ MS/MS spectrum, we observe an increase in ion stability. In Figure 5 precursor [M.]+ requires additional collision energy to generate fragment ions compared to the [M+H]+ species. This increase in stability results from delocalization of charge for the 303 Da species. In Figure 5c-d, fewer fragment ions are observed for the 303 Da [M.]+ species at 20 eV collision energy compared to Figure 5a-b (vertical dashed line is drawn for visual reference). Found in the Supporting Information, Figure S15 shows percent total ion current values of major and minor fragment ions formed from mass 303 Da depletion. In Scheme 1b, the major fragment ions observed to form directly from mass 303 Da are outlined: mass 302 ([M-1]+), mass 210 ([M-93]+), and mass 197 ([M-106]+). At 25 eV, fragment ion 302 Da forms from the loss of one hydrogen (9%, 25 eV) and mass 197 Da results from the loss of methylene-aniline (42%, 25 eV). Fragment ion 210 Da forms at higher collision energy (10%, 30 eV), resulting from the loss of aniline. Several minor fragment ions are also observed from the depletion of these major fragment ions: 209 ([M-93]+), 181 (M-29]+), 180 ([M-17]+), and 165 Da ([M-15]+). In Scheme 1b, species 209 Da forms directly from mass 302 Da through an M-93 loss of aniline (16%, 35 eV). At higher collision energy, mass 210 Da fragments into 181 Da due to loss of -NH and –CH2 (9%, 45 eV). In Figure 5c, the depletion of 197 Da can be monitored to illustrate the formation of fragment ion 180 Da (30%, 50 eV, loss of –NH2 and one-hydrogen). Increasing fragmentation energy to 50eV causes mass 180 Da to form mass 165 Da (9%, loss of -NH2). Additional structures of the major and minor fragment ions formed from the 303 Da species can be found in Supporting Information Figure S16.</p><!><p>The [M-H]+ species is the most stable precursor compared to [M+H]+ and [M.]+ (see Figure 5). The increase in stability is due to the fluorene-backbone, as seen in the 2-ring [M-H]+ species versus the [M+H]+ species, discussed previously.3 In Figure 3c, the MS/MS spectrum shows three major fragment ions deriving from the 302 Da precursor ion at 20 eV: 285 ([M-17]+), 209 ([M-93]+), and 106 Da ([M-196]+). The dashed line in Figure 5e-f at 20 eV is drawn for visual alignment to illustrate fragment ions formed at %RIC above 5%. Here we observe the increased stability of the [M-H]+ precursor ion, higher collision energy is needed to form fragment ions. Total percent ion current values of major and minor fragment ions formed from the 302 Da species can be found in Supporting Information Figure S17. The formation of mass 285 Da corresponds to M-17, loss of ammonia (4%, 20 eV), this species is not one of the more intense fragment ions for the mass 302 Da precursor, but loss of –NH3 occurs prominently in the MALDI spectra for all species. In Scheme 1c the proposed fragment ion pathways are illustrated for the 302 Da precursor. The major fragment ion formed from the 302 Da precursor is an ion at mass 209 Da (36%, 20 eV), formally M-93 corresponding to loss of aniline; it can be assumed to come from charge-remote fragmentation (Supporting Information Figures S7-S8). Fragment ion 106 Da has been consistently characterized for the MDA 2-ring, 3-ring, and 4-ring species. For the 3-ring MALDI MS/MS studies, mass 106 Da forms from a loss of 4,4'-MDA and one hydrogen (5%, 30 eV). There are five additional minor fragment ions of interest, all formed at higher collision energies: 208 ([M-1]+), 193 ([M-16]+), 192 ([M-1]+), 180 ([M-29]+), and 165 Da ([M-28]+). Fragment ion 208 Da (16%, 35 eV) forms from 209 Da due to a loss of one hydrogen localizing the charge on the ring. Fragment ion 193 Da (18%, 30 eV) forms from a loss of –NH2 directly from fragment 209 Da. Furthermore, the 193 Da ion depletes into mass 192 Da (8%, 30 eV) from loss of the bridge-hydrogen; in addition, higher collision energy causes mass 165 Da (30%, 50 eV) to form from the 193 Da species due to the loss of –CHN– from the protonated species. The 180 Da (26%, 50 eV) ion also forms at high collision energy and is formed from the 209 Da species from a loss of CH2=NH. Additional structures of the major and minor fragment ions formed from the 302 Da precursor can be found in the Supporting Information Figure S18.</p><!><p>The three 4-ring MDA precursors behave similarly to the 3-ring MDA. Here we observe [M+H]+ as the least stable precursor amongst [M.]+ and [M-H]+ species, forming more fragment ions at lower collision energy (Figure 3d-f); this is consistent with our previous 2-ring studies.3 The MALDI MS/MS spectra for the 4-ring MDAs show major fragment ion peaks at low collision energy for each precursor species: [M+H]+, [M.]+, and [M-H]+. It is interesting to note that an increase in fragment ions is observed in Figure 3d for the [M+H]+ species (15 eV) compared to [M.]+ and [M-H]+ in Figure 3e-f (20 eV). In Figure 6, we monitor the depletion of each precursor species as a function of applied collision energy from 0 to 50 eV in 5 eV increments. Figure 6a,c,e shows CID breakdown curves for the major fragment ions having a %RIC of 20% or more, and Figure 6b,d,f illustrates the minor fragment ions (less than 20% RIC) formed along the 0–50 eV ramp. Major fragment ions to be discussed form in high abundance at collision energies below 35 eV, additional CID curves for fragment ions formed at 35 eV or higher are provided as Supporting Information (Figure S19). Total percent ion current tables are provided in the Supporting Information (Figures S20-S22) for each precursor; these tables high-light fragment ion (%RIC) formation as CID energy is increased. The major fragment ions observed for each precursor species will be discussed in further detail.</p><!><p>The MS/MS spectrum in Figure 3d highlights the 409 Da [M+H]+ precursor forming three major fragment ions at low collision energy (15 eV): 316 Da ([M-93]+), 315 Da ([M-94]+), and 303 Da ([M-106]+). In Figure 6a-b, the depletion of 409 Da species is monitored over a 0–50 eV span at 5 eV increments, a dashed line is drawn for visual alignment. The three major fragment ions are observed to originate directly from the depletion of 409 Da. Proposed structures of these fragment ions are provided in Scheme 2a. The CID curves show that the most abundant fragment ion, mass 316 Da (37%, 15 eV), corresponds to the loss of aniline through charge-remote fragmentation. Based on the CID curves and Supporting Information Table S20, mass 209 Da forms directly from the 316 Da species through M-107 loss of methylene-aniline and one hydrogen (15%, 35 eV). The second major fragment ion at mass 315 Da (15%, 30 eV) also forms from the 409 Da species due to the loss of aniline and one hydrogen. The third major fragment ion, mass 303 Da has the same structure as the 3-ring MDA [M.]+ species discussed above. The charge is delocalized on the ring, and the 303 Da (14%, 35 eV) fragment ion forms from the loss of a methylene-aniline. Additional fragmentation of mass 303 Da leads to the formation of a minor fragment ion, mass 210 Da (M-93, 14%, 35 eV), which represents the loss of aniline through charge remote fragmentation. Higher collision energy fragment ions formed from the [M+H]+ precursor can be found in Supporting Information Figure S19.</p><!><p>As observed in Figure 3e, the 408 Da species is more stable than the 409 Da precursor. The collision energy is increased to 20 eV for the [M.]+ species and only a few fragment ions are observed compared to the [M+H]+ spectrum. In Figure 6c-d, 408 Da is monitored across an increasing collision energy gradient, here the [M.]+ species reaches 50% depletion at 25 eV. Scheme 2b illustrates the two major fragment ions derived from the 408 Da species: mass 315 Da ([M-93]+) and 302 Da ([M-106]+). Major fragment ion 315 Da initially forms due to loss of aniline (23%, 25 eV). This major fragment ion can further fragment into two minor fragment ions at higher collision energy: 314 Da ([M-1]+) and 106 ([M-209]+). Fragment 314 Da forms from the loss of one hydrogen at the bridge-carbon (16%, 45 eV), and fragment 106 Da (30%, 50eV) forms due to charge-remote fragmentation. MS3 data support 314 Da and 106 Da species originating from the 315 Da species, see Supporting Information Figure S6. The third major fragment ion formed from the 408 Da precursor is mass 302 Da (11%, 30 eV); this species is formed from the loss of a methylene-aniline and the structure resembles 3-ring MDA with the loss of a hydrogen from the bridged methylene. Further depletion of the mass 302 Da species increases the formation of mass 209 Da (30%, 50 eV) through loss of aniline (M-93). Additional supporting data for 408 Da fragment ions can be found in the Supporting Information (Figures S19 and S21).</p><!><p>The 407 Da [M-H]+ species is also more stable than the 409 Da [M+H]+ ion. In Figure 3f the MS/MS spectrum at 20 eV shows few fragment ions and a strong 407 Da precursor peak at 62% relative intensity. Figure 6e-f illustrates the major and minor fragment ions formed above 5% as the collision energy was monitored from 0–50 eV. As the 407 Da precursor depletes, four major fragment ions are observed to derive directly from the [M-H]+ species. Scheme 2c shows mass 314 Da ([M-93]+), 211 Da ([M-196]+), 209 Da ([M-198]+), and 106 Da ([M-301]+) all forming from the 407 Da [M-H]+ precursor. Fragment ion 314 Da (16%, 25 eV) is formed from the loss of aniline and then further depletes into minor fragment ion 297 Da ([M-17]+, 9%, 25 eV), formed from the loss of an ammonia. Additionally, ions 211 Da (6%, 30 eV) and 209 Da (24%, 35 eV) are both formed by fragmentation at the center CH2, causing the loss of methylene-dianiline. The difference in these fragment ion masses correspond to alternative structural rearrangement and loss of two hydrogens. The 106 Da fragment forms at high collision energy as seen in our previous studies;2 this ion forms in 37% abundance at 50 eV. Further exploration of high energy fragment ions formed due to 407 Da depletion can be found in Supporting Information (Figures S19). A complete breakdown of fragment ion %RIC over 0–50 eV ramp can also be found in Supporting Information (Figure S22).</p><!><p>In this study, 3-ring and 4-ring MDA have been extensively characterized using MALDI-MS, MALDI-IM-MS, and multi-stage fragmentation (MS/MS, MS3) techniques. The combination of MS/MS and IM-MS is demonstrated to be a successful tool in the structural characterization of these unique precursor species and their respective fragment ions. In previous work, we concluded that both the 3-ring and 4-ring MDA only form the [M+H]+ precursor in ESI, whereas in this current MALDI study we observe three unique precursor species as seen in the 2-ring studies: [M+H]+, [M.]+, and [M-H]+. Here we observe both the 3-ring and 4-ring MDA forming these three unique precursor species. Using energy-resolved ion fragmentation studies, the [M+H]+ species was found to be the least stable of the three precursor ions. The [M.]+ and [M-H]+ species were both more stable, generating fewer fragment ions at a higher collision energy. The IM spectra exhibited one narrow structural population for the 3-ring precursor species, whereas the 4-ring MDA precursors each had two unique conformational families present. IM-MS separations can identify the presence of multiple protonation sites coexisting in the gas-phase. Future studies will include the characterization of the 4-ring MDA IM-MS spectra. These findings provide insight into the complex nature of PUR precursors and their unique fragmentation behavior.</p>

PubMed Author Manuscript

DNA Microviscosity Converts Ruthenium Polypyridyl Complexes to Effective Photosensitizers

A unique radiative decay engineering strategy using DNA microviscosity for the generation of ruthenium polypyridyl complex (RPCs) mediated singlet oxygen for selective damage of DNA and killing cancer cells is reported. This investigation also demonstarte the effect of light-driven RPCs on bacterial growth arrest, through DNA nick, and differential localization in cancer and non-cancer cells. Moreover, upon binding with DNA, RPCs experience high local microviscosity, which causes significant enhancement of the excited state lifetime and thus generates singlet oxygen. The visible-light-triggered singlet-oxygen efficiently produce nick in DNA and inhibits bacterial growth. RPCs also localize inside the nucleus of the cancer cell and in the vicinity of the nuclear membrane of non-cancerous cells, confirmed by live-cell confocal microscopy. The results provide a facile platform for the novel antibiotic intended discovery combined with cancer therapy.

dna_microviscosity_converts_ruthenium_polypyridyl_complexes_to_effective_photosensitizers

Introduction:<!>Result and Discussion:<!>Conclusions:<!>UV-Visible and fluorescence measurements<!>Circular Dichroism and time-resolved measurements<!>Scanning Electron Microscopy<!>Confocal Microscopy<!>XRD<!>Methods: 2.1 UV-Visible and fluorescence measurements<!>Time-resolved measurement<!>Singlet oxygen detection experiment<!>Viscosity dependent lifetime measurement<!>DNA photocleavage assays<!>Scanning Electron Microscopy C4<!>Cell culture:

<p>In recent years, ruthenium-based mononuclear complexes have been extensively investigated for their versatile chemical tunability, [1] exciting photophysical and photochemical properties and long-lived electronically excited state. [2] The Ruthenium polypyridyl complexes offer access to facile chemical modification which facilitates a range of diverse ligands to co-ordinate, imparting a distinct functionality. Notably, the valency of this transition metal can be fundamentally saturated and hence, exhibits an inertness towards nonspecific targets in the cellular milieu. These complexes have been the choice of molecule as a metallodrug in pharmaceutics for chemotherapeutic applications because it can be tuned for better DNA interaction. [3] Besides, drug discovery and its specific delivery coupled with ruthenium complexes have proved to be an efficient route for targeting various genomic diseases like cancer. [4] Also, these complexes are of great interest in the field of tumor detection and have drawn attention in the development of new generation drugs with in vitro as well as in vivo applications. [5] Analogous to this, both ruthenium complexes and nanoparticles have also been explored for the antibacterial properties and light-induced activities. [6] Although, metal complex induced DNA cleavage and cancer cell cytotoxicity are reported, they suffer from a serious problem of UV light excitation or very low absorbance in visible region. [7] Moreover, they induce apoptosis in cancer cell via DNA damage, however, required prolong treatment time (24 h) with ruthenium complex, which surrogates several unknown hits and hence remains incomprehensible. [8] Even though, the pre-existing therapies have achieved remarkable success; their use has always confined by various side-effects due to non-specific targeting. Photodynamic therapy seems to be a proficient approach as it is devoid of harmful effects with targeted delivery.</p><p>Herein, we have extensively investigated the spectroscopic properties, single crystal XRD structure, mode of DNA binding and their efficacy as antibacterial and anticancer agents of four RPCs (C1, C2, C3, and C4; Scheme 1a, 1b, Table S1). The synthesis of RPCs has reported earlier. [9] The synthesized RPCs generate ROS via the radiative decay engineering mediated by local DNA microviscosity. Ultimately, this leads to light-induced DNA cleavage, and eventually, results in nicked circular DNA (Scheme 1c). The visible light-mediated activities of these RPCs lead to interference with DNA functionality in vivo and hence cell lysis. Extensive investigation of this class of molecules can be useful for novel broad-spectrum antibiotic and cancer therapy.</p><!><p>The absorption and emission maxima of C4 were measured to be 430 nm and 630 nm, respectively, with considerable overlap of emission and excitation spectra (Figure 1a). Moreover, we titrated the plasmid DNA with different concentrations of C4 and exposed to circularly polarised light. We found a significant change in emission maxima of DNA for the corresponding increasing concentration of C4 (Figure 1b) confirming their interaction. Thus, the luminescence intensity of RPCs upon addition of increasing concentrations of plasmid DNA was also measured. The plot exhibits an increasing trend in luminescence intensity, which further implies the DNA and RPCs interaction (Figure 1c). With this preliminary observation, we investigated the effect of Ethidium bromide (EtBr), a known DNA intercalator, addition to the mixture of C4 and DNA solution. Interestingly, with the gradual addition of EtBr, the luminescence intensity of C4 decreases (Figure 1d). Such decrease illustrates the displacement of C4 from DNA by EtBr due to the competitive binding. Hence, it confirmed the intercalation of C4 on DNA. These studies have been done for all the mentioned RPCs, and the results for C1, C2, and C3 are combined in SI (Figures S1-4). Furthermore, the measured luminescence lifetime RPCs shows a notable enhancement(ca.10 times) upon DNA binding (Figure 2a). Supported by the above results, EtBr displacement causes reduction in their luminescence lifetime (Figure 2b). The lifetime plot shows a regular trend against DNA and EtBr concentration (Figures 2c-d). This enhanced lifetime reveals the increased time upon intercalation of RPCs with DNA, resulting in the nick generation. However, the lifetime of metal complexes is not limited to DNA interaction, rather local viscosity of medium also plays the most important role in the considerable lifetime enhancement. [10] To validate this, we performed experiments using Ethylene Glycol (EG) as a source of viscous medium. The concentrations of EG were varied from 0-100% in water. The results show a high degree of lifetime dependence on viscosity. Clearly, the lifetime for C2 and C4 enhanced upto 10 folds for the final 100% EG concentration (Figures 3a-b). The distribution of lifetime data follows a linear trend against EG concentrations (inset Figures 3ab). The results for C3 are provided in SI (Figure S3c). Further, we checked the binding of RPCs with DNA concerning to major and minor groove binding (Figure 3c). We found that the lifetime of C4 drastically restored to original value upon addition of excess Hoechst, wellknown DNA minor groove binder, [11] to the C4-DNA solution. However, we do not observe any effect of spermine, which is a DNA major-groove binder. [12] Hence, conforming RPCs minorgroove binding. Our previous report on structurally similar RPCs, suggests the generation of ROS by RPCs. [9] Our hypothesis claims the ROS mediated DNA nick generation as a mode of metal complex action. These phenomena successively lead to a series of events when studied in vivo. For this, we monitored the fluorescence of 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ADBM-DMA) upon addition of RPCs and visible light irradiation. As expected, the luminescence intensity gradually decreases with time due to the endoperoxide formation and gets saturated in a time-frame of 15 minutes (Figure 3d). This confirms the generation of singlet-oxygen by the RPCs. However, there is an insignificant effect of visible light on the luminescence intensity of ADBM-DMA without RPCs (Figure S5). To understand the role of excited state lifetime on the singlet oxygen generation, we performed luminescence quenching experiment of C1-2 and C4 with molecular oxygen in H 2 O and D 2 O. As expected, we observed a significant fraction of 3 MLCT state is quenched by molecular oxygen (Figure S6, Table 2). For further clarification, we investigated the effect of designed RPCs induced DNA cleavage activity by the generation of nick in supercoiled (SC) DNA upon photo-irradiation. The ROS generated by metal complexes, upon visible-light irradiation, produces nicked circular (NC) and hence relaxes the DNA. The time-frame of photo-irradiation for optimum nick generation in supercoiled DNA was determined to be 1 h (Figure 4a). Also, RPCs were studied tocheck the extent of their activity on DNA. It is imperative to mention that under the same photoirradiation conditions and molecular concentration; C4 produced the maximum nick (Figure 4a-b). Moreover, effect of different concentrations of the same RPC was also verified for the calculation of optimal as well as maximum nick generation. The concentration of 10 µM (for C4) was found to be sufficient for the conversion of supercoiled DNA into the nicked circular (Figure 4c). However, there were no effects on supercoiling of DNA upon incubation with C4 under the dark conditions which further confirms the significance of photo-irradiation (Figure 4c Motivated by these findings, we extended our investigation in cellulo systems. Keeping the idea of ROS generation and DNA photocleavage, we explored the effect of these RPCs against the gram-negative bacteria, E. coli. These RPCs were incubated with freshly growing bacterial culture, at logarithmic phase, followed by 1h visible-light irradiation. The treated cultures were grown further under the optimal condition, and the optical density (OD) of the bacterial culture was measured after 4.5 hours. Particularly, C4 was found to comprise of most effective bacterial inhibition activity, as indicatedby arrest of OD with the progression of time (Figure 4d). In contrast, there is no growth arrest under the same experimental conditions except the dark instead of photoirradiation (Figure S19). To understand better the optimal concentration of RPCs for bacterial growth inhibition, we titrated different concentrations of RPCs. For C4, 50 µM concentration was found to be most effective against bacterial growth, as it showed significant inhibition. Further, we investigated the growth inhibition of different gram-positive and negative bacteria and found to have a similar effect, although the extent of growth arrest is different (Figure S20). For better insight, we captured the SEM images of bacteria with and without treatment of C4 under dark and light conditions. Interestingly, the bacteria treated with C4 and photo-irradiation have disrupted cell-membrane and distorted morphology (Figure 5c). However, the controls retain normal cell membranes and morphology (Figure 5a-b). Next, we investigated the effect of RPCs in the mammalian cells. We have performed the cellviability assay for both normal and cancer cell lines. The concentration used in this investigation was much lower in comparison to the measured IC 50 value (Figure S21). Interestingly, we found that the C4 mainly localizes to the nucleus of the skin cancer cell line, B16F10, after a short duration of incubation (Figure 5d-f). This bolstered our hypothesis of binding and generating the nick in the DNA and after that inducing the cytotoxic effects. We captured the time-lapse images of B16F10 cells after treatment with C4 and found that it leads to the cell lysis (see movie S1). On the contrary, C4 is mainly distributed in the cytoplasm and near the nuclear membrane of the normal cell lines, CHO (Figure S22). This phenomenon is attributed to the fact that there is perturbed permeability of the cell membranes of cancer cells than normal cells due to differential lipid peroxidation. [13] We further plotted the luminescence intensity distribution of RPCs inside the cells. The intensity profile illustrates localization of</p><p>RPCs in the inner region of the nucleus for B16F10 (Figure 5g-h) and outside the nuclear membrane for CHO cells (Figure 5i-j). Moreover, the distribution of C4 was further verified in other cell lines with different ROIs of B16F10 and BHK21 (Figure S23-24).</p><!><p>In summary, we have reported ruthenium-based metal complexes exhibiting versatile photodynamic characteristics. The luminescence lifetime of these compounds increases drastically as they experience enhanced local micro-viscosity upon DNA binding. RPCs generate singlet oxygen due to enhanced lifetime, which further produces visible lightdependent nick in the supercoiled DNA. Moreover, RPCs show antibacterial activities and get localized in the nucleus of cancer cells. Furthermore, the visible light-dependence emission property is appealing as the mode of activation is non-harmful under the physiological context and can be utilized for targeted cancer therapy and antibiotics.</p><p>Type: -Ecotron OD measurement: -Synergy H1 (BioTek), Version-Gen5 2.07</p><p>Multi-mode plate reader</p><!><p>Shimadzu UV-1800 dual-beam spectrophotometer was used for UV-Vis. measurements and Horiba jobin yvon fluorolog used for fluorescence measurements.</p><!><p>Circular dichroism spectra were recorded on a Jasco J-815 circular-dichrograph using 10 mm quartz cuvette containing 1 ml solution. CD spectra were measured in continuous mode with standard sensitivity (100 mdeg), 0.1 nm data pitch, scanning speed 200 nm/min, response 1.0 sec using TAE buffer (pH 8.5) containing 25 mM MgCl 2 . CD spectra of 5.0 nM DNA were recorded in absence and in presence of increasing concentration of metal complexes.</p><!><p>The images were captured in Carl Zeiss ultra-plus Scanning Electron Microscope using 5-20 KV HT and 10,000 times magnification.</p><!><p>The LASER of 488 for nm was used for the RPCs excitation and images were captured using Olympus Fluoview FV 3000 confocal microscope.</p><!><p>Single crystal X-ray structural studies were performed on a Bruker D8 venture instrument.</p><!><p>The UV-Vis and fluorescence properties of the Ruthenium polypyridyl complexes (RPCs)</p><p>were measured in water. The gradual increase in fluorescence intensity of metal complexes with the addition of varying concentration of DNA was monitored. Further, the intercalation of metal complex with DNA was confirmed by titrating the varying concentration of Ethidium bromide (EtBr) with the DNA and metal complex in bound state.</p><!><p>The instrument response function (IRF) was measured before and after fluorescence lifetime measurement using a dilute suspension of Ludox (from Sigma) colloidal silica. The emission polarizer was positioned at magic angle (54.7º) polarization with respect to excitation polarizer.</p><p>Exponential fitting function was employed by iterative deconvolution method using supplied software DAS v6.2. The quality of the fitted data was judged from the reduced chi-squared value (χ 2 ), calculated using the IBH software provided with the instrument.</p><!><p>The singlet oxygen detection was done using 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ADBM-DMA). 2 µM ADBM-DMA was added to the metal complexes and photoirradiated followed by monitoring of fluorescence emission. As a control, in the simultaneous experiment, the fluorescence emission of only ADBM-DMA after photo-irradiation was monitored to see the effect of light on the photophysical property of ADBM-DMA.</p><!><p>The lifetime dependence of metal complexes (5 µM) on viscosity of solution was monitored.</p><p>The lifetime was measured with the varying concentrations of Ethylene Glycol (EG). The range of solvents varies from 100% TAE buffer (pH 8.5) to 100% EG. The change in lifetime was plotted against viscosity.</p><!><p>Plasmid DNA was purified from bacterial JM109 cells using FavorPrep Plasmid Extraction Mini Kit. The concentration of DNA was measured at 260 nm and diluted to final concentration of ~50 ng/µl with distilled water. 150 µl of DNA samples from the stock was distributed in different wells of 96 well-plate. Then metal complexes were added in varying concentration of 1 µM, 2 µM, 5 µM and 10 µM followed by photo-irradiation for 1 hour. Photo-irradiated DNA samples were then run into 1% EtBr-agarose gel at 70V for 25 minutes. The bands were then visualized and intensities calculated using syngene tool software. The experiments were done thrice for each metal complex independently.</p><!><p>(final concentration = 50 µM) was added to the freshly grown aliquots of 1 ml E. coli culture (OD= 0.3± 0.03). It was photo-irradiated for 1 hour. In one of the aliquots, metal complex was added and kept in dark as control for photo-irradiation. The cells were pellet down @ 11000 rpm for 1 minute and washed thrice with distilled water. The final volume was made upto 1 ml with distilled water. 5 µl of the samples was taken for SEM imaging.</p><!><p>The B16F10, CHO and BHK21 cells were grown with regular supplementation of DMEM+10% FBS medium at 37°C and 5% CO 2 . For microscopy, the cells were seeded on glass bottom dishes. The C4 was added to cells and incubated for 15 minutes. Thereafter, the cells were washed with PBS followed by imaging.</p>

ChemRxiv

A prospective phase II study of 2-methoxyestradiol administered in combination with bevacizumab in patients with metastatic carcinoid tumors

Purpose Angiogenesis inhibition has emerged as a potentially promising treatment strategy for neuroendocrine tumors. 2-Methoxyestradiol (2ME2; Panzem\xc2\xae) is a natural derivative of estradiol with demonstrated anti-angiogenic activity in animal models. We performed a prospective, phase II study of 2ME2, administered in combination with bevacizumab, in patients with advanced carcinoid tumors. Methods Thirty-one patients with advanced carcinoid tumors were treated with 2ME2, administered orally at a dose of 1,000 mg four times daily. Patients also received bevacizumab 5 mg/kg intravenously every 2 weeks. Patients were observed for evidence of toxicity, tumor response, and survival. Results The combination of 2ME2 and bevacizumab was relatively easily tolerated and was associated with anticipated toxicities for these two agents. No confirmed radiologic responses (by RECIST) were observed. However, 68% of the radiologically evaluable patients experienced at least some degree of tumor reduction, and the median progression-free survival (PFS) time was 11.3 months. Conclusion 2ME2 and bevacizumab can be safely administered to patients with advanced carcinoid tumors. While major tumor regression was not observed with this regimen, the encouraging median progression-free survival time suggests that this regimen has some degree of anti-tumor activity and supports the further investigation of angiogenesis inhibitors in this disease.

a_prospective_phase_ii_study_of_2-methoxyestradiol_administered_in_combination_with_bevacizumab_in_p

Introduction<!>Patient population<!>Treatment program<!>Pharmacokinetics<!>Statistical considerations<!>Patient demographics<!>Exposure to study medication and treatment discontinuation<!>Pharmacokinetics<!>Toxicity<!>Efficacy<!>Discussion

<p>Carcinoid tumors are characterized by a clinical course that is usually more indolent than that of other malignancies but is usually fatal in patients with advanced, metastatic disease. While systemic treatment with alkylating agents has been associated with tumor regression and improved survival in patients with advanced pancreatic neuroendocrine tumors, patients with advanced carcinoid tumors have few standard systemic treatment options [1–3]. Somatostatin analogs generally control symptoms of flushing and diarrhea associated with carcinoid syndrome. Treatment with the somatostatin analog octreotide has also recently been shown to delay tumor progression, though is rarely associated with tumor regression [4]. Alpha interferon has been reported to have modest efficacy in carcinoid tumors, though its widespread use has been limited by the potential for toxicity [5]. In recent years, the development of new treatments for patients with carcinoid tumor has been an increasing focus of investigation.</p><p>The vascular nature of carcinoid tumors, together with evidence of high levels of VEGF and VEGFR expression, led to initial interest in exploring the efficacy of VEGF pathway inhibitors in this disease [6, 7]. Three such agents have been formally evaluated in prospective studies: the small molecule tyrosine kinase inhibitors sorafenib and sunitinib, and the monoclonal antibody bevacizumab. Sorafenib was evaluated in 50 patients with carcinoid and 43 patients with pancreatic neuroendocrine tumors. In a preliminary report of this study, responses were observed in 7% of the carcinoid patients and 11% of the patients with pancreatic NET [8]. Sunitinib was studied in a multi-institutional trial comprising 109 patients with advanced neuroendocrine tumors [9]. Partial responses were observed in 2% of the carcinoid cohort and 16% of the pancreatic NET cohort. Finally, bevacizumab was evaluated in a randomized phase II setting, in which 44 patients with advanced carcinoid tumors were randomly assigned to receive either bevacizumab or pegylated IFN-α-2b [10]. Four of 22 patients (18%) treated with bevacizumab were reported to have achieved confirmed radiographic partial responses, whereas none of the patients who received pegylated IFN-α-2b had a partial response.</p><p>2-Methoxyestradiol (2ME2; Panzem®) is a natural derivative of estradiol formed by sequential hydroxylation and O-methylation of estradiol at the 2-position [11]. 2ME2 does not have a high binding affinity to estrogen receptors and accordingly does not show direct estrogenic activity in both in vitro and in vivo [11]. In tumors, 2ME2 inhibits microtubule formation in endothelial cells. Additionally, 2ME2 inhibits expression of hypoxia induced factor (HIF) 1-alpha, resulting in decreased secretion of VEGF [12, 13]. Early-stage trials of 2ME2 in solid tumors and in prostate cancer suggested modest antitumor activity [14, 15]. 2ME2 was subsequently reformulated as a NanoCrystal® dispersion (NCD) to improve its bioavailability. In subsequent phase I studies with this new formulation, the maximum tolerated dose was defined at 1,000 mg orally four times daily, and in a phase II study enrolling 18 patients with platinum-resistant ovarian cancer or primary peritoneal carcinomatosis, treatment with 2ME2 NanoCrystal dispersion was associated with clinical benefit rate of 31% [16–18].</p><p>In light of the anti-angiogenic effects of both 2ME2 and bevacizumab, we performed a prospective study to evaluate the safety and antitumor efficacy of these two agents administered together in patients with advanced carcinoid tumors. Thirty-one patients with advanced carcinoid tumor received 2ME2, administered at dose of 1,000 mg by mouth four times daily, in combination with bevacizumab 5 mg/kg intravenously every 2 weeks. Patients were followed for endpoints of toxicity, tumor response, and survival.</p><!><p>All patients were required to have histologically documented, locally unresectable or metastatic carcinoid neuroendocrine tumor. Patients with small cell carcinoma or pancreatic endocrine tumors were not eligible. Measurable disease, as defined by RECIST, was required. Mandated laboratory requirements included aspartate aminotransferase (AST) and alanine aminotransferase (ALT) <2.5 times the upper limit of normal (<5 times upper limit of normal if liver metastasis was present), total bilirubin ≤2 mg/dL, serum creatinine ≤ 1.5 mg/dL, total white blood cell count >3,500/mm3, absolute neutrophil count (ANC) ≥1,500/mm3, international normalized ratio ≤ 1.5, platelet count ≥100,000/mm3. All patients were required to have Eastern Cooperative Oncology Group (ECOG) performance status <2. Patients with history of myocardial infarction or angina pectoris in the last 12 months, clinically apparent central nervous system metastasis, concurrent treatment with therapeutic doses of any anticoagulant, history of severe bleeding, uncontrolled severe hypertension, history of nephrotic syndrome, urine protein:creatinine ratio ≥1.0, or radiotherapy or chemotherapy within the previous 4 weeks were excluded. Prior treatment with chemoembolization, cryotherapy, or radio-frequency ablation was allowed if measurable disease was not affected.</p><!><p>The study was designed as a modified phase 2, single-arm, open-label trial. Because the drugs had not previously been administered in combination, patients were enrolled sequentially into two cohorts. The first cohort (Cohort 1) comprised 3 patients who received an oral dose of 1,000 mg Panzem® NCD four times daily and a concurrent IV administration of 5 mg/kg bevacizumab every 14 days, beginning on day 1. Patients were treated for a 28-day treatment period and then observed for 7 days. If no DLT or other significant toxicity was observed, they received subsequent 28-day cycles of treatment without additional 7-day observation periods. If DLT was observed, then the cohort was to be expanded to 6 patients prior to proceeding to full enrollment. Dose-limiting toxicity was defined as ≥ grade 3 non-hematologic, or grade 4 hematologic, treatment-related toxicity that did not resolve in 2 weeks (i.e., return to baseline), or an event that made continued treatment unsafe in the opinion of the investigator. Patients were evaluated with a physical examination, blood tests, and for toxicity every other week during the treatment period. Toxicity was graded according to the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), Version 3.0. Tumor response was evaluated at the end of every other 28-day treatment period for the duration of therapy using multi-phasic computed tomography (CT), or magnetic resonance imaging (MRI).</p><!><p>Steady-state plasma levels of 2ME2 were measured as part of the study. Blood samples for determination of steady state plasma concentrations of 2ME2 and its metabolite, 2ME1 (2-methoxyestrone), were collected prior to the first dose of Panzem® NCD on Day 1 of the initial treatment cycle and at any time during the regular dosing schedule for subsequent cycles.</p><!><p>The primary endpoint of our study was tumor response rate, with the goal of evaluating whether the overall response rate was ≥10% against the null hypothesis that the overall response rate was <10%. Assuming a response rate of 10%, the type 1 error was calculated to be 4.2% and the power was 81.6%. With this goal, 2 or more radiographic responses would have been required to identify an active regimen. Secondary endpoints of the study included assessment of toxicity, overall, and progression-free survival. Progression-free and overall survival (OS) estimates were calculated using Kaplan–Meier methodology. Progression-free survival was defined as the time from date of study entry to the first documentation of objective tumor progression or death; patients who were removed from the study without evidence of disease progression or death were censored at the time they were removed from the study. Toxicity and complications of treatment were assessed based on reports of adverse events, physical examinations, and laboratory measurements.</p><!><p>A total of 31 patients were enrolled and treated in the study. The baseline characteristics of the patient population are shown in Table 1. Patients had a median age of 57 and were relatively evenly distributed by gender. Twelve (39%) patients received concurrent therapy with octreotide during the course of the study. The small bowel intestine was the most common primary disease site. Patients had received a variety of prior therapies for their disease: fourteen (45%) patients had received prior chemotherapy, 7 (23%) had undergone previous radiation therapy, and 3 (10%) had received alpha interferon. In addition, nearly half (15/31, 48%) of the patients had received other forms of anticancer therapy, including other investigational agents.</p><!><p>Of the 31 enrolled patients, 23 completed 2 or more cycles of treatment. Of the 8 patients who discontinued treatment prior to completing 2 cycles, 6 discontinued due to adverse events and 2 for other reasons. At the time of data cutoff (12 months after enrollment of the last patient), 10 patients continued to receive study therapy and 21 patients had discontinued study therapy. Seven patients discontinued due to an adverse event, of which 4 were felt to be treatment-related, 12 discontinued due to withdrawal of consent, investigator discretion, or other reasons, and 2 due to disease progression.</p><!><p>Composite plasma concentration–time profiles were generated from blood samples collected during the study. The profiles demonstrated a steady-state Cmax of 63.53 ng/mL for 2ME2 and 700.50 ng/mL for its metabolite, 2ME1. These steady-state plasma concentrations were well above the target estimated minimum effective concentration of 3.33 ng/mL [19]. The Tmax for both 2ME2 and 2ME1 was 2.00 h. The estimated AUC0–24 h was 483.43 ng h/mL for 2ME2 and 7,112.59 ng h/mL for 2ME1. The half-lives of 2ME2 and 2ME1 could not be calculated from the available data.</p><!><p>Thirty-one patients were evaluable for toxicity. The most frequently reported treatment emergent adverse events were gastrointestinal and included nausea (18/31 any grade, 58%) and diarrhea (14/31 any grade, 45%). Adverse events are summarized in Table 2. Eighteen (58%) patients experienced at least one grade-3 treatment emergent adverse event during the study. The most common grade-3 events were hypertension (5/31, 16%) and diarrhea (2/31, 6%). Bleeding is a rare but well-known complication of anti-angiogenesis therapy, and three (10%) patients in our study experienced gastrointestinal bleeding. In two of these cases, the bleeding was attributed to gastroesophageal varices; in the third case, the source of bleeding was not identified. Only three (10%) patients reported grade-4 treatment-emergent adverse events. The grade-4 events included lung infection, suicidal ideation, and hypertension. The lung infection and suicidal ideation were considered unrelated to study drug, whereas the hypertension was considered probably related.</p><p>A total of 7 patients discontinued treatment due to treatment-emergent adverse events during the course of the study. In four cases (deep vein thrombosis, hypertension, hyperbilirubinemia, and grade 2 proteinuria), the adverse event was considered probably related to treatment. Other than the single patient who developed proteinuria, no significant changes in urine protein/creatinine ratios were observed in the patient population during treatment. One death occurred during the study due to a pulmonary infection that was considered unrelated to study drug.</p><!><p>Twenty-eight patients were evaluable for radiologic response. While no patients had radiologic partial or complete responses, of the 28 evaluable patients 27 (96%) had stable disease and 1 (4%) patient had progressive disease as their best response to therapy. Confirmed tumor responses by RECIST were not observed; however, 19 of the 28 evaluable (68%) patients had some degree of reduction in the sum of tumor LDs after screening, and two patients had reductions in LD sum ≥20% (Fig. 1). Biochemical response was assessed using plasma chromogranin A levels and 24-h urine collections of 5HIAA, measured at baseline and at the initiation of every subsequent 4-week cycle. Of 24 patients with elevated chromogranin A levels at baseline, only one experienced a response, defined as a >50% decrease. Similarly, of 19 patients evaluable for 5HIAA response, none experienced a >50% decrease during the course of study treatment.</p><p>While evidence of disease progression was not a requirement for study entry, 22 (71%) patients had documented evidence of progression within the 12 months prior to study entry. The overall median progression-free survival time in our study was 11.3 months (Fig. 2a). Median overall survival could not be estimated, as overall survival was >50% at the end of the observation period (Fig. 2b).</p><!><p>We found that treatment with the combination of 2ME2 and bevacizumab was both feasible and safe in patients with advanced carcinoid tumors. The adverse events associated with this regimen were consistent with the known profiles of both agents. The efficacy observed with the combination in patients with advanced carcinoid tumors is more difficult to assess in this single arm phase II study, although our data suggest some degree of antitumor activity.</p><p>Previous studies have suggested that combining angiogenesis inhibitors in patients with cancer has the potential for both significant efficacy and toxicity. The combination of sorafenib and bevacizumab was associated with impressive clinical activity in a phase I study in patients with renal cell carcinoma, but was also associated with a high incidence of hypertension and the development of microangiopathic hemolytic uremia [20]. High rates of grade 3 or 4 hypertension, proteinuria, and bleeding were also observed in a phase I trial of sunitinib and bevacizumab in patients with renal cell carcinoma, precluding further evaluation of the combination at standard doses of both drugs [21]. In contrast, the combination of 2ME2 and bevacizumab in our study appeared to be relatively well tolerated. Grade 3 or 4 hypertension developed in 6 patients, and 3 patients developed evidence of gastrointestinal bleeding. However, hypertension led to treatment discontinuation in only one patient; and 2 of the patients with gastrointestinal bleeding had a pre-existing condition (esophageal varices) that may have led to the bleed. Only a single patient in our study discontinued treatment due to proteinuria.</p><p>The naturally indolent nature of neuroendocrine tumors and the absence of observed major tumor responses in our single-arm phase II study make it difficult to definitively assess the antitumor activity of bevacizumab and 2ME2 in advanced carcinoid disease. Our observation that no patient treated with 2ME2 and bevacizumab experienced a partial or complete response by RECIST differs from a prior phase II study of bevacizumab and octreotide, in which a response rate of 18% was reported [10]. It is possible that our use of a different bevacizumab dosing regimen (5 mg/kg every 2 weeks rather than 15 mg/kg every 3 weeks) contributed to this difference. Two patients in our study experienced reductions of ≥20% in the sum of longest tumor diameters, and 19 (68%) patients experienced at least some degree of tumor shrinkage. The overall rate of PR + SD in our study was 96%, a value that is nearly identical to the PR + SD rate of 95% observed in the prior study of bevacizumab + octreotide, and superior to the PR + SD rate of 85% in the subgroup of carcinoid patients treated in a phase II study of sunitinib [9, 10].</p><p>Decreases in plasma levels of the neurosecretory protein chromogranin A have been associated with clinical improvement and improved prognosis in patients receiving somatostatin analogs, cytotoxic chemotherapy, and other anti-tumor agents [2, 22, 23]. We found that the combination of 2ME2 and bevacizumab had minimal effect on chromogranin A levels in treated patients: only a single patient experienced a significant reduction in this marker. A similar lack of correlation between chromogranin A response and clinical outcomes in patients with neuroendocrine tumor was also observed in phase II studies of bevacizumab and sunitinib [9, 10]. In both of these studies, evidence of antitumor effect was seen on radiologic imaging studies, suggesting that chromogranin A may not be a reliable surrogate marker of response for anti-angiogenic agents [9, 23].</p><p>Progression-free survival time has also been used as an endpoint to evaluate the potential efficacy of novel agents in patients with neuroendocrine tumors. The median progression-free survival time in our study was 11.3 months, a value that compares favorably to progression-free survival times reported in other, similar studies of novel agents in carcinoid tumors (Table 3). Two prior phase II studies, one evaluating everolimus in combination with octreotide and the second evaluating bevacizumab or interferon in combination with octreotide, reported median progression-free survival times of 14.4 months in patients with carcinoid tumors, a value that is superior to the progression-free survival time of 11.3 months observed in our study [10, 23]. However, both of these earlier studies required concurrent treatment with octreotide in all patients. Somatostatin analogs, including octreotide, are commonly used in patients with advanced carcinoid tumors as a means to control symptoms of hormonal hypersecretion such as flushing and diarrhea, but more recently have also been shown to be associated with improved time to tumor progression. In a placebo-controlled randomized study enrolling patients with midgut carcinoid tumors, the median time to tumor progression was 14.3 months in patients receiving octreotide, when compared to 6 months in the cohort receiving placebo [4]. The fact that only 12 (39%) of the patients treated in our study received concurrent octreotide may have contributed to the somewhat shorter PFS observed in our study.</p><p>While our study suggests that treatment with 2ME2 and bevacizumab is associated with minor tumor reductions and encouraging progression-free survival durations, a study of this regimen in the randomized setting would be necessary to more definitively assess this endpoint. An international randomized phase III study to confirm the activity of sunitinib in pancreatic neuroendocrine tumors has recently been stopped early, after preliminary results demonstrated that treatment with sunitinib was associated with a median progression-free survival duration of 11.1 months, when compared with 5.5 months in the placebo arm [24]. In a second, ongoing study, led by the Southwest Oncology Group, patients with advanced carcinoid tumors are currently being randomized to receive treatment with octreotide and either IFN-α-2b or bevacizumab, with a primary end point of progression-free survival.</p><p>In conclusion, our study demonstrates the feasibility of administering two anti-angiogenic agents, 2ME2 and bevacizumab in patients with advanced carcinoid tumors. Our observations of minor decreases in tumor size and an encouraging progression-free survival duration, supports the potential for activity of angiogenesis inhibitors in this disease. The lack of confirmed RECIST-defined responses in our study also highlights the challenges of assessing the antitumor activity of antiangiogenic agents and other novel, biologic therapies in this disease. Randomized phase II designs and the development of more reliable biomarkers for response may facilitate the future evaluation and development of similar regimens for patients with neuroendocrine tumors.</p>

PubMed Author Manuscript

High-Throughput Screening Assay Datasets from the PubChem Database

Availability of high-throughput screening (HTS) data in the public domain offers great potential to foster development of ligand-based computer-aided drug discovery (LB-CADD) methods crucial for drug discovery efforts in academia and industry. LB-CADD method development depends on high-quality HTS assay data, i.e., datasets that contain both active and inactive compounds. These active compounds are hits from primary screens that have been tested in concentration-response experiments and where the target-specificity of the hits has been validated through suitable secondary screening experiments. Publicly available HTS repositories such as PubChem often provide such data in a convoluted way: compounds that are classified as inactive need to be extracted from the primary screening record. However, compounds classified as active in the primary screening record are not suitable as a set of active compounds for LB-CADD experiments due to high false-positive rate. A suitable set of actives can be derived by carefully analysing results in often up to five or more assays that are used to confirm and classify the activity of compounds. These assays, in part, build on each other. However, often not all hit compounds from the previous screen have been tested. Sometimes a compound can be classified as \xe2\x80\x98active\xe2\x80\x99, though its meaning is \xe2\x80\x98inactive\xe2\x80\x99 on the target of interest as it is \xe2\x80\x98active\xe2\x80\x99 on a different target protein. Here, a curation process of hierarchically related confirmatory screens is illustrated based on two specifically chosen protein use-cases. The subsequent re-upload procedure into PubChem is described for the findings of those two scenarios. Further, we provide nine publicly accessible high quality datasets for future LB-CADD method development that provide a common baseline for comparison of future methods to the scientific community. We also provide a protocol researchers can follow to upload additional datasets for benchmarking.

high-throughput_screening_assay_datasets_from_the_pubchem_database

Introduction<!>Compound data repositories host libraries of molecular compounds and associated biological activities<!>False positive rate in primary HTS experiments is high<!>Hierarchical confirmatory screening experiments validate primary hit compounds<!>Previous studies underline importance of chemical data curation for LB-CADD modelling<!>Significance<!>Curation process based on hierarchy of confirmatory high-throughput screens validates active compounds<!>High-throughput screens validate active compounds associated with NPY \xe2\x80\x93 Y1 and Y2 HTS screens<!>Case study: curating primary cell-based high-throughput screening assay for antagonists of the Y1 receptor<!>Case study: curating primary cell-based high-throughput screening assay for agonists of the Y2 receptor<!>Uploading of curated datasets into PubChem<!>Conclusions

<p>The development of ligand-based computer-aided drug discovery (LB-CADD) methods for in silico (virtual) high-throughput screening (HTS) shows promising results for identifying potential hit compounds, i.e., compounds that share a biological activity of interest [1]. With the popularity gain of HTS in academia, the need for LB-CADD method development continues to increase [2,3]. The cost of an HTS screen correlates nearly linearly with the number of physically screened compounds. LB-CADD has the potential to reduce these costs in a resource-limited academic environment by helping to prioritize which compounds to include in a screening campaign. However, LB-CADD method development depends on the availability of reliable HTS assay datasets to study the relationship of ligand structure and biological activity. It is a challenge to identify suitable refined datasets for LB-CADD benchmarking that are available to the research community. Frequently in both industry and academia, proprietary datasets are not disclosed to the research community for use in LB-CADD benchmarking and methods development. Therefore, novel methods cannot be directly compared to existing algorithm implementations and scientific progress is difficult to gauge. In other research fields, e.g., machine learning, standardized datasets are available and serve as foundation for evaluation and benchmarking of novel algorithms. Examples are the MNIST database for hand-written digits and UCI Machine Learning Repository [4,5]. These datasets provide a common ground for testing new methods and allowing for easy comparison of novel and previously established approaches.</p><!><p>PubChem is a public repository providing HTS experiment results containing biological activities for several hundred thousand of compounds tested against different biological targets [6–8]. It provides a platform to host target-related HTS datasets. PubChem is maintained by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine, which is part of the National Institutes of Health (NIH). Over 1,000,000 bioassays for more than 9,000 protein targets can be accessed online contributed by more than 70 small molecule and RNAi screening centers and research laboratories. It is also supported by over 300 small molecule vendors contributing to the growing compound database of PubChem worldwide. Vendors include US government-funded institutions, research laboratories pharmaceutical companies, and collaborators hosting chemical biology databases. Other HTS repositories, such as ChEMBL or BindingDB, are alternatives to PubChem with different philosophies of annotation and evaluation of chemical biology datasets with their respective databases. A review of these HTS repositories can be found here [9–13].</p><!><p>Typically, primary HTS experiments categorize small molecules as hit, inactive, or unspecified about the desired biological activity. However, depending on the design of the HTS experiment, there are many other reasons why a compound might be designated as hit ranging from activity of the compound an undeclared target in the cell to optical interference. Therefore, primary screens are only a first iteration that reduces the available compound library to a smaller set that can be interrogated in more detail. As compounds are tested without replication (singleton?) and the cut off for activity is typically loose to minimize the number of false negatives, the false positive rate can be high. Although outliers are common in HTS experiments, statistically robust methods not sensitive to outliers are necessary for hit selection, e.g., z*-score, SSMD*, B-score, and quantile-based methods [14]. Confirmatory screens act as a validation filter by testing hit compounds with multiple replications of the experiment, recording concentration response curves, test hit compounds with an identical assay setup but in the absence of the putative target protein, and sometimes exclude even compounds that act on the target protein but not selectively.</p><!><p>The biological assay database of PubChem allows for the deposition of primary as well as confirmatory HTS experiments. Due to the requirement from funding agency on data sharing, primary screening results from NIH funded HTS projects were often deposited to PubChem prior the deposition of confirmatory assays and counter screens. Confirmatory assays seek to establish the relationship between chemical structure and a defined biological outcome (SAR). Confirmatory assays applications range from validating active compounds identified in the primary screen, over the target confirmation through orthogonal assays, and determination of specificity through testing against other subtypes of the target protein or related proteins. For molecular probe development, confirmatory assays are used to investigate a smaller subset of often similar compounds to investigate the SAR around the given scaffold further. A hierarchy of confirmatory assays is established when results of dependent confirmatory screens are analysed. In progressed stages of the hierarchy, concentration response experiments provide values for half maximal effective concentration (EC50) or inhibition (IC50) in addition to the determined binary active/inactive outcome. Despite of multiple update mechanisms provided by the PubChem system, datasets regarding the same HTS assay project but deposited under different time lines are sometimes not sufficiently summarized. Upon completion of the HTS project, a curation process is necessary to incorporate all experimental data from different stages of the assay project and provide a dataset with the ultimate bioactivity outcomes.</p><!><p>In a previous study, we assembled nine datasets from HTS campaigns representing major families of drug target proteins for benchmarking LB-CADD methods see (Table 1). Emphasis was placed on biological target diversity and the high quality HTS activity obtained through confirmatory screen validation. These collated datasets provided the foundation for an extensive LB-CADD benchmarking study using the cheminformatics framework BCL: ChemInfo [15]. For the present manuscript, we collaborate with PubChem to make these datasets easily accessible for all researchers.</p><p>These datasets were selected with the goal to cover a wide-range of protein target classes. Each target class is represented by a sampled chemical space, spanned by the screened molecules evaluated within related HTS assay experiments. Primary and confirmatory screens were curated from PubChem and this curation process represent a tool for more systematic benchmarking of novel LB-CADD algorithms. For this manuscript, each curated dataset was re-assembled and aligned by CIDs before being uploaded into PubChem. Datasets marked with an asterisk in Table 1 have been modified with respect to our previous study due to compound alignment by common substructure overlap rather than PubChem identifier (CID).</p><!><p>LB-CADD is particularly attractive in the resource-limited environment of academia as it reduces the cost and increases quality of drug discovery and/or probe development. Quantitative structure-activity-relationship (QSAR) models developed in LB-CADD are only as good as the data quality used for training such models. Thus, there is a pressing need to develop and systematically employ HTS assay record curation protocols helpful in the pre-processing of any chemical dataset. This manuscript highlights difficulties when working with HTS experimental data in the public domain and illustrates the curation process on two chosen examples targets as well as the re-upload of the new datasets into PubChem.</p><p>Establishing a dataset "gold standard" for benchmarking novel LB-CADD methods is important for testing performance of new algorithms in respect to the complexity of the chemical space and for different biological targets. It also counters a trend that newly developed methods are tested on proprietary datasets which creates difficulties when reproducing results and reduces transparency when comparing methodological advances in LB-CADD method development. As chemical space differs in complexity for each protein target, it is imperative for new LB-CADD methods to be benchmarked on representative high quality datasets. The here described curation process has the potential to provide a wide range of higher quality datasets freely accessible to the research field.</p><!><p>The following curation process evaluates the description of PubChem assays, identifies the PubChem assay ID (AID) of the primary screen and discusses the validation and classification of active compounds from confirmatory screens. Confirmatory screens can be subdivided into the categories "confirming" and "descriptive". "Confirming" assays validated a compound as active at a declared molecular target (e.g., testing the compounds in the presence and absence of the declared target). The application of "confirming" assays results in identification of a set of validated hits.</p><p>The second sub-category is "descriptive". Typically, "descriptive" assays occupy a position in the hierarchy downstream from the confirmatory assays. An example of a descriptive assay is a "counter screen" against another molecular target. Since the compound activity has been validated, it is viewed as a validated hit. Additional data add to our understanding of the compound's activity. e.g., a compound could be demoted from "active" to "inactive" based on a descriptive assay. However, this would be in the context of a previously declared intent (e.g., antagonists of the NPY Y1 receptor) and a gating criterion (e.g., 50-fold selective against Y1). Such criteria are commonly used but need to be highlighted in the context of curating a data set. Here, validated hits are active at the declared target but can be declared "inactive" within the context of the curated dataset when additional "descriptive" data is taken into consideration. To construct a final dataset, the inactive compounds are taken from the corresponding primary assay. However, the authors would like to emphasize that this manuscript does not endorse or vouch for the applied HTS methods, given assay results or interpretations of the mentioned assays below. The here described curation process merely utilizes the assay outcomes given by the assay providers and the screening facilities.</p><!><p>PubChem provides publicly available biological assay results for a diverse set of protein targets. For the scope of this manuscript, we chose neuropeptide Y (NPY) receptor type 1 and 2, (Y1 and Y2). These receptors are members of a larger family of NPY receptors (Y1, Y2, Y4, Y5) which are part of the family of G-protein-coupled receptors (GPCR) [16,17]. As their name suggests, the receptors are effectors of the neuropeptide neurotransmitter NPY, studies have implicated these receptors in diverse biological events, including feeding, alcoholism, anxiety and depression, pain perception, immunity and inflammation, vascular remodeling hypothermia, and bone and energy metabolism [18–25]. Due to the varied role of these receptors in human disease and physiology, the identification of high-affinity selective probes that target each receptor subtype may provide novel tools for the study of NPY-related pathologies.</p><!><p>In this case study, the PubChem assay AID1040 tests compounds for their ability to act as antagonists of the NPY receptor Y1. A cell line transfected with Y1 and a cyclic-nucleotide gated channel (CNGC) was used to measure Y1 antagonism by the test compound. The cells were treated with the β-adrenergic receptor agonist, isoproterenol, to activate adenylate cyclase, thus increasing cytosolic cyclic adenosine monophosphate (cAMP) concentrations, and therefore increasing CNGC activity. Elevated CNGC activity decreases the cell membrane potential, which is measured using a membrane potential-sensitive fluorescent probe. Because the Y1 receptor is Gi-coupled, addition of the NPY counteracts isoproterenol action resulting in a decrease in CNGC activity. A tested compound that is an Y1 antagonist will counteract NPY action, thus the isoproterenol-evoked high level of cAMP will be maintained and high CNGC activity will be preserved. This primary assay AID1040 tested 196,255 compounds and identified 1,990 actives. A subset of 1,195 hit compounds from the set of 1,990 active compounds was investigated further by the following two confirmatory screens. AID1254 repeated the primary screen experiment to validate activity for the hit compounds. AID1255 tested selectivity of hit compounds by removing antagonists of the Y2 receptor. This assay used a cell line transfected with the Y2 receptor and a cyclic-nucleotide gated channel (CNG) was used to measure receptor antagonism through CNGC opening. This assay serves as an elimination of "false positives" in this context that could result from modulation of other biological protein targets. The findings of AID1255 resulted in 332 compounds active against Y2. 252 compounds were ultimately confirmed through AID1254 as active and selective. The following two HTS screens (AID1277 and AID1278) represent a second level of validation and further investigated a smaller fraction of just 63 compounds. AID1277 determines concentration response curves for a subset of compounds identified as active in the previous experiments. Multiple criteria for testing the compounds had to be fulfilled. The compounds were active against the primary screen (AID1040). Compounds confirmed inactive by the confirmatory screen AID1254 were excluded. Additionally, these compounds had to be inactive when assessing Y2 antagonism through AID1255. The final set of active compounds is comprised of 801 active molecules, taken from the actives of AID1040, subtracting inactive compounds from AID1254, subtracting actives from AID1255, subtracting inactive from AID1277, and actives from AID1278 as shown in Figure 1. 'Active' compounds within this context are defined as a combination of active and selective compounds for Y1.</p><!><p>This study investigated small molecules for antagonism of NPY receptor Y2. A cell line transfected with Y2 and CNGC using a primary screening assay similar to the assay described for Y1 receptor, above. This primary screen AID793) tested 140,092 molecules for activity and identified 1,384 hit compounds. The confirmatory screen AID1257 evaluated a subset of 707 from the 1,384 molecules in more detail. It confirmed activity of compounds that were identified as actives in the primary screen AID793 with the same experimental assay setup. 707 compounds were tested in more detail and 479 molecules were confirmed inactive, and thus subtracted from the initial set of active compounds. On the other hand, AID1256 was designed to identify non-selective antagonists among the actives of the primary screen because of inhibition of the Y1 receptor. The same set of 707 compounds was screened and 135 compounds were removed as non-selective. The next stage of confirmatory screens evaluated a more specific subset of 119 compounds. Assay AID1279 determined whether compounds are active against the primary screen (AID793), activity for antagonism towards Y2 had to be confirmed in AID1257, and whether the compound showed activity in the cell-based HTS assay measuring Y1 antagonism (AID 1256). Out of the 119 actives molecules 74 compounds were confirmed and thus excluded from the pool of overall actives. The second assay (AID1272) screened the same 119 compounds as AID1279 but evaluated each molecule by different criteria: The compounds had to be active in the primary screen AID793. This activity had to be confirmed in AID1257. And lastly, these compounds had to be inactive with respect to measuring Y1 antagonism (AID 1256). A total of 119 compounds were screened and 47 inactive compounds were confirmed and removed. Next, a layer of counter screens AID2210, AID2212, AID2224, involved in this series evaluated 89 compounds for cross-findings among actives for agonism of Y1 and antagonism for Y2 and inhibition of cyclic nucleotide gated ion channel (CNGC) activity. Active compounds found through those assays were excluded from the set of final actives. Finally, as assays for late stage results from probe development efforts to identify antagonists of NPY-Y2, AID2211 and AID2220 were set up with the same conditions as AIDs 793, 1256, 1257, 1272, and 1279. Non-selective Y2 agonists and compounds acting as Y1 agonists were excluded. Figure 2 shows a detailed flow chart depicting the individual compound subtractions.</p><p>In summary, the assembly of the final actives dataset, an ensemble of 699 active compounds was determined by selecting the actives from the primary screen and excluding inactive compounds of AID1257 and AID1272, as well as subtracting actives from AID1256, AID1279, AID2210, AID2211, AID2212, AID2220, and AID222.</p><!><p>PubChem provides access to biological assay data e.g., through its Power User Gateway (PUG) [26]. Data queries can be sent via XML to request AID data for molecule in a specific format (e.g., SDF, SMILES) as well as the associated biological assay data containing metadata, and activity related data. Every compound is uniquely identified by its compound identifier (CID) or substance identifier (SID). Sets of molecules can be downloaded in respect to a given AID. These identifier in conjunction with the activity categorization of a compound allows for the curation of sets of molecules of confirmatory screens as discussed, the two case studies (see above).</p><p>Through the hierarchical relationship of primary and confirmatory assay experiments, compounds can be aligned by their respective CID. Dependent on the outcome on each hierarchy level, compounds can be classified as active or inactive depending on the result of the last involved confirmatory screen. The ensemble of molecules that satisfies all levels of the HTS hierarchy represents the final curated dataset.</p><p>The PubChem Upload system (pubchem.ncbi.nlm.nih.gov/upload) offers a mechanism to submit the newly curated set of compounds into PubChem. After specifying which compounds are involved by specified by SID identifiers the aligned hierarchy of compound activities through all involved HTS results can be uploaded. Once the submission was successful and approved by a PubChem curator the newly curated dataset is accessible to the public and can be shared with the research community.</p><!><p>High-quality HTS datasets are important for LB-CADD method development. However, results of various validation experiments for an assay project are often reported separately in PubChem and final set of inactive, inconclusive, and conformed active compounds is mostly lacked in the database. The goal of this work is to provide an overview of a curation process, starting from primary screens and their associated confirmatory screens, building a hierarchical structure through multiple related assay experiments. It needs to be emphasized that the applied HTS methodologies, the given assay results, and interpretations are taken 'as is'. Thus, curation process relies on a high-quality standard for experimental data given by assay providers and the screening facilities. The assembly and upload of the curated dataset to the PubChem database is discussed based on two specifically chosen protein target use-cases. The upload of curated datasets into PubChem is described and thus supports the development of a publicly available database for benchmarking LB-CADD methods. Ultimately, availability of such datasets will eliminate the need to test LB-CADD methods on proprietary datasets allowing ready reproduction and comparison of results. Furthermore, such curation projects help to enhance the utility of HTS data in the PubChem database by summarizing and excluding false positives and experimental artifacts at various assay stages, and thus to highlight confirmed biological compounds.</p>

PubMed Author Manuscript

Disruption of endocytosis through chemical inhibition of clathrin heavy chain function

Clathrin-mediated endocytosis (CME) is a highly conserved and essential cellular process in eukaryotic cells. Although classical genetics has been used to understand CME, its highly dynamic and vital nature is difficult to approach with such tools. In contrast, small molecules can acutely and reversibly perturb CME, however, the few chemical CME inhibitors that have been applied to plants are ineffective or show undesirable side effects. Here, we identify the previously described endosidin9 (ES9) as an inhibitor of clathrin heavy chain (CHC) function in both Arabidopsis and human cells through affinity-based target isolation, in vitro binding studies and X-ray crystallography. Moreover, we present a chemically improved ES9 analog, ES9-17, which lacks the undesirable side effects of ES9, while retaining the ability to target CHC. ES9 and ES9-17 have expanded the chemical toolbox to probe CHC function, and present chemical scaffolds for further design of more specific and potent CHC inhibitors across different systems.

disruption_of_endocytosis_through_chemical_inhibition_of_clathrin_heavy_chain_function

<!>Affinity purification identifies Arabidopsis thaliana CHC1 as potential ES9 binder<!>ES9 binds CHC<!>ES9 binds the N-terminal domain (nTD) of CHC<!>Identification of nonprotonophoric ES9 analog<!>ES9-17 is a CME inhibitor<!>ES9-17 targets CHC<!>Discussion<!>Methods<!>Plant material and growth conditions<!>Generation of hemagglutin (HA)-tagged AP2S and AP2M constructs and Arabidopsis PSB-D cell culture transformation<!>Chemical treatments, chemical labeling and imaging in Arabidopsis\n<!>ATP measurements<!>Affinity purification with biotinylated small molecules<!>LC\xe2\x80\x93MS/MS Analysis<!>CETSA<!>DARTS<!>Molecular docking<!>Cloning, expression and purification of human nTD CHC1 and Arabidopsis nTD CHC1/2<!>X-ray data collection<!>Differential scanning fluorimetry (DSF) assay<!>Transferrin uptake in HeLa cell cultures<!>Cell viability assay<!>Root Growth Assay<!>TEM<!>Statistical tests and generation of graphs<!>Life sciences reporting summary<!>Supplementary Material

<p>Clathrin-mediated endocytosis (CME) is a major route for internalization of plasma membrane (PM) proteins and molecules from the extracellular enviroment 1,2 , but its dynamic and essential nature makes it difficult to dissect using classical genetics approaches. Chemical inhibitors of endocytosis are an attractive alternative to the current methods for disrupting protein functions. However, despite the extensive structural and biochemical knowledge about CME in eukaryotic cells 3 , the development of chemicals that interfere with this process is still at a relatively early stage. Until now, a few small molecules were shown to target the CME machinery in mammalian, yeast or plant systems 4 . Some of the most commonly used small-molecule CME inhibitors in mammalian systems are Pitstop2 (ref. 5), targeting the N-terminal domain (nTD) of the clathrin heavy chain (CHC), Dynasore 6 and the Dynasore-based series of small molecules called Dyngo 7 , both affecting the dynamin function. Recently, a natural product, Ikarugamycin, has been used to inhibit CME in different systems, but neither its potency nor specificity toward CME have been extensively examined 8 . Since none of the above mentioned molecules showed consistent effects in plant cells, plant cell biology has taken advantage of TyrphostinA23 (TyrA23), a CME-inhibiting small molecule 9 . However, TyrA23 has recently been described as a protonophore in Arabidopsis thaliana, and its inhibition of endocytosis was shown to occur through non-specific cytoplasmic acidification 9 . Therefore, CME research in plants would benefit from novel, potent small molecule inhibitors to dissect endocytosis to improve our understanding of the many physiological processes that rely on it.</p><p>Previously, the small molecule endosidin9 (ES9) was identified as an endocytosis inhibitor in different model systems 9 . Although ES9 is primarily a protonophore, its observed interference with CME did not seem to originate solely from cytosol acidification. For example, in Drosophila melanogaster, ES9 blocked synaptic vesicle recycling, closely mimicking the phenotype in mutants defective in clathrin or dynamin functions, while in Arabidopsis, ES9 was found to retain its ability to inhibit endocytosis at an increased apoplastic pH, in marked contrast to other protonophores such as TyrA23 (ref. 9). These results suggested that, despite its protonophore activity, ES9 might target proteins involved in CME. Here, we identified CHC as the protein target of ES9. Structural activity relation (SAR) analysis of ES9 aimed at disconnecting the protonophoric and CME-inhibiting activities discovered the improved ES9-17 analog, which inhibited endocytosis, but without its former protonophore activity. In vitro target validation strategies, including cellular thermal shift assays (CETSA) 10 and Drug Affinity Responsive Target Stability (DARTS) 11 further confirmed CHC as a target of ES9-17. Altogether ES9 and ES9-17 expand the current chemical toolbox for CME inhibition and present promising scaffolds for further development of chemical probes targeting CHC across different systems.</p><!><p>Although the small molecule ES9 (1) had been previously characterized as a protonophore and an unspecific inhibitor of CME 9 , not all ES9-induced phenotypes were explained by its protonophore activity, implying that ES9 might inhibit CME through a direct interaction with its machinery 9 . To explore this possibility, we performed a SAR analysis of ES9 to identify a suitable position for derivatization with a linker and a biotin tag for carrying out an affinity-based target identification (Supplementary Fig. 1a; Supplementary Note 1). A small collection of commercially available and in house synthesized ES9 analogs were investigated for their ability to inhibit the uptake of the lipophilic styryl endocytic tracer dye N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl)hexatrienyl)pyridinium dibromide (FM4-64) 12 in Arabidopsis root epidermal cells at a concentration of 50 μM and upon 30 min treatment (Supplementary Fig. 1a–c). The partial structure or possible hydrolysis products ES9-3 (2) and ES9-7 (3) lacked activity in this endocytosis inhibition assay. Similarly, methylation of the acidic NH position of ES9-6 (4) resulted in FM4-64 uptake and, thus, loss of activity. Replacement of the bulky bromine substituent on the thiophene ring by a hydrogen produced a simplified analog ES9-2 (5) that retained activity, hinting at a suitable anchoring place that can tolerate the introduction of a linker moiety for a biotin label attachment. Replacement of the thiophene ring itself by its phenyl bioisostere gave the active phenylsulfonamide analog ES9-8 (6) that readily allowed the further derivatization of the bromine-occupied position. Thus, a hydroxyl-terminated linker was introduced at this position giving the analog ES9-9 (7) which indeed retained its activity in the endocytosis inhibition assay. Next, a biotin tag was introduced to generate a biotin-coupled variant of the active ES9-9, named ES9-10 (8). However, ES9-10 lost the ability to inhibit FM4-64 uptake, likely because ES9-10 was prevented from entering the cell by either the biotin moiety requiring transport 13 or the cell wall. A linker-biotin probe without the ES9 moiety, ES9-13 (9) (Supplementary Fig. 1a–c), was included as a negative control in further pull-down experiments.</p><p>To identify possible targets of ES9, we prepared protein extracts for affinity purification from PSB-D wild type Arabidopsis cell cultures. Proteins bound to ES9-10 or ES9-13 were analyzed by means of mass spectrometry. In total, three biological replicates were examined for the ES9-10 affinity purification and the ES9-13 control, each represented by three technical repeats. Proteins were listed based on the number of times they were identified in the different biological and technical replicates, and based on the number of peptide-to-spectrum matches. To reduce the number of the putative ES9-10 interactors we filtered the obtained data as previously described 14,15 . Only proteins found more than once in the three biological replicates in the ES9-10 pull-down experiments, and not in the ES9-13, were considered as potential hits, resulting in a list of 11 candidates (Supplementary Data Set 1), among which the Arabidopsis thaliana CHC1 isoform is an essential CME component 16 .</p><!><p>Although we cannot exclude that other proteins identified in the ES9-10 pull-down might play a role in CME, we chose to focus on CHC, as CHC1 was found as one of the top candidates, and has a well described role in CME. To validate the possible interaction between ES9 and CHC1 in Arabidopsis, we employed the cellular thermal shift assay (CETSA) that monitors target engagement based on small molecule-induced changes in the thermal stability of protein targets 10 . The aggregation temperature (Tagg) of CHC was assessed through immunoblot analysis using anti-CHC antibody, which cross-reacts with the two Arabidopsis CHC isoforms, CHC1 (AT3G11130) and CHC2 (AT3G08530), in Arabidopsis cell culture lysates treated with DMSO for 30 min and heated for 2 min at 12 different temperature points (30-65°C). Thermal denaturation of CHC under control (DMSO) conditions indicated a Tagg of 45±0.23°C (mean ± standard error of the mean, SEM) (Fig. 1a). For further isothermal dose-response fingerprint (ITDRFCETSA) experiments of CHC in the presence of ES9, we set the temperature at 46°C to ensure a sufficient shift in the denaturation temperature and determined the half maximum effective concentration (EC50) to be 120.5±1.09 μM (mean±SEM) (Supplementary Fig. 2a). In contrast, the highly abundant protein ATP synthaseβ (ATPβ), chosen as a control, was largely unaffected, even at high compound concentrations. Although the EC50 for the thermal denaturation of CHC in the presence of ES9 was 120 μM, a higher concentration of 250 μM ES9 was used for CETSA to achieve sufficiently sized Tagg shifts. The presence of 250 μM ES9 for 30 min and heating for 2 min at 12 different temperature points (30-65°C) generated a lower Tagg of 43 ±0.29°C (mean ± SEM) than that of the vehicle control (45±0.23°C, mean±SEM), thus resulting in a Tagg shift of 2°C (Fig. 1a). Thermal denaturation of the control proteins, tubulin and ATPβ, showed negligible Tagg shifts in the presence of either 250 μM ES9 or the vehicle (Fig. 1b; Supplementary Fig. 2b). Moreover, in the presence of the inactive analog ES9-6 (250 μM), the Tagg for CHC did not differ when lysates were either treated with DMSO (44.76±0.47°C) or ES9-6 (44.49±0.27°C) (mean±SEM) (Supplementary Fig. 2c). The results obtained with CETSA were further corroborated by means of DARTS 11 study, used to determine the potential of ES9 to protect CHC from protease digestion (Fig. 1c). In the presence of ES9 (250 μM), CHC was significantly stabilized at one pronase concentration when compared to the DMSO-treated control samples. Such stabilization was not observed for the control protein ATPβ (Fig. 1c). Taken together, our data indicate that CHC likely is the target of ES9 in Arabidopsis cells.</p><!><p>In an attempt to predict the binding site of ES9 on CHC, ES9 was docked to the only available structures of the human CHC1 nTD in a complex with either Pitstop1 (pdb2XZG) or Pitstop2 (pdb4G55) 5 . The obtained prediction with pdb4G55 suggested that the binding site for ES9 on Arabidopsis CHC1 might be the same as that for Pitstop2 in human CHC1 (Supplementary Fig. 3a). Residues Arg64, Phe91 and Gln89 in the binding site were set as flexible during docking and the first nine predicted positions were all similar, except for minor reorientations of the flexible residues. To confirm this prediction, the highly homologous β-propeller nTDs of the human CHC1 and the two Arabidopsis CHC1 and CHC2 isoforms (Supplementary Fig. 3b) were produced and the respective protein-ES9 interactions were analyzed in vitro by means of differential scanning fluorimetry (DSF). Upon ES9 binding to the nTD of the Arabidopsis CHC1, DSF detected a shift in the thermostability of CHC1, resulting in a change in its melting temperature (ΔTm) (Fig. 1d; Supplementary Table 1). In contrast, the thermal denaturation curve for the inactive analog ES9-6 (160 μM) overlapped with that of the (DMSO) control (Fig. 1d). The protein stability of the nTD in the presence of ES9, assessed as ΔTm, was similar for the CHC1 and CHC2 of Arabidopsis and the human CHC1 (Supplementary Fig. 2d,e; Supplementary Table 1). Altogether, our results demonstrate that ES9 binds to the nTD of CHC both in Arabidopsis and in human.</p><p>To understand the molecular basis of the CME inhibition, the binding site of ES9 in the nTD of CHC was mapped by determining the structure of the human nTD of CHC1 in a complex with ES9 by protein X-ray crystallography (Fig. 2a). Crystals with the clathrin nTD diffracted up to a resolution of 1.6Å and the structure was solved by molecular replacement with the CHC1 nTD as a search model (Supplementary Table 2). The CHC1 nTD forms a seven-bladed β-propeller, each with four antiparallel strands. The electron density for ES9 could be identified between the first and second blades (Fig. 2b). Modeling of ES9 into this electron density revealed that the ES9-binding site on the CHC nTD overlaps with the binding site for the CME adaptor proteins harboring a clathrin box motif 17 . ES9 is positioned in the cavity formed by six amino acids (Arg64, Ile93, Phe91, Gln89, Leu 82, and Ile66). The conformation of ES9 is stabilized by electrostatic interactions between its benzonitro moiety and Arg64, and between its thiophene group and the carboxyl oxygen of Ser67 (Fig. 2c,d). All the six amino acids involved in binding of ES9 to nTD are conserved between human CHC1 and Arabidopsis CHC1 and CHC2 (Supplementary Fig. 3b). Thus, the crystal structure analysis corroborated the previous docking predictions that ES9 targets the nTD of CHC in a similar fashion as the Pitstop2 (ref. 5).</p><!><p>Although ES9 binds CHC, its protonophore activity 9 limits the use of this small molecule as a specific CME inhibitor. With the aim to uncouple CHC binding of ES9 from its protonophore activity, we carried out a focused SAR analysis to identify ES9 analogs that lack protonophore activity, but retain the ability to inhibit FM4-64 uptake (Supplementary Fig. 4a; Supplementary Note 1). As a weak acid that gives a delocalized anionic charge via electronic conjugation within a large hydrophobic compound is a known molecular architecture that allows protonophore activity, resulting in lipohilic anions 18 , we attempted to influence the acidity of the sulfonamide of ES9. For several analogs with expected differences in acidity and/or charge delocalization, we assessed both the FM4-64 uptake inhibition and the impact on the mitochondrial membrane potential as visualized with MitoTracker Red CM-H2XRos dye 19 in Arabidopsis root epidermal cells 9 when applied at concentration of 50 μM (Supplementary Fig. 4b–d). Transfer of the nitro group from the para to the meta position in ES9-29 (10) is expected to make the sulfonamide less acidic, while preserving specific contacts with target biomolecules. However, ES9-29 retained its protonophore activity, indicating that the sulfonamide was still fairly acidic. Other substitutions on the phenyl ring, like in ES9-15 (11) and ES9-16 (12) led to loss of the FM-64 uptake inhibition activity. Altogether, elimination of the nitro group in ES9 should strongly shift the equilibrium toward the protonated form, and inhibit resonance of the charge to the phenyl ring but it could affect the binding affinities for biomolecular targets. In fact, we found that ES9-14 (13) (Supplementary Fig. 4a) and ES9-17 (14) (Fig. 3a) both remained active with respect to FM4-64 uptake inhibition and did not abolish MitoTracker Red CM-H2XRos staining of the mitochondria at 50 μM (Supplementary Fig. 4b–d). Although, ES9-17 inhibited the FM4-64 uptake in Arabidopsis root epidermal cells with an EC50 of 13 μM (Fig. 3b), a concentration of 30 μM was used for further cellular analysis to ensure a substantial reduction in endocytosis (Fig. 3c). In addition, CME inhibition with 30 μM ES9-17 proved to be reversible, as FM4-64 uptake was clearly recovered after 120 min washout with control medium in two independent experiments (Supplementary Fig. 4e).</p><p>To rule out the protonophore activity of ES9-17, we assessed its capacity to affect cellular ATP in dark-grown Arabidopsis PSB-D cell cultures when used at 30 μM together with ES9 (10 μM) and the inactive analog ES9-6 (50 μM). As expected and in contrast to the sharp ATP decrease after treatment with 10 μM ES9 (ref. 9), neither 30 μM of ES9-17 nor 50 μM ES9-6 depleted ATP (Fig. 3d). All small molecules did not interfere with increasing fluorescein diacetate (FDA) fluorescence over time, indicating that they had no cytotoxic properties (Supplementary Fig. 5a). Previously, the protonophore activity of ES9 was shown to be unspecific to the mitochondria, because ES9 acidified the cytoplasm probably through dissipation of the proton gradients over the PM 9 . Therefore, we assessed whether ES9-17 affected the cytoplasmic pH. Arabidopsis seedlings were preincubated with Lyso Tracker Red DND 99 (ref. 20) to label membranes delineating acidic compartments, followed by a 30-min incubation with DMSO, 10 μM ES9 or 30 μM ES9-17 (Fig. 3e). As anticipated, 10 μM of ES9 relocated Lyso Tracker Red DND 99 predominantly to the cytosol when compared to the DMSO control, whereas 30 μM of ES9-17 failed to induce a similar staining pattern. Furthermore, unlike ES9, application of ES9-17 (30 μM for 30 min) did not compromise the motility of the actin cytoskeleton (ABD2 of Fimbrin-GFP) 21 , microtubules (GFP-MAP4) 22 , the Golgi (ST-mRFP) 23 and the trans-Golgi network (TGN)/early endosome (EE) compartments (VHA-a1-GFP) 24 (Supplemental Fig. 5b–e).</p><!><p>To characterize the potential of ES9-17 as a CME inhibitor, we evaluated the internalization of several PM-localized cargos subjected to CME in Arabidopsis and in human HeLa cells. We assessed whether CME-mediated uptake of the brassinosteroid (BR) receptor 25 BR INSENSITIVE1 (BRI1) would be affected by ES9-17. Arabidopsis seedlings expressing GFP-tagged BRI1 (BRI1::BRI1-GFP) (ref. 26) were pretreated for 1 h with 50 μM cycloheximide (CHX) to reduce the amount of newly synthesized proteins, followed by treatments with DMSO or ES9-17 (30 μM) for 30 min and then with 50 μM Brefeldin A (BFA) and FM4-64 for 30 min. In the presence of ES9-17, BRI1-GFP as well as FM4-64 failed to label the BFA bodies (Fig. 4a), hinting at an inhibition of uptake from the PM. The lack of BRI1-GFP-labeled BFA bodies was not due to a limitation of BFA body formation, because BFA bodies marked by the TGN/EE marker VHAa1-GFP 24 were formed (Supplementary Fig. 6a). Furthermore, fluorescently labeled Alexa fluor 674 castasterone (AFCS), which binds to BRI1 and consequently enters the cell through CME 25 , failed to stain the vacuole in root epidermal cells when applied to Arabidopsis seedlings expressing BRI1-GFP 26 after pretreatment with 30 μM ES9-17 for 30 min (Supplementary Fig. 6b). To corroborate the CME inhibition by ES9-17, we examined another PM-localized cargo, which is subjected to CME, the leucine-rich repeat receptor kinase PEP RECEPTOR1 (PEPR1) 27 . Treatment of Arabidopsis root cells expressing RPS5A::PEPRI-GFP with 100 nM of the Pep1 peptide for 10 s after pretreatment with DMSO for 30 min induced PEPR1-GFP internalization (Fig. 4b). However, when roots were pretreated with 30 μM ES9-17 for 30 min and elicited with 1 μM Pep1, PEPR1-GFP was still predominantly localized to the PM even 90 min after the elicitation, implying an inhibitory effect of ES9-17 on the CME-mediated PEPR1 uptake (Fig. 4b).</p><p>To link the reduction of cargo internalization caused by ES9-17 to its possible effects on PM recruitment of the CME machinery we evaluated the dynamic behavior of clathrin, CLC1-GFP 9 and CHC1-GFP 9 , and one representative of the CME adaptor proteins in plants, the TPLATE muniscin-like (TML) subunit of the TPLATE complex (TPC) 28 in root epidermal cells of Arabidopsis after ES9-17 application. In the presence of DMSO, the majority of the PM-localized foci had an average residence life time of 31.98 sec, 44.62 s and 36.72 s for CLC1-GFP, CHC1-GFP and TML-GFP, respectively (Fig. 4c; Supplementary Fig. 6c, d). When seedlings were treated with 30 μM ES9-17, the residence life time of CLC1, CHC1 and TML in the PM increased substantially with an average of 73.99 s, 83.55 s and 68.66 s, respectively.</p><p>Since ES9 was found to bind both Arabidopsis and human CHC, we assessed if ES9-17 could inhibit transferrin uptake in HeLa cells, a well-known clathrin-mediated process 29 . Treatment of HeLa cells with 30 μM ES9-17 for 30 min appeared to reduce the uptake of transferrin, but not to an extent as observed with 20 μM Pitstop2 (Supplementary Fig. 7a). In parallel with the inhibitory treatments, a cell proliferation assay ascertained that the resulting inhibition of the transferrin uptake was not due to the cytotoxicity of the compounds (Supplementary Fig. 7b). In an effort to compare ES9-17 with Pitstop2 we sought to assess Pitstop2 activity in Arabidopsis but failed to detect CME inhibition. Pitstop2 did not block FM4-64 uptake when applied at 30 μM for 30 min (Supplementary Fig. 7c), and similarly failed to inhibit the internalization of the CME cargo, BRI1-GFP in the presence of BFA (Supplementary Fig. 7d). Higher concentrations of Pitstop2 appeared to affect overall FM4-64 fluorescence, but did not result in uptake inhibition (Supplementary Fig. 7e). Moreover, the CETSA thermal denaturation curves for CHC and ATPβ in the presence of either DMSO (Ø) or 250 μM Pitstop2 were very similar. The Tagg with DMSO (Ø) and Pitstop2 for CHC was 44.57±0.29°C and 44.5±0.34°C (mean±SEM), respectively and 55.03±1.41°C and 54.63±1.05°C (mean±SEM) for ATPβ, respectively (Supplementary Fig. 7f). Taken together, ES9-17 affected dynamic behavior of core components of CME, and the uptake of different cargoes. In addition, ES9-17 affected transferrin uptake in HeLa cells, similar to Pitstop2, while Pitstop2 proved to be inactive in Arabidopsis with respect to CME inhibition.</p><!><p>As ES9-17 inhibited several clathrin-dependent processes and the parent molecule ES9 was found to bind CHC, we hypothesized that CHC might also be the target of ES9-17. To test this hypothesis, we made use of the target validation approaches CETSA and DARTS (Fig. 5a). ITDRFCETSA analysis at 46°C indicated an EC50 of 123±1.13 μM (mean±SEM) for ES9-17 (Supplementary Fig. 8a), which is very close to the EC50 observed for ES9. Similarly, as observed for ES9, a shift in Tagg for CHC was detected in the presence of ES9-17. Arabidopsis cell culture lysates treated with DMSO (Ø) for 30 min and heated for 2 min at 12 different temperature points (30-65°C) resulted in a Tagg for CHC of 44.96±0.2°C (mean±SEM), whereas in the presence of 250 μM ES9-17 the Tagg was 42.71±0.2°C (mean±SEM), thus generating a Tagg shift of 2.2°C (Fig. 5a). In contrast, ES9-17 failed to induce a shift in Tagg for the control proteins, tubulin and ATPβ (Fig. 5b; Supplementary Fig. 8b). These results were supported by the DARTS assay, in which cell lysates were treated with ES9-17 (250 μM) at particular dilutions of pronase (Fig. 5c). At pronase concentrations of 1:6000 and 1:4000, a 2-fold and 1.5-fold stabilization was observed for CHC in ES9-17-treated cell lysates, respectively, of which only the 1/6000 dilution was found to be statistically significant (one-way ANOVA with Dunett's multiple comparisons test), whereas the control proteins, ATPβ (Fig. 5c) and selected TPC 28 and adaptor protein complex-2 (AP-2) subunits 30 , were not stabilized (Supplementary Fig. 8c–e). To further strengthen the specificity of ES9-17, we assessed its possible interaction with the coatomer subunit γ (AT4G34450, γ-COP), since this protein ranked second in the ES9 affinity purification (Supplementary Data Set 1). The DARTS assay with ES9-17 failed to demonstrate significant protection of the γ-COP from protease digestion (Supplementary Fig. 8f), concluding that ES9-17 does not bind γ-COP. Previously we observed that ES9 affects Golgi cisternae morphology and induces the formation of Golgi-endoplasmic reticulum (ER) hybrid compartments 9 indicative of a non-functional coat protein I (COPI) trafficking. In contrast, transmission electron microscopy (TEM) analysis of Arabidopsis Col-0 root tips treated with ES9-17 (30 μM) did not reveal striking changes in Golgi and ER morphology (Supplementary Fig. 8g), though occasionally ES9-17 appeared to induce larger multivesicular bodies (Supplementary Fig. 8g, white triangle). Altogether our results showed that ES9-17 did not affect the COPI function in Arabidopsis.</p><p>To confirm the results obtained with ES9-17 in CETSA and DARTS assays, we assessed genetically whether ES9-17 interacts with CHC. In contrast to ES9, of which the protonophore activity prevents meaningful genetic studies, ES9-17 lacks the protonophore characteristics and, hence, can be used to evaluate the increased sensitivity or resistance of mutant alleles. Double knockout mutant lines in CHC cannot be used, because CHC has an essential function. Therefore, several single mutant alleles for CHC1 and CHC2 (ref. 16) were examined for their higher sensitivity to ES9-17 than that of the wild type. Primary root growth for 5-day-old Arabidopsis (accession Columbia-0) seedlings was inhibited with an EC50 of 9 μM (Supplementary Fig. 8h). To assure a sufficient root growth inhibition, we set the concentration of ES9-17 at 12 μM. Seedlings of chc1-4 (Supplementary Fig. 8i), chc2-1 and chc2-2 (ref. 16) mutant lines displayed a significantly increased sensitivity to ES9-17 compared to the Col-0 seedlings (one-way ANOVA with Dunett's multiple comparisons test) (Fig. 5d). Root growth did not significantly vary for the different mutant alleles when compared to Col-0, when grown under control conditions (Supplementary Fig. 8j) (one-way ANOVA with Dunett's multiple comparisons test). Taken together and similar to the results obtained with ES9, the results obtained with CETSA, DARTS and CHC mutant alleles point toward CHC as a protein target of ES9-17 in Arabidopsis.</p><!><p>In plants, CME is one of the most studied pathways for internalization of membrane-associated and soluble cargos from the PM and the extracellular environment 1 . As loss-of-function, CME mutants are often lethal or have no phenotypes because of gene redundancy 16 , methods to perturb CME largely rely on the use of inducible expression of mutant forms of critical proteins involved in endocytosis, such as clathrin and auxillin 31,32 , and siRNA-mediated depletion of adaptor proteins 28 . The drawbacks of such approaches are the low gene induction efficiency, the construct silencing and the considerable time needed to deplete existing complexes of these proteins in a cell (2-5 days), while the cell may adapt and even alter its gene expression, without certitude that only CME is impacted. Prolonged loss of a protein, such as clathrin, might impair post-Golgi trafficking 33 , with possible defects in secretion and vacuolar targeting as a consequence. Therefore, application of small molecule inhibitors of CME combined with live-cell imaging can greatly facilitate studies of the CME machinery and dynamics. An attractive feature of chemical inhibitors is that they can be applied acutely to reveal the direct block of a particular process and that their effect is reversible 34 .</p><p>Over the years, different small molecule effectors of endocytosis and endosomal function in Arabidopsis have been described, including Secdin 15 and several compounds from the endosidin (ES) series 35–38 . The identification and characterization of the CME inhibitor ES9 (ref. 9) revealed that the mode of action of this compound is mediated via its mitochondrial uncoupling and protonophore activities, therefore causing ATP depletion and cytoplasmic acidification. This mode of action appeared to be shared with the until then, most commonly used plant CME inhibitor, TyrA23. Therefore, in contrast to mammalian systems, in which the small CME inhibitors Pistop2 and Dynasore are well established 5,6 , plants lack small molecules that specifically target the CME machinery. While Dynasore has been used as a CME inhibitor in plants 39 , reports on the activity of Pistop2 in plant cells are lacking. Our results revealed that Pitstop2 is inactive as a CME inhibitor in Arabidopsis. This dissonance in activity behavior can be rationalized using structural considerations based on a key amino acid difference between Arabidopsis CHC1/CHC2 and human CHC1, and in light of the structurally characterized binding pocket of Pitstop2 in human CHC1. The hallmark of Pitstop2 binding by human CHC1 centers on the snug accommodation of its naphthalene ring by an evenly distributed cushion of β-branched residues (Ile52, Ile66, Ile80, Ile93 and Val50) 5 . However, Arabidopsis CHC1 and CHC2 display a Leu at position 80 (Supplementary Fig. 3b), which would be expected to introduce an irreconcilable steric hindrance that would prevent Pitstop2 binding.</p><p>Even though ES9 acts as a protonophore, evidence suggested that ES9 may have CME inhibiting effects beyond those resulting from cytoplasmic acidification 9 , prompting an affinity-based target identification strategy that identified the Arabidopsis CHC1 as a possible ES9-binding protein. Interestingly, both CHC1 and CHC2 equally interacted with ES9 in subsequent in vitro target validation strategies, such as CETSA, DARTS, and DSF, despite the fact that only CHC1 was found as a meaningful ES9 interactor and CHC2 was detected in the background lists (Supplementary Data Set 1). The interaction with CHC was further confirmed by the crystal structure of the human nTD of CHC1 with ES9, revealing that ES9 binds the same clathrin box as the Pitstop2 molecule 5 , positioned between blades 1 and 2 of the nTD β-propeller.</p><p>Regardless of the binding of ES9 to CHC, its usefulness as a specific CME inhibitor is compromised by its pronounced protonophore characteristics. Here we have described ES9-17 as an improved analog of ES9, lacking the nitro group, that was capable of inhibiting CME without protonophore activity. Docking predictions and the crystal structure of the nTD of human CHC1 in complex with ES9 suggested that the nitro group establishes an electrostatic interaction with Arg64. As a consequence, ES9-17 might bind the nTD of CHC in a different than ES9 orientation and affinity (Supplementary Fig. 3a). Nevertheless, ES9-17 inhibited the uptake of FM4-64 and of several CME cargos in Arabidopsis, validating ES9-17 as an inhibitor of endocytosis. Moreover, and similar to the previously reported CME inhibitors from the Pitstop family 5 , ES9-17 increased the dwell time of the endocytic foci at the PM, in contrast to the general freeze of CME dynamics observed with ES9 (ref. 9). In addition, ES9-17 did not acidify the cytoplasm and, thus, did not hamper endomembrane compartment motility and cytoplasmic streaming, as visualized by the TGN/EE, Golgi and cytoskeleton dynamics and the overall Golgi, TGN/EE and BFA body morphology.</p><p>The target validation strategy for ES9-17 indicated binding to CHC in CETSA and DARTS assays similar to what we describe for ES9. Both the temperature shift in the CHC denaturation and the EC50 concentration for ES9-17 and ES9 were essentially the same, as was the protection from protease digestion in the DARTS assay, strongly suggesting that ES9-17 also targets CHC. Currently we can only speculate why we observed a sizable difference in EC50 values for FM4-64 inhibition and in vitro validation strategies such as CETSA for both ES9 and ES9-17. Possibly, accessory proteins present in a biological context might increase affinity, but equally, in vitro conditions might increase the apparent EC50 values. Furthermore, ES9 appeared to be able to inhibit FM4-64 uptake with a higher potency (EC50 of 5.16 μM) 9 than that of ES9-17 (EC50 of 13 μM) likely due to being both a protonophore and a CHC inhibitor.</p><p>Notably, Golgi morphology in presence of ES9-17 appeared the same as in the control, unlike ES9, which induces substantial morphological changes in the Golgi 9 . These morphological changes are in part the result of the protonophore characteristics but are possibly also the result of targeting the γ-COP subunit of the coatomer complex, which ranked second after CHC in the ES9 affinity purification list. The lack of γ-COP protection from protease digestion in presence of ES9-17, as observed with the DARTS assay, further highlight the specificity of ES9-17 in terms of CME inhibition. Together with the genetic data, our results make for compelling evidence that ES9-17 is a specific CME inhibitor in Arabidopsis.</p><p>With ES9-17, several new opportunities arise in the quest to understand clathrin-mediated trafficking. For example, ES9 and ES9-17 offer a chemical scaffold to further improve on and identify more potent inhibitors of CHC function. In doing so we would further increase our understanding of the molecular aspects of CHC function in Arabidopsis. As we established that ES9 binds the same pocket as Pitstop2, it is reasonable to assume that the mode of ES9 inhibition would be similar to that of Pitstop2, that is, interfering with the recruitment of accessory proteins harboring a clathrin box motif 5 . Furthermore, although the core function of CHC in CME is known, we have little understanding of the specific roles of CHC1 and CHC2 in Arabidopsis. Rendering either CHC1 or CHC2 insensitive to ES9-17, yet still biologically functional, might help to deconvolve their function. The ability to selectively inhibit CHC1 or CHC2 function might help to highlight their different functions in the endomembrane system, or at the tissue and developmental level.</p><p>In summary, with ES9-17 we have presented for the first time a small molecule inhibitor of CME in Arabidopsis. While its activity is not limited to Arabidopsis, other inhibitors of CME, such as Pitstop2, lack activity in Arabidopsis, making ES9-17 to our knowledge the only small molecule probe for CME in Arabidopsis, which in addition is chemically different from the Pitstop2 family. It therefore allows to dynamically and reversibly inhibit CME pharmacologically, and together with ES9 offers a scaffold and platform for the further rational development of additional ES9 analogs. Such analogs might possess an increased affinity toward CHC or allow the distinction between CHC1 and CHC2, and serve as a platform to allow detailed dissection of the CHC function in Arabidopsis and other systems, both in a CME context and beyond.</p><!><p>Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper.</p><!><p> Arabidopsis thaliana (L.) Heyhn. (accession Columbia-0 [Col-0]) seedlings and other lines were stratified for 2 days at 4°C and grown vertically on agar plates containing half-strength Murashige and Skoog (½MS) medium supplemented with 1% (w/v) sucrose for 5 days at 22°C in a 16-h/8-h light/dark cycle, prior to use. The Arabidopsis mutants and transgenic lines used were: chc2-1 (ref. 16), chc2-2 (ref. 16), TML::TML-GFP/tml-1 (ref. 28), CLC1::CLC1-GFP/Col-0 (ref. 9), RPS5A::CHC1-GFP/Col-0 (ref. 9), VHA-a1::VHA-a1-GFP/Col-0 (ref. 24), 35S::ST-mRFP/Col-0 (ref. 23), RPS5A::PEPR1-GFP/pepr1pepr2 (ref. 27), BRI1::BRI1-GFP/Col-0 (ref. 26) 35S::GFP-MAP4/Ler 22 and 35S::Fimbrin-GFP/Col-0 (ref. 21). The T-DNA insertion line chc1-4 (SALK_110063) was obtained from the Nottingham Arabidopsis Stock Centre. The T-DAN insertion of chc1-4 was confirmed by PCR by using left primer flanking SALK insertion (5'-CAAGTGACGCATCACAACATG-3'), right primer (5'-ACCATTGCTCAAAACATACGC-3') and T-DNA specific primer, LBA1 (5'-TGGTTCACGTAGTGGGCCATC-3') (Supplementary Fig. 8i).</p><!><p>Entry clones pDONRRP4-1R-RPS5A 9 , pDONRRP2R-P3-HAstop and pDONR211-AP2S (AT1G47830) or pDONR211-AP2M 30 (AT5G46630) were used together with pH7m34GW in multisite Gateway reactions (Life Technologies) to generate the RPS5A::AP2S-HA and RPS5A::AP2M-HA constructs. The AP2M-HA and AP2S-HA expression vectors were used to transform Agrobacterium tumefaciens C58. The PSB-D wild type cell cultures were subsequently transformed as described 14 .</p><!><p>ES9 and analogs were acquired through Chembridge (http://www.chembridge.com/) or synthesized. They were dissolved in DMSO (Sigma-Aldrich) and 50°mM stocks were stored at -20°C in glass vials. ES9-17 was freshly prepared from lyophilized powder (stored at -20°C) prior to use. Brefeldin A, cycloheximide and Pitstop2 (Sigma-Aldrich) were dissolved in DMSO. Endocytosis was visualized with 2 μM FM4-64 (Life technologies). Washout experiments involved a 30 min pretreatment of seedlings in liquid ½ MS medium with ES9-17 (30 μM), followed by an additional 30 min treatment in presence of FM4-64 (2 μM). Subsequent substitution of treatment medium with medium without treatment, but with FM4-64, constituted the washout. Seedlings were imaged at indicated time points. The experiment was performed twice independently. Repeat 1 was washed with regular ½ MS and FM4-64 (2 μM), while repeat 2 was washed with ½ MS plus DMSO and FM4-64 (2 μM). Staining and imaging of mitochondria and acidic compartments in the seedlings treated with the small molecules was performed as described previously 9 . To observe the internalization of AFCS, seedlings expressing BRI1-GFP were treated with ES9-17 for 30 min followed by 20 μM of AFCS for 20 min. Seedlings were then quickly washed and mounted for microscopic observations for 20 min. To follow the PEPR1-GFP internalization, seedlings expressing PEPR1-GFP were treated with ES9-17 for 30 min and FM4-64 (2 μM) was added for further 15 min. Seedlings were then elicited with Pep1 100 nM and imaged at different time points. Imaging for AFCS uptake and PEPR1 internalization was performed with an SP8 confocal laser scanning microscope with 40×water immersion lens. Life-time of endocytic foci were measured in seedlings upon treatment with ES9-17/DMSO for 30 min as described previously 9 . Seedlings expressing VHA-a1-GFP and ST-mRFP were treated with or without drug for 30 min followed by time lapse images with 6 time points for 90 s, 10 s interval. Images were taken with Olympus FV10 ASW confocal laser scanning microscope with a 60× water immersion lens (NA 1.2) and 3× digital zoom.</p><p>The cytoskeletal dynamics of Arabidopsis root cells with and without drug treatments were performed with the Perkin Elmer Spinning disc using a 60x water corrected Plan Apo (NA 1.2) objective. Time lapse series were taken for 12 min, 5 time points per minute (MAP4) or 5 min, 1 time points per second (Fimbrin). Images were processed by means of the ImageJ (Fiji) software package. More specifically, for the MAP4 and Fimbrin superposed multi-color images, the background was subtracted using a rolling ball radius of 50 pixels and a walking average of 4 was subsequently applied to the time series. Colored projections were generated by superposing six different time points spread evenly over the duration of the acquisition. The six time points were merged using the merge channels tool of Fiji where the grey channel was left blank. All experiments described for Pitstop2 in Arabidopsis were performed as described for ES9-17. For the quantification of the cytosolic/PM signal intensity ratio, non-saturated images were converted in ImageJ to 8-bit and regions of interest (ROIs) were selected based on the PM or cytosol localization. Histograms listing all intensity values per ROI were generated and the averages of the 100 most intense pixels were used for calculations. Three cells were quantified per seedling and averaged.</p><!><p>ATP and FDA measurements in three-day-old wild type PSB-D Arabidopsis cell cultures were performed as described previously 9 .</p><!><p>PSB-D wild type cell cultures were harvested by separating them from the medium, flash frozen in liquid nitrogen and ground with a Retsch® MM400. Cell material was weighed and extraction buffer (EB) (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% NP-40 [Sigma-Aldrich] with 1 tablet/10 mL cOmplete ULTRA protease inhibitor cocktail, EDTA free [Roche]) was added in 2:1 ratio (200 μl extraction buffer for 100 mg material). The protein concentration was determined with the Bradford method (Quick start Bradford 1× Dye reagent [Bio-Rad]). Lysate was prepared by removing endogenous biotin, with Streptavidin Sepharose High Performance beads (GE Healthcare; hereafter referred to as beads). Washed beads (50 μl, and washed 3× with EB) were applied to 1-ml lysate and incubated at 4°C for 1 h on a rotary wheel. A second batch of beads was washed 3x with EB, and biotinylated small molecules (2 μl of 50 mM stock per 50 μl beads) were added with the last wash. The mixture was left at room temperature for 15 min, supernatant of the last wash removed, and transferred to 4°C until further use. Supernatant constituting the biotin cleared lysate was collected and added to beads incubated with the biotinylated small molecules. The subsequent mixture was incubated for a minimum of 2 h, or overnight at 4°C on a rotary wheel, followed by centrifugation whereafter the mixture was spun down (4°C), the supernatant removed, and beads were washed 3 times (50 mM Tris-HCl, pH 8, 150 mM NaCl). Appropriate amounts of the LDS sample buffer (4× LDS sample buffer supplemented with 10× sample reducing agent; Novex, Life Technologies) were added and samples were incubated for 10 min at 70°C, followed by a 2-min centrifugation at maximum speed (18 000 x g). The resulting supernatant was collected and run on 4-12% Bis-Tris protein gels with MES buffer (NuPage Novex gels and buffer from Life technologies). Gels were stained with SYPRO Ruby protein gel stain (Molecular probes, Invitrogen) and stained gel regions were excised and cut in approximately 3 pieces before submission to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.</p><!><p>Gel pieces containing the proteins of interest were cut from the gel and transferred to Biopure® Eppendorf tubes (Eppendorf AG, Hamburg, Germany). After two consecutive 15-min wash steps with water/acetonitrile (1/1, v/v) (both HPLC analytical; Mallinckrodt Baker B.V., Deventer, The Netherlands), the gel pieces were completely dried in a centrifugal vacuum concentrator, subsequently rehydrated in 10 μl of a 0.02 μg/μl of a sequencing-grade modified trypsin stock solution (Promega Corporation) and, completely submerged in freshly prepared 50 mM ammonium bicarbonate solution. The overnight digestion at 37°C was stopped by acidification with TFA. After centrifugation (16 000 x g for 5 min), the peptide mixture was removed from the gel pieces, transferred to a new Eppendorf tube, completely dried in a centrifugal vacuum concentrator and redissolved in 20 μl of 0.1% (w/v) TFA in water/acetonitrile (98/2, v/v) (loading solvent). 10 μl of the 20 μl obtained peptide mixtures mixtures were introduced into an LC-MS/MS system through an Ultimate 3,000 RSLC nano LC (Thermo Fisher Scientific) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific). The sample mixture was first loaded on an in-house made trapping column (100 μm internal diameter [I.D.] × 20 mm, 5 μm beads C18 Reprosil-HD; Dr. Maisch, Ammerbuch-Entringen, Germany). After flushing from the trapping column, the sample was loaded on an in-house made analytical column (75 μm I.D. × 150 mm, 5 μm beads C18 Reprosil-HD; Dr. Maisch) packed in the needle (PicoFrit SELF/P PicoTip emitter, PF360-75-15-N-5; New Objective, Woburn, MA, USA). Peptides were loaded with loading solvent (0.1% (v/v) TFA in water/acetonitrile, 2/98 (v/v)) and separated with a linear gradient from 98% solvent A' (0.1% (v/v) formic acid in water) to 40% solvent B′ (0.1% (v/v) formic acid in water/acetonitrile, 20/80 (v/v)) in 30 min at a flow rate of 300 nL/min, followed by a 5-min wash, reaching 99% solvent B'. Two packing and two analytical columns were configured in tandem LC mode. Switching between two flow paths, an analysis and a regeneration flow path, allows column washing and re-equilibration off-line; thus, while one column is re-equilibrated, the system injects a sample on the other column. The mass spectrometer was operated in data-dependent, positive ionization mode, automatically switching between MS and MS/MS acquisition for the 10 most abundant peaks in a given MS spectrum. The source voltage was 3.4 kV and the capillary temperature was 275°C. One MS1 scan (m/z 400-2,000, AGC target 3 × 106 ions, maximum ion injection time 80 ms) acquired at a resolution of 70,000 (at 200 m/z) was followed by up to 10 MS/MS scans (resolution 17,500 at 200 m/z) of the most intense ions fulfilling predefined selection criteria (AGC target 5 × 104 ions, maximum ion injection time 60 ms, isolation window 2 Da, fixed first mass 140 m/z, spectrum data type: centroid, underfill ratio 2%, intensity threshold 1.7×E4, exclusion of unassigned, 1, 5-8, >8 charged precursors, peptide match preferred, exclude isotopes on, dynamic exclusion time 20 sec). The HCD collision energy was set at 25% Normalized Collision Energy and the polydimethylcyclosiloxane background ion at 445.120025 Da was used for internal calibration (lock mass).</p><p>From the MS/MS data in each LC run, Mascot Generic Files (mgf) were created with the Mascot Distiller software (version 2.4.3.3; Matrix Science). These peak lists were then searched with the Mascot search engine and the Mascot Daemon interface (version 2.4, Matrix Science). Spectra were searched against the TAIR10 database. Variable modifications were set to pyro-glutamate formation of amino-terminal glutamine, acetylation of the protein N-terminus, methionine oxidation and propionamide cysteine formation. Mass tolerance on precursor ions was set to ± 10 ppm (with the Mascot C13 option set at 1) and on fragment ions to 20 mmu. The instrument setting was on ESI-QUAD. The enzyme was set to trypsin/P, allowing one missed cleavage, whereas cleavage was allowed also when lysine or arginine was followed by proline. Only peptides that were ranked first and scored above the threshold score, set at 99% confidence, were withheld. All data were managed by ms_lims 40 and analyzed with R (http://www.R-project.org) embedded in KNIME. The data were filtered by removing all peptides smaller than eight amino acids and only the proteins containing at least two peptides in one of the experiments were taken into account for data analysis.</p><!><p>The protocol is largely based on the previously published procedure 10 with minor adjustments. Wild type or transgenic Arabidopsis PSB-D cell cultures were harvested, flash frozen in liquid nitrogen and ground with a Retsch® MM400. Cell material was added at a ratio of 2:1 in extraction buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% NP-40 with 1 tablet/10 mL cOmplete ULTRA protease inhibitor cocktail, EDTA free (Roche)). Cell material was allowed to thaw on ice for 15 min with occasional mixing. Samples were centrifuged for 30 min (18 000 x g at 4°C. Lysates were pooled per cell type and the protein concentration was determined with the Bradford method (Quick start Bradford 1× Dye reagent (Bio-Rad)). Pooled lysates were treated with small molecules or mock (DMSO) and incubated for 30 min at room temperature on a rotary wheel. The treated lysates were aliquoted in 60-μl fractions in PCR tubes, treated for 2 min at 12 temperature points (30, 35, 40, 42, 44, 46, 48, 50, 52, 55, 60, and 65°C) in a Bio-Rad thermal cycler, allowed to cool down and centrifuged (18,000 x g) for 30 min at 4°C. The supernatant (50 μl) was taken and processed for standard Western blot analysis to detect the proteins. All antibodies were diluted in TBS-T with 5% (w/v) skimmed milk. Anti-CHC (1/3.000, AS10 690 Agrisera) anti-AtpB (1/4.000, AS05085 Agrisera) were detected with horseradish peroxidase (HRP)-linked anti-rabbit IgG (1/10.000, GE Healthcare), anti-tubulin (1/15.000) with HRP-linkked anti-mouse IgG (1/10.000, GE Healthcare), anti-TPLATE (1/1.000) and HA with anti-HA-HRP (1/4.000, Abcam). Blots were developed with Western Lightning® Plus–ECL, Enhanced Chemiluminescence Substrate (Perkin-Helmer) and imaged with a Bio-Rad ChemiDoc XRS+ molecular imager. Band intensity was measured with the Bio-Rad Image Lab software package.</p><!><p>The DARTS protocol was adopted as previously described 11,15 . Lysates were prepared as described for CETSA. Lysates treated with ES9-17 (250 μM) or mock (DMSO) were incubated for 30 min at room temperature. After incubation, lysates were split into equal aliquots for pronase (Roche, 10 165 921 001) digestion. Pronases were diluted from a 10 mg/ml stock solution in dH2O. The 1/100 starting dilution of pronase that was used for all the subsequent dilutions as indicated was obtained by dissolving 12.5 μl pronase stock solution in 87.5 μl 1× TNC buffer (500 mM Tris-HCl, pH 8, 500 mM NaCl, 100 mM CaCl2, 10× stock). All dilutions were prepared with 1× TNC buffer. Digestion (30 min) was started with 1-min intervals and stopped by addition of the sample buffer at a 1× final concentration (4× LDS sample buffer with 10× sample reducing agent; Novex, Life Technologies) in the same sequence as the digestion had been started. Western blotting, protein detection and quantification were as described for CETSA. The anti-γ-COP (anti-Sec21p) (1/1.000, AS08327, Agrisera) was detected with anti-rabbit IgG, HRP-linked antibody (1/10.000, GE Healthcare).</p><!><p>Water and all ligands of the human clathrin nTD crystal structure pdb-entry 4G55 in complex with Pitstop2 (ref. 5) were manually deleted from the pdb-text file. The emptied structure was subjected to a local minimization with the GROMOS96 (43B1 parameter set) 41 implementation within the Swiss-PdbViewer 42 , and polar hydrogens were added. The ES9 ligand was drawn three-dimensionally with Avogadro 1.1.1 (ref. 43) and was minimized with the built-in MMFF94s force field 44 . The AutoDockTools 1.5.4 suite 45 was used for pdbqt-format preparations of proteins and ligands. Dockings were done with AutoDock-Vina 1.1.0 (ref. 46) with exhaustiveness set at 64; residues Arg64, Phe91 and Gln89 were set as flexible. The grid-box size was x = 20, y = 22 and z = 20 Å, centered at x = 50.0, y = -10.2 and z = 24.5. PyMOL (Molecular Graphics System, Version 1.7 Schrödinger, LLC) was used for visualization.</p><!><p> Arabidopsis nTD CHC1 (residues 1-377), CHC2 (residues 1-378) and human nTD CHC (residues 1-363) were cloned into the pGEX4T-1 vector and transformed into the competent Escherichia coli BL21 (DE3) cells. Transformed cells were cultured in Luria-Bertani medium supplemented with carbenicillin (100 μg/ml)at 37°C until an O.D. of 0.6 was reached and expression was induced by the addition of 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG). Cell cultures were kept for another 4 h at 37°C. Next, cells were harvested by centrifugation at 6000 x g,, resuspended in lysis buffer (10 mM Tris-HCl, pH 8.3, 500 mM NaCl, 10% (w/v) glycerol and 5 mM DTT) supplemented with protease inhibitors (Roche) and lysed by sonication. The proteins were purified by affinity purification using a GSTrap FF 1-ml column. The column was first equilibrated with equilibration buffer (10 mM sodium phosphate buffer, 150 mM NaCl, pH 7.5) and the sample applied to the GSTrap column. The column was subsequently washed with equilibration buffer and the bound protein was eluted with the elution buffer (10 mM NaH2PO4, 150 mM NaCl, 10 mM reduced glutathione, pH 7.5) and collected in 1-ml aliquots. The GST tag was cleaved by overnight thrombin digest at 20° C, after which the sample was again applied to a GSTrap column to remove the cleaved GST as well as uncleaved GST-CHC. The flow through was collected and further polished by size exclusion chromatography with a SD75 16/600 column (GE Healthcare) equilibrated on HBS (20mM HEPES pH 7.4 150mM NaCl). Protein concentration (A280) was measured with a Nanodrop 1000 (Thermo Scientific) and aliquots at a concentration of 2 mg/ml were stored at –80°C.</p><!><p>Purification and crystallization of nTD were carried out as previously described 5 . X-ray data were collected at beamline BL14.2 at BESSY-II (Berlin, Germany) and processed in XDS and Xscale 47 . Data collection statistics are shown in Supplementary Table 3. The phase problem was solved by molecular replacement using phaser 48 using the 1.7 Å structure of nTD (PDB ID:4G55) as a model. All water molecules and ligand atoms were omitted from the starting model. Subsequent cycles of refinement to 1.6 Å resolution were performed in PHENIX 49 . Structure file of ES9 was generated using the Dundee PRODRG2 server 50 and manually fitted to the electron density. All structural figures were produced with PyMOL (Molecular Graphics System, Version 1.7 Schrödinger, LLC). The data were deposited in the PDB under ID: xxxx.</p><!><p>For the DSF assay, the Light cycler 480, Real-time PCR system (Roche) was used as described previously 49 . The purified nTD of human CHC1 and Arabidopsis CHC1/2 was diluted in a buffer containing 20 mM Hepes, pH 7.4, 150 mM NaCl. Each well of a 96-well microplate contained a concentration of 1.6 μM protein, 5x Sypro Orange (Invitrogen), 2.5 μl of compound and buffer up to a total volume of 25 μl. Thermal scanning 10 to 95°C at 1.5°C/min) was done with a real-time PCR setup and the fluorescence intensity was measured every 10 sec. The software Light cycler 480Sw 1.5.1 was utilized for calculating melting temperature.</p><!><p>Maintenance and imaging of HeLa cells for transferrin uptake was performed as described previously 9 .</p><!><p>For the WST-1 assay, HeLa cells were grown in a 96-well plate and were incubated with the compounds for 30 min followed by addition of 10 μl WST-1 reagent (Sigma-Aldrich) to 100 μl of medium in a 96-well plate. Absorbance was measured at 450 nm versus a 690 nm reference by means of a plate reader.</p><!><p>Seeds were sown on ½ MS solid medium, stratified for 2 days at 4 °C in the dark, and placed vertically in the light. At 5 days after germination, seedlings were transferred to solid ½ MS medium without sucrose supplemented with 100 mM D-sorbitol with ES9-17 or DMSO and incubated for another 2 days, after which the plates were scanned and root growth was measured. For measurements, scanned images were processed and evaluated with ImageJ. Fold change in root growth was measured as a ratio of root length on 2 days after treatment with ES9-17/DMSO to the root length at the start of the treatment.</p><!><p>Five-days-old Arabidopsis thaliana Col-0 seedlings, grown on solid ½ MS medium, were treated by immersing them in liquid ½ MS supplemented with DMSO and 30 μM ES9 17 for 30 min. Root tips were subsequently excised, immersed in 20% (w/v) BSA and frozen immediately in a high-pressure freezer (Leica EM ICE; Leica Microsystems, Vienna, Austria). Freeze substitution was carried out using a Leica EM AFS (Leica Microsystems) in dry acetone containing 1% (w/v) OsO4 and 0.5% glutaraldehyde over a 4-days period as follows: -90°C for 54 h, 2°C per hour increase for 15 h, -60°C for 8 h, 2°C per hour increase for 15 h, and -30°C for 8 h. Samples were then slowly warmed up to 4°C, rinsed 3 times with acetone for 20 min each time and infiltrated stepwise over 3 days at 4°C in Spurr's resin and embedded in capsules. The polymerization was performed at 70°C for 16 h. Ultrathin sections were made using an ultra-microtome (Leica EM UC6) and post-stained in in a Leica EM AC20 for 40 min in uranyl acetate at 20°C and for 10 min in lead stain at 20°C. Sections were collected on formvar-coated copper slot grids. Grids were viewed with a JEM 1400plus transmission electron microscope (JEOL, Tokyo, Japan) operating at 60 kV.</p><!><p>All statistical tests and graphs other than boxplots were done and generated with Graphpad Prism 6. Dose-response curves with a log-transformed x-axis were generated using nonlinear regression with a log(inhibitor) vs. response model, and setting a variable slope (four parameters) and top and bottom constraints to 1 and 0 respectively. Other dose-response and CETSA curves were generated with a Boltzmann sigmoid equation with top and bottom constraints set to 1 and 0 respectively, when applicable. Boxplots were generated with the online tool BoxPlotR (http://boxplot.tyerslab.com/) from the Tyers and Rappsilber laboratories.</p><!><p>Further information on experimental design and reagents is available in the Life Sciences Reporting Summary.</p><!><p>Any supplementary information, chemical compound information and source data are available in the online version of the paper.</p>

PubMed Author Manuscript

Cs₂CO₃-Promoted reaction of tertiary bromopropargylic alcohols and phenols in DMF: a novel approach to α-phenoxyketones

The reaction of bromopropargylic alcohols with phenols in the presence of Cs 2 CO 3 /DMF affords α-phenoxy-α'-hydroxyketones (1:1 adducts) and α,α-diphenoxyketones (1:2 adducts) in up to 92% and 24% yields, respectively. Both products are formed via ring opening of the same intermediates, 1,3-dioxolan-2-ones, generated in situ from bromopropargylic alcohols and Cs 2 CO 3 .

cs₂co₃-promoted_reaction_of_tertiary_bromopropargylic_alcohols_and_phenols_in_

Introduction<!>Entry<!>Results and Discussion<!>Conclusion<!>Experimental<!>ORCID ® iDs

<p>Due to the relative stability, ease of handling and the presence of reactive sites, bromoacetylenes are widely applied in synthetic organic chemistry. They are known to be involved in various transformations including homo-and cross-coupling [1][2][3][4][5][6][7], addition [1,8,9], cycloaddition [1,10,11] and other reactions. Of particular synthetic value is the addition to the triple bond of bromoacetylenes to provide vinyl adducts, which can undergo numerous transformations. For example, bromoacetylenes were demonstrated to add imidazoles, imidazolines [12], and benzimidazoles [13,14] to give vinyl bromides. Sulfonamides reacted with bromoacetylenes to deliver N-bromovinyl-p-toluenesulfonamides that under Heck reaction conditions afforded N-(p-toluenesulfonyl)pyrroles [15]. The CsF-promoted nucleophilic addi-tion of isocyanides to bromoacetylenes furnished the functionalized bromovinyl amides followed by Pd-catalyzed formation of 5-iminopyrrolone [16]. Sequential nucleophilic addition/intramolecular cyclization of amidine with bromoacetylenes led to imidazoles [17]. Also, M 2 CO 3 -catalyzed (M = K or Cs) addition of phenols to bromoacetylenes produced bromovinyl phenyl ethers, which were converted into 4H-chromen-4-ones, benzo[b]furans, etc. [18][19][20][21]. The latter reaction attracted our attention and prompted us to explore the interaction of phenols and bromopropargylic alcohols under the reported conditions. The bromopropargylic alcohols are readily available from acetylenic alcohols and hypobromite [22] or N-bromosuccinimide [23]. The presence of the hydroxy group expands the synthetic</p><!><p>Alkali metal carbonate (equiv) potential of these bromoacetylenes. Thus, we have recently demonstrated a highly selective hydration/acylation of tertiary bromopropargylic alcohols with carboxylic acids promoted by alkali metal carbonates [24]. The reaction proceeds via the ringopening of 1,3-dioxolan-2-one intermediates formed with hydroxy and alkynyl groups of bromopropargylic alcohol and alkali metal carbonate. In the light of the above, it was unclear, in which direction would proceed the reaction of bromopropargylic alcohols and phenols. In the present paper, we report on the results of these studies.</p><!><p>Initially, bromopropargylic alcohol 1a and phenol (2a) were chosen as the model substrates for our investigation (Table 1). Completion of the reaction was monitored by IR and 1 H NMR spectroscopy by the disappearance of the bands at 2196-2212 cm -1 (-C≡C-Br) and signals of the bromopropargylic alcohol 1a, respectively. Under the conditions previously used [18][19][20][21] for the addition of phenols to bromoacetylenes (K 2 CO 3 or Cs 2 CO 3 , DMF, 110 °C), the reaction turned out to be non-selective: along with the expected bromovinyl phenyl ether 3a (3-9%) and phenoxyhydroxyketone 4a (25-39%), diphenoxyketone 5a was isolated in 9-24% yield (Table 1, entries 1-3). At 50-55 °C, the reaction slowed down and became more selective (Table 1, entries 4 and 5). With Cs 2 CO 3 (1 equiv) at 50-55 °C, the reaction proceeded for 3 h, the yield of the phenoxyhydroxyketone 4a increased up to 55% and 5-phenoxymethylene-1,3-dioxolan-2-one 7, one of the probable intermediates, was isolated in 5% preparative yield (Table 1, entry 4), whereas the use of 2 equiv of Cs 2 CO 3 led to slightly more selective reaction (Table 1, entry 5). Further lowering the temperature reduces the selectivity toward phenoxyketone 4a. At room temperature, the full conversion of bromopropargylic alcohol 1a took 15 h and yields of phenoxyketones 4a and 5a decreased (Table 1, entry 7). In the presence of K 2 CO 3 (1 equiv) at 50-55 °C, the same reaction was completed for 8 h, the yields and selectivity being not improved (Table 1, entry 10). In these cases, 5-phenoxymethylene-1,3-dioxolan-2-one 7 was also isolated in 6-9% preparative yield. Hydrocarbonates CsHCO 3 and KHCO 3 were also tested in the reaction, which gave 5-bromomethylene-1,3-dioxolan-2-one 6a as a major product in 29-36% yield ( 1, entries [16][17][18]. The efforts to increase the yield of diphenoxyketone 5a using 2 equivalents of phenol (2a) in the reaction with bromopropargylic alcohol 1a (Table 1, entries 8 and 9) failed.</p><p>Employing the reaction conditions similar to those given in entries 4 and 6 (Table 1), we examined the substrate scope of the process relative to other phenols (Scheme 1). It was found that the electronic character of the substituents and the steric hindrance affected the reaction outcome. Bromopropargylic alcohol 1c having a tert-butyl group reacted with phenol (2a) in DMF for 3 h to give phenoxyhydroxyketone 4l in only 34% yield, 5-bromomethylene-1,3-dioxolan-2one 6b (5%) being isolated (Scheme 3). In DMF/H 2 O (3 h), the conversion of 1c was incomplete (50%) and phenoxyhydroxyketone 4l was obtained in 39% yield. So, the steric hindrances of the bulky groups noticeably affect the reaction.</p><p>The reaction of secondary and primary bromopropargylic alcohols (4-bromobut-3-yn-2-ol and 3-bromoprop-2-yn-1-ol) and phenol (2a) with 1 equiv of Cs 2 CO 3 , DMF, 50-55 °C, for 3 h did not gave any products, the competitive polymerization of bromopropargylic alcohols 1 being predominant.</p><p>Finally, chloroacetylenic alcohol was involved in the reaction with phenol (2a, 1 equiv Cs 2 CO 3 , DMF, 50-55 °C, 3 h) to afford the corresponding product 4a in 29% isolated yield (Scheme 4).</p><p>We tested aniline and 2-naphthylamine as nucleophiles (DMF, 50-55 °C) in the reaction of bromopropargylic alcohol 1a (Scheme 5). But such a protocol turned out to be ineffective providing no desired products.</p><p>Several control experiments were performed to gain insight into the reaction mechanism (Scheme 6). When the reaction of 5-bromomethylene-1,3-dioxolan-2-one 6a and phenol (2a) was carried out with KOH, the conversion of the starting 6a was 55% and crude product contained phenoxyketone 4a, diphenoxyketone 5a and 5-phenoxymethylene-1,3-dioxolan-2-one 7.</p><p>Using 2 equivalents of phenol (2a) in the reaction of 5-bromomethylene-1,3-dioxolan-2-one 6a (Cs 2 CO 3 , DMF, 110 °C, 20 min) gave phenoxyketone 4a and diphenoxyketone 5a in 40 and 16% yields, correspondingly. These results confirm that compound 6a is the main intermediate to form phenoxyketones. Next, we carried out the experiment using CO 2 gas with DBU as a base. In comparison with reactions without CO 2 (Table 1, entries 17 and 18), bromopropargylic alcohol 1a with free CO 2 gas in the presence of 100 mol % of DBU and phenol (2a) (DMF, 50-55 °C, 3 h) afforded phenoxyketone 4a, 5-bromomethylene-1,3-dioxolan-2-one 6a and 5-phenoxymethylene-1,3-dioxolan-2-one 7 in 27, 4 and 19% yields, respectively. This result suggest that Cs 2 CO 3 acts as a source of CO 2 for the formation of 5-bromomethylene-1,3dioxolan-2-one 6a. Obviously, the formation of phenoxyhydroxyketone 4 proceeds via 1,3-dioxolan-2-one 6 generated from bromopropargylic alcohol 1 and Cs 2 CO 3 . Then, Br-substitution/hydration of 6 and the release of CO 2 give product 4 (Scheme 7).</p><p>Apparently, diphenoxyketone 5 results from decarboxylative conversion of 1,3-dioxolan-2-one 7 leading to intermediate A, nucleophilic attack of phenolate at the less sterically hindered carbon of the above zwitterion A and subsequent protonation of anion B (Scheme 8).</p><p>Based on these plausible mechanisms for the formation of phenoxyketones, it can be assumed that a decline of the Cs + concentration after Cs 2 CO 3 convertion to CsBr (because of the very poor solubility of CsBr in DMF) has an influence on the rate of diphenoxyketone formation. In addition, the suppression of the di(nitrophenoxy)ketone formation can be due to the lower basicity of a reaction mixture since nitrophenols 2d,e are more acidic than phenols 2a-c,f-i (pK a values: 9.99 [25,26] phenol (2a), 9.40 [27] α-naphthol (2b), 9.57 [27] β-naphthol (2c), 7.18 [25,26] p-nitrophenol (2d), 7.23 [25,26] o-nitrophenol (2e), 10.28 [25,26] p-cresol (2f), 10.27 [25,26] p-methoxyphenol (2g), 9.36 [25,26] p-bromophenol (2h), 10.19 eugenol (2i)). Addition of water to the reaction mixture also reduces the pH of the medium and simultaneously increases the concentration of hydroxide ions, therefore, diphenoxyketones 5 were not produced and dihydroxyketones 8 were formed as side products in these cases.</p><p>Among the approaches to produce α-phenoxyketones, the most common methodologies are base-catalyzed alkylation of the corresponding phenols with halo- [28][29][30] and mesyl [31][32][33] ketones (Scheme 9), the preparation of which are not always selective and high-yielded. The ring opening of ArOCH 2 -epox-Scheme 10: α-Ketol rearrangement of phenoxyketones 4a and 4f.</p><p>ides [34,35], the SmI 2 -catalyzed reductive coupling of acid halides with ketones [36,37] and acetolyses of α-phenoxy-αdiazoketones [38] were also employed.</p><p>Recently, F. P. Cossío et al. [39] have described a method for the preparation of benzo</p><!><p>We have shown that the main direction of the reaction of bromopropargylic alcohols and phenols in Cs 2 CO 3 /DMF is the hydration/phenoxylation of bromopropargylic alcohols to afford phenoxyketones. This step-economical process takes place under mild reaction conditions using simple readily available starting materials. The synthesized phenoxyketones are of interest as valuable building blocks for the production of other important molecules (e.g., amino alcohols, diols, etc.) [40][41][42][43][44][45][46] and potential pharmaceuticals. α-Hydroxyketones are structural subunits of natural products [47][48][49] and compounds possessing immunosuppressant [50], antidepressant [51], amyloid-β protein production inhibitory [52], urease inhibitory [53], farnesyl transferase inhibitory (kurasoin A and B) [54,55], antitumor and antibacterial (doxorubicin, olivomycin A, chromomycin A 3 , carminomycin I, epothilones) [56][57][58] activities.</p><!><p>General information</p><!><p>Olesya A. Shemyakina -https://orcid.org/0000-0001-7371-3982</p>

Beilstein

Assessment of Coproduction of Ethanol and Methane from Pennisetum purpureum: Effects of Pretreatment, Process Performance, and Mass Balance

To overcome the structural complexity and improve the bioconversion efficiency of Pennisetum purpureum into bioethanol or/and biomethane, the effects of ensiling pretreatment, NaOH pretreatment, and their combination on digestion performance and mass flow were comparatively investigated. The coproduction of bioethanol and biomethane showed that 65.2 g of ethanol and 102.6 g of methane could be obtained from 1 kg of untreated Pennisetum purpureum, and pretreatment had significant impacts on the production; however, there is no significant difference between the results of NaOH pretreatment and ensiling-NaOH pretreatment in terms of production improvement. Among them, 1 kg of ensiling-NaOH treated Pennisetum purpureum could yield 269.4 g of ethanol and 144.5 g of methane, with a respective increase of 313.2% and 40.8% compared to that from the untreated sample; this corresponded to the final energy production of 14.5 MJ, with the energy conversion efficiency of 46.8%. In addition, for the ensiling-NaOH treated Pennisetum purpureum, the energy recovery from coproduction (process III) was 98.9% higher than that from enzymatic hydrolysis and fermentation only (process I) and 53.6% higher than that from anaerobic digestion only (process II). This indicated that coproduction of bioethanol and biomethane from Pennisetum purpureum after ensiling and NaOH pretreatment is an effective method to improve its conversion efficiency and energy output.

assessment_of_coproduction_of_ethanol_and_methane_from_pennisetum_purpureum:_effects_of_pretreatment

Introduction<!>Raw Material<!><!>Raw Material<!>Experimental Design and Process<!><!>Pretreatment<!>Enzymatic Hydrolysis and Ethanol Fermentation<!>Anaerobic Digestion<!>Analytical Methods<!>Analysis of Mass and Energy Flow<!>Data Calculation and Statistical Methods<!>Enzymatic Hydrolysis and Fermentation of Pennisetum purpureum under Process I<!><!>Enzymatic Hydrolysis and Fermentation of Pennisetum purpureum under Process I<!><!>Anaerobic Digestion Performance of Pennisetum purpureum under Process II<!><!>Anaerobic Digestion Performance of Pennisetum purpureum under Process II<!>Anaerobic Digestion Performance of Stillage Samples of Pennisetum purpureum under Process III<!>Variation in the Characteristics of Samples during the Process<!><!>Variation in the Characteristics of Samples during the Process<!><!>Material Flow under Different Production Processes<!><!>Material Flow under Different Production Processes<!><!>Material Flow under Different Production Processes<!><!>Energy Flow under Different Production Processes<!><!>Conclusions<!><!>Author Contributions<!>

<p>Bioenergy and biofuels production originating from biomass has drawn increasing attention due to its advantages of protecting the environment and relieving the urgent demand for fossil energy.1 Lignocellulosic biomass, including hardwood, softwood, and herbaceous plants, with an annual yield of up to 200 billion tons globally, offers an inexpensive and abundant resource for biofuels production.2,3 However, the inherent complex structure of lignocellulosic biomass, including high cellulose crystallinity, carbohydrates-lignin complexity, and high lignin content, resists the attack on carbohydrates by microorganisms or enzymes during biological conversion, resulting in low conversion efficiency and thus a low biofuel production.4,5 In order to promote the bioconversion efficiency, pretreatment prior to the biological conversion process is considered to efficiently destroy the complex structure, and various pretreatment methods have been developed.6</p><p>Pretreatment methods are usually classified into physical, chemical, and biological categories and their combination.7,8 Among them, physical pretreatment includes mechanical pulverization, ultrasound, and radiation; chemical pretreatment includes acid/alkaline pretreatment,9,10 oxidation pretreatment, and ionic liquid pretreatment;11 while biological pretreatment mainly consists of enzyme pretreatment and microbial/fungi pretreatment.12 Each has its own merits and drawbacks. Among biological pretreatment methods, ensiling pretreatment has received wide research interests due to its advantages in low external energy demand, preserving the nutrients of raw material, and providing a long-term stable supply of material.13 The main effect of ensiling pretreatment is that hemicellulose is hydrolyzed to monosaccharides such as glucose and xylose, which are then converted to short-chain organic acids such as lactate and acetate through anaerobic microbial fermentation; as such the recalcitrance of lignin-carbohydrate complexity is reduced, increasing the accessibility of cellulose to microorganisms and enzymes.14,15 Moreover, intermediates produced from ensiling pretreatment such as lactic acid can be easily converted into gaseous biofuel (such as biogas). Zhao et al. reported that, compared with raw switchgrass, biomethane production from the anaerobic digestion (AD) of ensiling-treated switchgrass was improved by 33.6%.16 It was also reported that ensiling pretreatment increased the ethanol yield from sugar beet pulp by nearly 50%.17 Except for ensiling pretreatment, other biological pretreatments have also been proven to improve the fermentation performance of biomass. For example, the biogas production from microalgae increased by more than 21% after enzymatic pretreatment;18 Wyman et al. reported that the biogas production from corn stover pretreated with white-rot fungi increased by 19%.19 Chemical pretreatment methods refer to the use of chemicals (such as alkaline, acids, and ionic liquid) during the process, which have been widely accepted to increase the methane yield in the AD process;20 among these methods, alkaline pretreatment, with NaOH as the most employed and effective chemical reagent,21 has drawn extensive attention with following advantages: (1) it can be carried out under mild conditions (atmospheric pressure under 100 °C);3,7 (2) the hydroxide ion (OH–) can remove most lignin by breaking the ester bonds between xylan and lignin, and partial hemicellulose by weakening the hydrogen bond between cellulose and hemicellulose,22 which facilitates the accessibility of cellulose to microorganisms and enzymes. Kang et al. investigated the effect of NaOH pretreatment on the digestion performance of Pennisetum Hybrid, reporting that the methane yield increased from 249.3 mL/g volatile solid (VS) to 301.7 mL/g VS under the condition of 35 °C for 24 h.23 Kataria et al. reported that the ethanol yield from NaOH treated Kans grass under the condition of 120 °C for 120 min increased to 0.38 g/g substrate.24 To the best of our knowledge, although both ensiling and NaOH pretreatment are popular methods employed for improving bioenergy and biofuel production from raw lignocellulosic material, the effect of integrated ensiling-NaOH pretreatment on biofuel production has not been studied yet. Integrated ensiling pretreatment and NaOH pretreatment can not only ensure the long-term supply of raw materials but also remove lignin and improve biofuel production efficiency.</p><p>Pennisetum purpureum, a kind of herbaceous plants, has characteristics of high biomass production, strong adaptability to the environment, and high cellulose content, making it a potential candidate for biofuel production.25 Previous studies have proved that Pennisetum purpureum is a promising feedstock for ethanol production, with a yield of 4.3 mg/mL.26 Stillage, the residues after ethanol fermentation, contains high nondegraded carbohydrates content and other organic matters, which has a high chemical oxygen demand (COD) load and is considered to have a considerable pollution potential, especially for industrial-scale bioethanol plant;27 it is reported that the amount of stillage produced is about 10 times that of bioethanol production.28 Thus, treating stillage with the well-established AD technique can not only reduce pollution but also produce renewable biogas, so as to make full advantage of pentose and other organic matters that cannot be utilized in ethanol fermentation.29 Kaparaju et al. found that the methane yield of 324 mL/g VS was obtained when the wheat straw stillage was used as raw material with a concentration of 12.8 g VS/L.30 Alkan-Ozkaynak et al. used corn stillage as raw material for AD and found that the biogas yield was 763 mL/g VS. These suggest that stillage is a potential substrate for AD.31 Moreover, Liu et al. found that the energy conversion efficiency of sugarcane bagasse after sequential bioethanol and biogas production increased up to 59.5%; this was only 35.7% for ethanol production and 23.8% for biogas production, respectively.32 Several studies comparing the energy output of pretreated feedstocks in ethanol production alone, methane production alone, and ethanol–methane coproduction are presented in Table 1; it can be concluded that the energy produced by the coproduction process is significantly higher than that of the single production process. However, the energy output of feedstocks after ensiling pretreatment or NaOH pretreatment in these three production processes is yet analyzed in detail. Herein, it is plausible to speculate that the coproduction of ethanol and biogas/biomethane from ensiling, NaOH and combined pretreated Pennisetum purpureum would greatly improve its conversion efficiency; as such it would make this feedstock more feasible for biofuel production on an industrial scale, which necessitates further study to verify this hypothesis. In addition, the introduction of material flow analysis (MFA) into a biofuel production process could provide some useful and insightful information regarding the material utilization efficiency and digestion efficiency, making it possible to concisely control the process.33 Previous studies focused on the mass and energy flow of ethanol–methane coproduction, however, with the carbon and nitrogen flow yet not investigated.</p><p>Therefore, the objectives of this study were to (1) investigate the effects of ensiling pretreatment, NaOH pretreatment, and combined pretreatment on the physicochemical structure and ethanol-methane coproduction conversion efficiency of Pennisetum purpureum; (2) analyze and compare the material and energy flow of Pennisetum purpureum under different processes, and (3) calculate and compare the energy conversion efficiency of Pennisetum purpureum to optimize the feasible pretreatment and biological conversion process.</p><!><p>Pennisetum purpureum, were harvested from Zengcheng district, Guangzhou City (China), in September 2019, with a plant height of 2–3 m. The harvested samples were chopped to 1–2 cm by hay cutter and smashed with a pulverizer, and then a part of the samples was frozen at −20 °C before use. The other part was vacuumed and sealed in plastic silo bags and ensiled at ambient temperature for 90 days. The composition of all samples is listed in Table 2.</p><!><p>Total solid (TS) is calculated based on wet weight; others are based on the dry weight.</p><!><p>NaOH was purchased from Macklin Bio-Chem Technology Co. Ltd. The commercial cellulase with an activity of 144 FPU/g powder was purchased from Imperial Jade Biotechnology Co. Ltd. (Ningxia, China), which was extracted from the fermentation broth of Penicillium sp.. Saccharomyces cerevisiae Y2034 used for ethanol production was obtained from the National Center for Agricultural Utilization Research (U.S.A.),39 which can only utilize hexose with an ethanol fermentation efficiency of 85–90%.</p><!><p>To compare the effects of ensiling pretreatment, NaOH pretreatment, and their combination on the bioconversion efficiency of Pennisetum purpureum, three processes were designed in this study, which was illustrated in Figure 1. Process I was performed to produce ethanol via enzymatic hydrolysis and fermentation, and process II was performed to produce biogas through AD from untreated and pretreated Pennisetum purpureum, respectively. Process III integrated AD of stillage (including both solid and liquid fractions) from Process I after ethanol fermentation to achieve coproduction of ethanol and biogas.</p><!><p>Simplified flowchart for three processes (AD: anaerobic digestion).</p><!><p>Ensiling pretreatment was conducted in a vacuumed and sealed plastic silo bag, and then stored at ambient temperature for 90 days.</p><p>According to a previous study where the ethanol production from NaOH pretreated sugarcane bagasse increased to nearly 20 g/L, the alkaline pretreatment condition was chosen to be 2% NaOH (w/v), a solid-to-liquid ratio of 1:20 (w/v) based on total solids (TS), at 80 °C for 2 h with 150 rpm.40 Sequential ensiling and NaOH pretreatment were set up under the same condition, with the ensiled samples underwent the NaOH pretreatment. After pretreatment, the samples were centrifuged for solid–liquid separation. The solid fraction was washed with distilled water to a neutral pH and then stored at −20 °C for the enzymatic hydrolysis and fermentation. The liquid fraction was stored to determine the concentration of total carbon (TC) and total nitrogen (TN).</p><!><p>Before fermentation, enzymatic hydrolysis of the pretreated samples was performed to achieve the conversion of polysaccharides into monosaccharides. The sterilized pretreated samples were added into the aseptic 0.05 M acetate buffer (pH 4.8) at a solid concentration of 10%; the buffer contains 5 g/L yeast extract, 5 g/L peptone, 5 g/L KH2PO4, 0.2 g/L (NH4)2SO4, and 0.4 g/L MgSO4·7H2O. The enzymatic hydrolysis experiment was carried out at the cellulase loading of 20 FPU/g cellulose of substrate and shaken at the condition of 50 °C and 150 rpm for 72 h. The hydrolysates (including both solid and liquid fractions) after enzymatic hydrolysis were inoculated with the activated yeast strain which was cultivated at 30 °C and 150 rpm for 16 h in a medium containing 20 g/L peptone, 10 g/L yeast extract, and 20 g/L glucose; the reaction system was then placed in a shaker incubator at 30 °C and 150 rpm for 72 h. For high-performance liquid chromatography (HPLC) analysis, samples were taken at intervals of 12 h during enzymatic hydrolysis and 24 h during ethanol fermentation.</p><p>All fermentation slurries were distilled with a rotary vacuum evaporator at 70 °C and −0.1 MPa for 7 min to separate the ethanol from the fermented liquid.</p><!><p>The anaerobic digestion experiments of untreated or pretreated samples (process II) and stillage (process III) were performed at an automatic methane potential test system (Bioprocess Control Sweden AB, AMPTS II).41 The untreated or pretreated samples that performed anaerobic digestion directly were denoted AD. For example, AD of samples after ensiling pretreatment, NaOH pretreatment, and ensiling-NaOH pretreatment are coded AD-ensiling, AD-NaOH, and AD-ensiling-NaOH, respectively. While the stillage (including both solid and liquid fractions) from the process I performed AD was denoted ensiling, NaOH, and ensiling-NaOH after ensiling, NaOH, and ensiling-NaOH pretreatment. The inocula were added according to the VS content of the solid part after distillation the untreated/pretreated sample, and the ratio of samples to inoculum was 1:2 based on VS. The inocula-only (400 mL) reactors were used as controls. The actual methane yield was calculated by eq 1. All reactors were flushed with nitrogen gas for 5 min to guarantee an anaerobic condition. The temperature was controlled at 37 ± 1 °C. Each condition was set in triplicate. The entire period of this experiment was 30 days.1where Yactual is the specific methane yield (mL/g VS) from samples, Ysample is the sample measured methane yield (mL/g VS), Ycontrol is the methane production of the inocula-only (mL/g VS), Vsample is the volume of inocula added in the sample reactor (mL), and 400 is the volume of inocula added in the control reactor. The inocula for biogas production were granule sludge which were cultivated with cellulose and peptone in the laboratory before use; the pH value, TS content and VS content of the inocula were 7.5, 1.7 ± 0.1%, and 1.5 ± 0.0%, respectively.</p><!><p>The content of TS, VS, C, and N in the solid fraction from different processes was determined according to the previously described method.42 The samples were dried at 105 °C to a constant weight and then placed in a muffle furnace and incinerated at 550 °C for 2 h to calculate TS and VS. C and N content in the solid fraction were analyzed by Vario EL cube (elementar, Germany). A calorimeter (IKA C2000, Germany) was used to measure the calorific value (CV) of the samples.43 The TC and TN concentrations of the liquid fraction after NaOH pretreatment, ethanol fermentation, and AD were analyzed by Vario TOC (elementar, Germany) at 850 °C with an oxygen flow rate of 230 mL/min. The carbohydrates (glucan and xylan) and lignin contents of all samples were analyzed in accordance with the description of National Renewable Energy Laboratory (NREL).44 The monomeric sugars and ethanol concentration were tested by the HPLC system (Waters e2698, U.S.A.) equipped with SH1011 (Shodex) at 50 °C with 5 mM H2SO4 as the mobile phase at a flow rate of 0.5 mL/min. The surface morphologies of untreated and pretreated samples were imaged by scanning electron microscopy (SEM, Hitachi S4800) at an accelerating voltage of 2.0 kV.45</p><!><p>The flow of mass, element (C and N), and energy was systematically evaluated by MFA according to the study by Brunner and Rechberger.46 A software for substance flow analysis (STAN 2.6.801) was used to establish the MFA system model, and the data were processed and optimized by IAL-IMPL2013 algorithm to achieve material balance. The results of MFA were presented by graphics according to the study by Niu et al.43</p><!><p>Glucan recovery and lignin removal were calculated as per eqs 2 and 3, respectively, as follows:23where m1 and m2 are the mass of the sample before and after pretreatment (g), G1 and G2 are the percentages of glucan in the sample before and after pretreatment (%), and L1 and L2 are the percentages of lignin in the sample before and after pretreatment (%).</p><p>Enzymatic hydrolysis yield and ethanol yield were respectively calculated as follows:45where Cglucose is the concentration of glucose during enzymatic hydrolysis (mg/mL), Cethanol is the concentration of ethanol during fermentation (mg/mL), glucan content is the mass of glucan in samples (mg), V is the volume of fermentation liquid (mL), 0.9 is the dehydration coefficient of glucose converted to glucan, 0.51 is the theoretical coefficient for converting glucose into ethanol, and 1.11 is the theoretical coefficient of conversion of glucan to glucose.47</p><p>A software of SPSS 19.0 was applied to analyze the statistical difference of the data, and a one-way analysis of variance was used. The variance level was 0.05%.</p><!><p>The concentration of monomeric sugars and ethanol during the respective enzymatic hydrolysis and fermentation process (process I) are shown in Figure 2. During the enzymatic hydrolysis, it was observed that the concentrations of glucose, xylose, cellobiose, and arabinose varied for all samples. With the time prolonged, the concentration of glucose increased significantly, while the xylose concentration was almost unchanged. For the untreated sample, the glucose concentration yielded at 16.7 g/L after 72 h enzymatic hydrolysis (Figure 2A). For ensiling pretreated sample, the glucose concentration was 16.9 g/L at the end of enzymatic hydrolysis (Figure 2B), with an insignificant increase of 1.2% when compared with that from the untreated sample (p > 0.05). For the NaOH pretreated sample, the glucose concentration was 52.8 g/L (Figure 2C), which increased by 216.7% compared to that from the untreated sample (p < 0.05). For the ensiling-NaOH pretreated sample, the glucose concentration was 53.7 g/L after 72 h enzymatic hydrolysis (Figure 2D), resulting in an increase of 222.0% compared to that from the untreated sample (p < 0.05), while the xylose and arabinose concentrations were almost constant throughout the enzymatic hydrolysis.</p><!><p>Monomeric sugars and ethanol concentration of untreated and pretreated Pennisetum purpureum. A: untreated sample; B: ensiling treated sample; C: NaOH treated sample; D: ensiling-NaOH treated sample.</p><!><p>After the enzymatic hydrolysis experiments (72 h), the hydrolysates (including solid and liquid fraction) were added into a fermentation reactor with activated yeast strain for ethanol fermentation. As also shown in Figure 2, the ethanol concentration dramatically increased after 24 h incubation. After 24 h incubation, the ethanol concentration was 5.4 g/L for the untreated sample (Figure 2A), 5.5 g/L for the ensiling treated sample (Figure 2B), 22.8 g/L for the NaOH treated sample (Figure 2C), and 25.4 g/L for the ensiling-NaOH treated sample (Figure 2D); these accounted for 84.2–94.4% of the total final ethanol concentration, which was 6.4 g/L for the untreated sample, 6.6 g/L for the ensiling treated sample, 24.8 g/L for the NaOH treated sample, and 26.9 g/L for the ensiling-NaOH treated sample, respectively. Meanwhile, the glucose concentration sharply decreased to 0.12–0.62 g/L, while the xylose concentration remained unchanged. These results agreed with Wang et al.,48 who reported that the yeast strain can timely ferment glucose into ethanol but cannot assimilate xylose. In addition, the ethanol concentration from the ensiling-NaOH treated sample was increased by 318.8% compared to that from the untreated sample, 310.9% compared to that from the ensiling treated sample, and 8.6% compared to that from the NaOH treated sample (p < 0.05). It was worth mentioning that the ethanol yield from the NaOH treated sample and the ensiling-NaOH treated sample was three times that from the ensiling treated sample, indicating that NaOH pretreatment and sequential ensiling and NaOH pretreatment of Pennisetum purpureum both are effective pretreatment methods to increase the ethanol yield. Meanwhile, it also suggested that ensiling-NaOH pretreatment is a feasible method that can not only realize the continuous supply of raw material but also improve the fermentation performance. This can be attributed to the removal of lignin after NaOH pretreatment, as the lignin removal was 57.2% for ensiling treated samples, and the relative glucan content was up to 62.1% (Table 3). In addition, the changes in the surface structure after pretreatment can also explain the increased efficiency of enzymatic hydrolysis and ethanol yield. The untreated and ensiling treated sample exhibited a smooth and orderly surface structure, but after NaOH pretreatment, the surface became rough and shaggy (Supporting Information), which increases the accessibility of cellulose. The ethanol concentration produced in this study are comparable with elephant grass and bagasse, with a respective yields of 26.125 and 15.0 g/L.49</p><!><p>Letters indicate that the values in the same column are significantly different (p < 0.05).</p><!><p>The daily and cumulative methane yield of untreated and pretreated Pennisetum purpureum (process II) are shown in Figure 3. For untreated samples (AD-untreated), the highest daily methane yield was 46.8 mL/g VS/d (Figure 3A) on day 2; this increased to 60.1 mL/g VS/d after ensiling pretreatment (AD-ensiling). For NaOH treated samples (AD-NaOH), the daily methane yield peaked at 95.4 mL/g VS/d on day 2, with an increase of 104.1% compared to that of untreated samples (p < 0.05). After ensiling-NaOH pretreatment (AD-ensiling-NaOH), the highest daily methane yield was 89.0 mL/g VS/d on day 2, which was increased by 90.3%, when compared to that from the untreated sample (p < 0.05). As shown in Table 4 and Figure 3B, the specific methane yield of the untreated sample was 204.6 mL/g VS; this increased to 208.9 mL/g VS after ensiling pretreatment (p > 0.05). After NaOH pretreatment, the specific methane yield was 274.5 mL/g VS, corresponding to an increase of 34.1% compared to that of the untreated sample (p < 0.05). The specific methane yield of ensiling-NaOH treated samples was 266.1 mL/g VS, which increased by 30.1% when compared with that of the untreated sample (p < 0.05). These results agreed with Costa et al.,50 who investigated the effect of NaOH pretreatment on the digestion performance of sugar cane bagasse and reported that the methane production from NaOH treated samples increased to 313.4 mL/g dry substrates.</p><!><p>Letters a–g indicated that the values in the same column are significantly different (p < 0.05).</p><p>Daily methane yield (A) and specific methane yield (B) of samples.</p><!><p>It was noticed that the specific methane from the NaOH treated sample was significantly higher than that from the untreated and ensiling treated sample (p < 0.01). This might be explained by the different lignin content in untreated and pretreated samples (Table 3). Herrmann et al. reported that higher lignin content has a negative impact on specific methane yield from lignocellulosic biomass such as Miscanthus and ryegrass, due to its cross-linkage with homocellulose, which reduces the biodegradability of homocellulose.51 Similar results have been reported by Sambusiti et al.,52 who found that the specific methane yield of NaOH treated sorghum increased by 18.80% with a lignin reduction of 63%. Under the conditions of 4 g NaOH/100 g TS and 55 °C for 24 h, the methane yield of sunflower stalk varieties increased by 29–44%, and the lignin removal was 23.3–36.3%.53 Kang et al. also found that the lignin removal was 58.9% when the Pennisetum Hybrid was pretreated with 2% NaOH at 35 °C for 24 h, and the methane yield was increased by 21.0% under this condition.23</p><!><p>The daily and cumulative methane yield from the AD of stillage after ethanol fermentation (process III) are also shown in Figure 3. For untreated samples (untreated), the highest daily methane yield was 69.5 mL/g VS/d (Figure 3A) on day 1; this was 69.9 mL/g VS/d after ensiling pretreatment (ensiling). For NaOH treated samples (NaOH), the daily methane yield peaked at 236.5 mL/g VS/d on day 1, with an increase of 240.2% compared to that from the untreated sample. After ensiling-NaOH pretreatment (ensiling-NaOH), the highest daily methane yield was 198.8 mL/g VS/d on day 1, which increased by 186.1% when compared to that from the untreated sample. As shown in Table 4 and Figure 3B, the cumulative methane yield of the untreated sample was 246.9 mL/g VS; this increased to 251.7 mL/g VS after ensiling pretreatment (p > 0.05). After NaOH pretreatment, the specific methane yield was 398.6 mL/g VS, corresponding to an increase of 61.4% compared to that of the untreated sample (p < 0.05). The specific methane yield of the ensiling-NaOH treated sample was 454.8 mL/g VS, which increased by 84.2% when compared with that of the untreated sample (p < 0.05). In addition, compared with the corresponding sample directly perform AD under process II, the methane yield of stillage samples under process III was respectively increased by 20.7% for the untreated sample, 20.5% for the ensiling treated sample, 45.2% for the NaOH treated sample, and 70.9% for the ensiling-NaOH treated sample. These results were similar to the literature in which the methane yield of stillage was 13.4–34.0% higher than that of pretreated barley straw.54 The enhanced maximum daily methane yield and cumulative methane yield could be explained by that the stillage contains more soluble pentose such as xylose and other degradable compounds, which can be easily converted to methane during the AD process.55 A previous study reported that utilization of xylose for biogas production could outpace its use for ethanol in terms of energy recovery; as such the AD of stillage achieved the highest energy recovery from acetic acid-pretreated corn stover after ethanol fermentation.56 Therefore, to better understand the mass balance and energy conversion efficiency of the substrate under different processes, it necessitates the comparisons in terms of mass flow including C and N content and energy recovery in this study.</p><!><p>The variation in the contents of TS, VS, C, N, TC, TN, and CV of Pennisetum purpureum during the coproduction process is shown in Table 5. Each step during the coproduction process has been analyzed. For the solid part of Pennisetum purpureum, the carbon contents were 44.7% for the untreated sample and 43.9% for ensiling treated sample, and after NaOH pretreatment, the values respectively decreased to 42.7% and 42.9% (p < 0.05). For the untreated sample, the carbon content in the solid part decreased to 41.0% after enzymatic hydrolysis and ethanol fermentation and further decreased to 38.2% after AD. For the ensiling treated sample, the carbon content in the solid part decreased to 40.5% after enzymatic hydrolysis and ethanol fermentation and further decreased to 38.6% after AD (p < 0.05). The carbon content in the solid part decreased to 39.5% for NaOH treated sample and 37.9% for ensiling-NaOH treated sample after enzymatic hydrolysis and ethanol fermentation, and further decreased to 37.6% and 35.4% after AD (p < 0.05), respectively. The nitrogen content in the solid part of Pennisetum purpureum was 0.7% for the untreated sample and 0.8% for ensiling treated sample and completely removed after NaOH pretreatment (p < 0.05), indicating that the NaOH pretreatment used in this study had an obvious effect on nitrogen removal. A significant increase of nitrogen content in the solid part was observed after enzymatic hydrolysis and ethanol production (p < 0.05); the nitrogen content increased to 2.1% for the untreated sample and 2.0% for the ensiling treated sample. Similarly, the values also increased to 2.3% for NaOH treated sample and 2.2% for ensiling-NaOH treated sample, respectively; this could be attributed to (1) the addition of cellulase, inoculum, and seed medium and (2) the protein was dissolved under alkaline condition and then precipitated in enzymatic hydrolysis and ethanol fermentation process (acidic condition). After AD, the nitrogen content in the solid part decreased to 2.0% for the untreated sample, 2.0% for the ensiling treated sample, 2.2% for the NaOH treated sample, and 2.1% for the ensiling-NaOH treated sample, respectively. The decreased nitrogen content could be attributed to the growth of methanogens during AD process which needs nitrogen, and consequently, the protein was degraded into free ammonia remaining in the liquid part.57,58</p><!><p>TS: total solid; VS: volatile solid; CV: calorific value; TC: total carbon; TN: total nitrogen. Letters a–d indicated that the values in the same column are significantly different (p < 0.05).</p><!><p>The TC concentration in the liquid fraction was 16 957.2 mg/L for the untreated sample and 10 645.6 mg/L for the ensiling treated sample after enzymatic hydrolysis (p < 0.05) and then increased to 17 145.3 and 11 056.6 mg/L after ethanol fermentation, respectively. After AD, the TC concentration significantly decreased to 1177.8 mg/L for the untreated sample and 851.7 mg/L for ensiling treated sample (p < 0.05). The TC concentration in the liquid fraction was 7240.0 mg/L for the NaOH treated sample and 5428.1 mg/L for the ensiling-NaOH treated sample and then increased to 30 989.9 and 33 125.6 mg/L after enzymatic hydrolysis and ethanol fermentation (p < 0.05). After AD, the TC concentration decreased to 1536.8 mg/L for NaOH treated sample and 1381.4 mg/L for ensiling-NaOH treated sample (p < 0.05). The TN concentration in the liquid part of Pennisetum purpureum was 1778.3 mg/L for the untreated sample and 1524.8 mg/L for ensiling treated sample after enzymatic hydrolysis and ethanol fermentation and then decreased to 915.0 and 829.7 mg/L after AD (p < 0.05), respectively. The TN concentration in the liquid fraction was 404.9 mg/L for NaOH treated sample and 471.8 mg/L for the ensiling-NaOH treated sample (p < 0.05) and then respectively increased to 1527.9 and 1678.1 mg/L after enzymatic hydrolysis. During the fermentation process, the TN concentration decreased from 1696.1 mg/L after ethanol fermentation to 1123.6 mg/L after AD for NaOH treated sample and from 2040.0 to 1043.3 mg/L for ensiling-NaOH treated sample (p < 0.05).</p><p>As shown in Figure 4A and Table 3, the contents of glucan, xylan, and lignin in the untreated sample were 36.9%, 18.4%, and 10.9%. For the NaOH treated sample, the solid loss was 54.3%, of which the lignin removal was 63.5%. For the ensiling-NaOH treated sample, the solid loss was 42.0%, of which the lignin removal was 57.2%. This suggested that the mass loss in this stage is mainly due to the removal of lignin, which is consistent with the result reported by Scholl et al.59 During the enzymatic hydrolysis process, for ensiling treated sample, the glucan content sharply decreased from 37.7% to 18.1% (Figure 4B); for the NaOH treated sample, the glucan content dropped from 27.2% to 13.2%, and the glucan recovery was 73.6% (Figure 4C); for the ensiling-NaOH treated sample, the glucan content decreased from 36.0% to 16.7%, with a glucan recovery of 95.4% (Figure 4D). The significant decrease in glucan content (p < 0.05) could be explained by the hydrolysis of glucan into glucose (Figure 2). It should be pointed out that the lignin content was almost the same during enzymatic hydrolysis, ethanol fermentation, and the AD process (p > 0.05); this is because that lignin is poorly degradable in those processes.60</p><!><p>Compositions and lignin removal of Pennisetum purpureum. A: untreated sample; B: ensiling treated sample; C: NaOH treated sample; D: ensiling-NaOH treated sample.</p><!><p>Combining the content of TS, VS, C, N, TC and TN during the three processes, the mass flow including C and N flow could be achieved (Figures 5–7).</p><!><p>Mass (A, D, and G), carbon (B, E, and H), and nitrogen (C, F, and I) flow of untreated, NaOH treated, and ensiling-NaOH treated Pennisetum purpureum in ethanol production (process I). A–C represent the untreated sample; D–F represent the NaOH treated sample; G–I represent the ensiling-NaOH treated sample.</p><!><p>For process I, the flow of mass, carbon and nitrogen element are shown in Figure 5. After NaOH pretreatment (Figure 5D,G), the mass recovery was 45.7% for the untreated sample and 58.0% for ensiling treated sample; this further respectively decreased to 24.9% and 30.0% after enzymatic hydrolysis and ethanol fermentation. Considering the overall reaction system volume, it was observed that 65.2 g ethanol could be produced from 1 kg dry untreated sample, which increased to 248.1 g ethanol after NaOH pretreatment and 269.4 g ethanol after ensiling-NaOH pretreatment. For the carbon flow (Figure 5E,H), after NaOH pretreatment, 43.6% of carbon in the untreated sample and 56.7% of carbon in the ensiling treated sample remained in the solid residue, with 30.8% and 24.7% of carbon flowing into the liquid fraction, respectively; 13.2% of carbon in the untreated sample (accounting for 30.3% of the residue after NaOH pretreatment) and 18.6% of carbon in the ensiling treated sample (accounting for 32.8% of the residue after NaOH pretreatment) flowed into ethanol after enzymatic hydrolysis and fermentation. For the nitrogen flow (Figure 5F,I), all of the nitrogen in the untreated sample and ensiling treated sample flowed into the liquid fraction after NaOH pretreatment. Subsequently, all of the nitrogen flowed into the enzymatic hydrolysis process were from cellulase and seed medium.</p><p>The mass, carbon and nitrogen flow of process II are shown in Figure 6. After NaOH pretreatment (Figure 6D,G), the mass recovery was 45.7% for the untreated sample and 58.0% for the ensiling treated sample; this further respectively decreased to 28.1% and 34.6% after AD. Considering the overall reaction system volume, it was observed that 140.0 g of methane could be produced from 1 kg of dry untreated sample, which increased to 194.3 g methane after NaOH pretreatment and 189.1 g methane after ensiling-NaOH pretreatment. For the carbon flow (Figure 6E,H), after NaOH pretreatment, 14.9% of carbon in the untreated sample (accounting for 34.1% of the residue after NaOH pretreatment) and 18.7% of carbon in the ensiling treated sample (accounting for 33.1% of the residue after NaOH pretreatment) flowed into methane after AD. For the nitrogen flow (Figure 6F,I), all of the nitrogen in the untreated sample and ensiling treated sample flowed into the liquid fraction after NaOH pretreatment. Subsequently, all of the nitrogen flowing into the AD process was from the inoculum; after methane production process, 53.7% and 51.9% of the nitrogen in the inoculum flowed into the liquid fraction for the NaOH treated and ensiling-NaOH treated sample, respectively.</p><!><p>Mass (A, D, and G), carbon (B, E, and H), and nitrogen (C, F, and I) flow of untreated, NaOH treated and ensiling-NaOH treated Pennisetum purpureum in methane production (process II). A–C represent the untreated sample; D–F represent the NaOH treated sample; G–I represent the ensiling-NaOH treated sample.</p><!><p>The mass, carbon and nitrogen flow of ensiling-NaOH treated Pennisetum purpureum samples under process III are shown in Figure 7. After NaOH pretreatment (Figure 7D,G), the mass recovery was 24.9% for the untreated sample and 30.0% for ensiling treated sample after enzymatic hydrolysis and ethanol fermentation; this further respectively decreased to 16.9% and 20.9% after AD. Considering the overall reaction system volume, it was observed that 65.2 g of ethanol + 102.6 g of methane could be produced from 1 kg of dry untreated sample, which increased to 248.1 g of ethanol + 139.0 g of methane after NaOH pretreatment and 269.4 g of ethanol + 144.5 g of methane after ensiling-NaOH pretreatment. This result was similar with the previous study conducted by Du et al.,35 who reported that 121.6 g of ethanol and 110.6 g of methane could be obtained from 1 kg of dried Pennisetum purpereum. For the carbon flow (Figure 7E,H), after NaOH pretreatment, 13.2% of carbon in the untreated sample (accounting for 30.3% of the residue after NaOH pretreatment) and 18.6% of carbon in the ensiling treated sample (accounting for 32.8% of the residue after NaOH pretreatment) flowed into ethanol after enzymatic hydrolysis and fermentation; meanwhile, 10.6% of carbon in the untreated sample (accounting for 24.4% of the residue after NaOH pretreatment) and 14.3% of carbon in the ensiling treated sample (accounting for 25.3% of the residue after NaOH pretreatment) flowed into methane after AD. For the nitrogen flow (Figure 7F,I), all of the nitrogen in the untreated sample and ensiling treated sample flowed into the liquid fraction after NaOH pretreatment. Subsequently, all of the nitrogen flowed into the enzymatic hydrolysis and fermentation process were from cellulase and seed medium, and the nitrogen during the AD process was from the inoculum.</p><!><p>Mass (A, D, and G), carbon (B, E, and H), and nitrogen (C, F, and I) flow of untreated, NaOH treated and ensiling-NaOH treated Pennisetum purpureum in ethanol-methane coproduction (process III). A–C represent the untreated sample; D–F represent the NaOH treated sample; G–I represent the ensiling-NaOH treated sample.</p><!><p>The energy flow under different production processes is shown in Figure 8. The energy values of ethanol and methane are 27.1 and 50.0 kJ/g, respectively.61 In this study, the energy value of raw Pennisetum purpureum is 16.8 MJ/kg, and only the energy output from ethanol and methane was calculated. As shown in Table 6, the energy production of 1.8 (10.5%), 7.0 (41.8%), and 6.9 (41.2%) MJ from 1 kg of untreated sample could achieve under processes I, II, and III; the energy production of 1.7 (9.5%), 6.9 (38.1%), and 6.7 (37.2%) MJ from 1 kg of ensiling treated sample could achieve under process I, II, and III; the energy produced from 1 kg of NaOH treated sample were 6.7 (39.9%), 9.7 (57.7%), and 13.7 (81.3%) MJ under three processes, and that from 1 kg of ensiling-NaOH treated sample was 7.3 (41.9%), 9.5 (54.3%), and 14.5 (83.4%) MJ under three processes, respectively. These results were similar to the literature when using other biomass for ethanol and methane production; for example, the energy yields of 5.1–5.2 and 8.8–9.3 MJ/kg were reported for oat straw and sugarcane bagasse.36,62 For the ensiling treated sample, the energy recovery from process III was 291.8% higher than those of process I, while the energies produced from processes II and III were similar. For NaOH treated sample, the energy production from process III increased by 103.4% compared with that from process I, and increased by 40.8% compared with that from process II. For the ensiling-NaOH treated sample, the energy production from process III increased by 98.9% compared with that from process I, and increased by 53.6% compared with that from process II. Comparing the three pretreatment methods and processes, the highest energy production was from the ensiling-NaOH treated sample in process III, which was 14.5 MJ/kg, with an energy conversion efficiency of up to 46.8% (accounting for 83.4% of residues after ensiling-NaOH pretreatment).</p><!><p>Energy conversion efficiency (%) = [ethanol and/or methane energy output]/[Pennisetum purpurem energy output] × 100%.</p><p>Energy flow of three processes of Pennisetum purpureum. A, D: process I; B, E: process II; C, F: process III. A–C represent the NaOH treated sample; D–F represent the ensiling-NaOH treated sample.</p><!><p>Ethanol production, methane production, and coproduction of ethanol and methane were comparatively investigated from Pennisetum purpureum after ensiling, NaOH and ensiling-NaOH pretreatment. Results showed that there were no significant differences between NaOH pretreatment and ensiling-NaOH pretreatment in terms of the enhancement in ethanol production and methane production. However, the highest energy output was obtained via the coproduction of ethanol and methane process after ensiling-NaOH pretreatment; the production of ethanol and methane from 1 kg of ensiling-NaOH treated Pennisetum purpureum was 269.4 g of ethanol and 144.5 g of methane, respectively, which resulted in an energy output of 14.5 MJ with the energy conversion efficiency of 46.8%. These results demonstrated that the coproduction of ethanol and methane from Pennisetum purpureum outpaced the single ethanol and methane production, which may provide useful information for optimally exploiting its use for renewable biofuels production.</p><!><p>Figure S1: SEM (scanning electron microscope) images of untreated and pretreated Pennisetum purpureum (PDF)</p><p>sc1c02010_si_001.pdf</p><!><p># P.W. and X.K. contributed equally to this paper.</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Solid-State NMR Spectroscopy Provides Atomic-level Insights Into the Dehydration of Cartilage

An atomic-level insight into the functioning of articular cartilage would be useful to develop prevention strategies and therapies for joint diseases such as osteoarthritis. However, the composition and structure of cartilage, and their relationship to its unique mechanical properties are quite complex and pose tremendous challenges to most biophysical techniques. In this study, we present an investigation of the structure and dynamics of polymeric molecules of articular cartilage using time-resolved solid-state NMR spectroscopy during dehydration. Full-thickness cartilage explants were used in magic-angle spinning experiments to monitor the structural changes of rigid and mobile carbons. Our results reveal that the dehydration reduced the mobility of collagen amino acid residues and carbon sugar ring structures in glycosaminoglycans but had no effect on the trans-Xaa-Pro conformation. Equally interestingly, our results demonstrate that the dehydration effects are reversible, and the molecular structure and mobility are restored upon rehydration.

solid-state_nmr_spectroscopy_provides_atomic-level_insights_into_the_dehydration_of_cartilage

Introduction<!>Preparation of bovine articular cartilage<!>NMR spectroscopy<!>Results and Discussion<!>RINEPT experiments reveal that GAGs in cartilage are highly mobile<!>Time-resolved MAS experiments to monitor slow dehydration process are feasible<!>Dehydration reduces the mobility but without denaturing the molecular components and their structure in cartilage<!>RINEPT reveals the reduction in the mobility of GAGs<!>Removal of GAGs does not alter collagen structure but influence its flexibility<!>Conclusions

<p>It is important to understand the mechanical and shock-absorbing strengths of cartilage in order to develop strategies to avoid rheumatic diseases and to slow their progression.1–3 While these intrinsic properties defining the function of cartilage should stem from the structural and dynamical organizations of its molecular constituents (~75% water, ~20% collagen, ~5% proteoglycans, and chondrocytes) (Figures 1A & 1B),1,4–7 it has been a major challenge to measure the molecular properties from an intact cartilage. The high-abundance of water, in particular, has been thought to play a major role in controlling the structure and dynamics of macromolecules and hence the function of cartilage in healthy and osteoarthritic joints.8,9 In this study, we report the effect of dehydration and rehydration of water in an intact cartilage at atomic-level resolution. To address this issue, we have developed a novel cell that controls the rate of dehydration during high-resolution nuclear magnetic resonance (NMR) experiments. Ramp-CP (cross-polarization)10 or INEPT (insensitive nuclei enhanced by polarization transfer)11,12 NMR experiments are used to identify molecular segments undergoing slow or fast molecular motions, and to monitor the changes in the dynamical structures of different molecules in cartilage.</p><p>At the whole organ level, changes in the concentration or structure of water are implicated in cartilage thinning or lesion formation which can be visualized clinically by T2 or T2* weighted magnetic resonance imaging (MRI) of joints.9,13–24 At the molecular-level, cartilage becomes temporarily dehydrated during compression because water is squeezed out of extracellular matrix, causing negatively charged glycosaminoglycans (GAGs) to rearrange conformation, generating a swelling osmotic pressure to counter the compressive load.6,24,25 We hypothesized that dehydration primarily affects mobile atoms in GAGs and collagen, and that the effects of dehydration on molecular structure is reversible. The chemical structure of cartilage extracellular matrix molecules, such as collagen and GAGs, has been studied via microscopy,27 light scattering,28 viscometry,29 and atomic force microscopy (AFM).3,26 The NMR method used in this study enables the determination of molecular structure of these polymer-like components in situ, does not require extensive specimen preparation, and allows measurement of rigid and mobile molecular structures on a single specimen. By comparison, osmotic dehydration used in NMR and MRI studies requires 20–48 hours equilibration time.6,24 We expect that this approach can be applied to the analysis of any material where water is an important structural molecule including biological tissues, hydrogels, and thin films.</p><!><p>Bovine articular cartilage specimen from a healthy young cow (about 6 months old) were obtained from a USDA-approved local slaughterhouse (Boyers Meat Processing, Canton, MI, USA) within 3 hours of death and frozen at −20 °C until used. Full-thickness cartilage specimens, including the calcified cartilage layer, were removed from undamaged sites on the femoral condyles using a sterile scalpel with care to not extract bone tissue and cut into ~1 mm × 2 mm rectangles. D2O-equilibrated cartilage was prepared by soaking bovine cartilage in calcium-buffered saline solution prepared with deuterium oxide (99.9% from Sigma Aldrich, St. Louis, MO, USA) for 72 hours with occasional mild shaking until use at −4°C. For the proteoglycan depletion, a cartilage specimen was incubated with 4 mM guanidine-HCl to remove the bulk of proteoglycan at room temperature for 48 hours, washed with excess PBS, and incubated in PBS enriched with 1 mg/ml trypsin (Sigma, St. Louis, MO, USA) for 12 hours at 25 °C with constant stirring to remove residual proteoglycans.</p><!><p>NMR experiments were carried out on a Varian VNMRJ 600 MHz solid-state NMR spectrometer equipped with a 4-mm triple-resonance MAS probe at room temperature (25 °C) under 10 kHz magic-angle spinning condition. A specially designed rotor (see Figure S1) was used for NMR measurements under a controlled dehydration process. 1H MAS spectra were recorded using a single pulse excitation. 13C MAS spectra were obtained using two different pulse sequences Ramp-CP and refocused-INEPT (RINEPT). A 2 ms contact time was used in the Ramp-CP pulse sequence, whereas 1 ms refocus delays were used in the evolution and refocusing periods of the RINEPT pulse sequence. A 80 kHz TPPM (two-pulse phase-modulation)30 was applied to decouple protons during signal acquisition.</p><!><p>It is highly important to preserve the frictionless motion between articular surfaces rendered by cartilage in order to avoid joint diseases like osteoarthritis. This unique property interestingly stems from the structural organization of molecular components constituting articular cartilage, particularly water, collagen and glycosaminoglycans. Therefore, there is a significant interest in understanding the high-resolution structural changes in collagen and GAGs (chondroitin sulfate, keratan sulfate and hyaluronan) due to dehydration and rehydration processes of cartilage.31,33 In this study, we report 13C MAS solid-state NMR measurements from an intact bovine cartilage at natural-abundance. Though cartilage contains different types of molecules with a varying abundance, its NMR spectrum can be simplified based on the difference in their time scale of motions as explained below.</p><!><p>Intact bovine cartilage explants were placed into a specially constructed NMR rotor, and the rotor was spun at the magic-angle. Two types of experiments that transfer magnetization from 1H to 13C were applied on this specimen: cross-polarization and RINEPT to selectively enhance 13C signal from rigid and mobile molecules from the cartilage tissue, respectively. The proton to carborn-13 polarization transfer from was achieved by the combined effects of C-H scalar (or J) and dipolar couplings in the RINEPT pulse sequence. The scalar coupling is a constant under the MAS condition, while dipolar coupling depends on the orientation of the related chemical bonds with respect to the external magnetic field. Modulation of CH and 1H-1H dipolar coupling under MAS and the distribution of dipolar couplings in the specimen will render the magnetization transfer extremely inefficient in the RINEPT pulse sequence. Therefore, to achieve a better magnetization transfer from proton to 13C, the strong C-H dipolar coupling (~35 kHz in a rigid specimen) needs to be suppressed to the order of J coupling (< 100 Hz); the strong 1H-1H dipolar couplings influencing the evolution of transverse magnetization also need to be suppressed for a better efficiency of the RINEPT sequence. The suppression of C-H and 1H-1H dipolar couplings could be due to the internal molecular motion or by fast MAS spinning. Under our 10 kHz MAS experimental condition, the 13C chemical shift spectral lines observed from the RINEPT experiment therefore should arise from highly mobile molecular constituents of cartilage; where the dynamics should have suppressed the C-H (and also 1H-1H) dipolar couplings to the order of the J-coupling during the RF-free delays used in RINEPT (tau=0.5 ms). On the other hand, the cross relaxation rate in the Ramp-CP pulse sequence depends on the strength of the C-H dipolar coupling. For the molecules with a weak dipolar coupling, the cross relaxation rate is too slow and insufficient for the 13C signal enhancement during the spin-lock time of the ramp-CP pulse sequence. Therefore, the spectral lines observed using the ramp-CP pulse sequence should arise from immobile molecular constituents of cartilage. Thus, by comparing spectra obtained using Ramp-CP and RINEPT pulse sequences, rigid and mobile structures present in cartilage can be identified.</p><p>As shown in Figures 1C and 1D, the two 13C chemical shift spectra of cartilage are significantly different; all experimentally measured chemical shift values and resonance assignment are summarized in Tables S1 and S2. The 13C Ramp-CP spectrum (Figure 1C) exhibits peaks (such as the peaks at 75, 105, and 182 ppm) primarily from collagen with some contribution from the rigid glycosaminoglycan's chain. On the other hand, the 13C spectrum obtained using the RINEPT pulse sequence (Figure 1D) consists of peaks from the sugar ring carbons of the mobile GAGs. Therefore, our NMR results suggest that cartilage GAGs undergo fast motions (microsecond time scale) whereas the motion of collagen falls in a slower time scale (millisecond time scale). The close resemblance of the 13C RINEPT spectrum (Figure 1D) with the 13C-Ramp-CP MAS spectrum of a pure powder specimen of chondroitin sulfate (Figures 1F) indicates that this type of glycosaminoglycan is more abundantly present in cartilage than hyaluronan (Figures 1E). Specifically, the spectral lines observed in the 50–60 and 70–80 ppm regions in Figures 1D and 1F are similar. This finding is in excellent agreement with previous reports in the literature.32,34–36 The large scale of observed motions for GAGs is mainly because more water molecules are associated with highly charged GAGs than with collagen. A recent study reported the feasibility of 1H-based HRMAS experiments on bovine patellar cartilage demonstrating the high mobility of GAG molecules in a fully-hydrated cartilage specimen.37 These results are also in excellent agreement with previous solid-state NMR studies based on the measurement of C-H dipolar couplings and 13C chemical shift anisotropy that predicted only 10–20% of water molecules are associated with collagen even though cartilage contains 70–80% water.5,31,38</p><!><p>Since water plays an important role in the function of cartilage and the fraction of water molecules associated with collagen is far lower than that associated with GAGs, it is important to understand the water-dependent structural dynamics of these biopolymer constituents. To this end, we performed time-resolved MAS experiments to monitor the effects of dehydration and rehydration on cartilage. The dehydration of cartilage was achieved during specimen spinning because a 0.1 mm diameter hole was drilled into the cap of the sample rotor, which generated a negative pressure inside the rotor (Figure S1 given in the Supplementary Information). The dehydration rate was easily controlled by using a polyethylene insert inside the end cap to adjust the size and length of the hole on the cap. 1H (Figure 2), 13C (Figures 3–6), and 31P (data not included as they did not provide any useful information) MAS NMR experiments were performed to investigate the water-dependent structural changes in normal bovine cartilage. The hydration level in the cartilage was monitored from the intensity of the water 1H peak observed at 4.7 ppm from a series of 1H MAS spectra of cartilage obtained using a single pulse excitation (Figure 2). As shown in Figure 2, an exponential decay of water content was observed, and spectra of fully-hydrated cartilage (that is the specimen before dehydration) and rehydrated cartilage were identical (Figure 3). The reduction in the signal intensity, the appearance of spinning side bands from the water resonance, and the appearance of additional peaks in the spectra indicate the disappearance of free water and the decreased molecular mobility due to dehydration. These experimental results suggest that the dehydration process of a cartilage specimen inside an MAS rotor can be controlled and therefore the measurement of the extent of water on individual molecules can be probed as discussed below.</p><p>Changes in the intensity and width of peaks in 13C spectra were used as NMR markers to measure any dehydration-induced changes in the molecular structure of cartilage as explained below. For comparison, another series of 13C NMR spectra was measured from cartilage with the distal end cap tightly sealed to prevent evaporation. No spectral changes were observed for even after 24 hours of spinning the specimen (data not shown). Since the main component in the mineral of cartilage is hydroxyapatite, 31P NMR was used to probe the phosphate mineral in calcified cartilage during the dehydration process; but 31P spectra dominated by a peak from the phosphate buffer did not provide any valuable information (data not included). 13C spectra of cartilage with deuterium exchange and Raman spectroscopy (Figure S2) on cartilage were also performed to monitor the dehydration process.</p><!><p>Since Ramp-CP and RINEPT experiments can be used to monitor the dynamics of molecules in cartilage, a series of 13C spectra were obtained as a function of a controlled dehydration process from a cartilage specimen. At the same time, 13C isotropic chemical shift values can be used to understand the backbone conformation. The recorded 13C Ramp-CP and RINEPT spectra on cartilage are given in Figures 3 and 4 respectively. The observed GAGs peaks in the Ramp-CP spectra are indicated using dashed lines. The dehydration of the cartilage specimen did not change the observed isotropic chemical shift values in Ramp-CP spectra (Figure 3) suggesting that the main triple-helical conformation of collagen does not depend on the amount of water associated. For example, the presence of peaks in the carbonyl region (~170 ppm) and a 17.5 ppm peak from 13Cβ Ala strongly suggest the triple-helical structure of collagen in cartilage remains even when it was dehydrated.5 In addition, a 5.2 ppm difference between 13Cβ (30.5 ppm) and 13Cγ (25.3 ppm) chemical shifts of Pro residue observed in this study confirms the presence of a trans-Xaa-Pro conformation in collagen.13 However, the broadening of spectral lines observed with dehydration suggests a reduction in the dynamics of collagen and possibly a slight increase in the conformational disorder. The 13Cγ (25.3 ppm) peak from hydroxyproline is highly sensitive to the hydration level as this residue plays an important role due to its extra hydrogen-bonding capability. The excellent agreement between our results obtained from an intact cartilage and that from collagen fibrils38 suggests that the structural and dynamical properties of collagen are similar in both types of specimens. The increase in collagen flexibility observed in these specimens, as more water molecules bind to polar groups of collagen when the hydration level is increased, not only contributes to the viscoelastic and mechanical properties of cartilage but also the key in the calcium phosphate biomineralization process.</p><p>The increasing appearance of peaks at 75, 105 and 182 ppm in the spectra suggest that the mobility of GAGs is considerably reduced so that the cross-polarization is more efficient due to stronger C-H dipolar couplings as water is removed from cartilage (indicated by dashed lines in Figure 3). For example, the signal intensity of GAGs at ~75 ppm increased and overlapped with the peak of Hyp Cγ from collagen at ~ 71 ppm as a consequence of dehydration as shown in Figure 3E. Further, two broad peaks from C2–C6 peaks of GAGs appeared at 71 ppm and 76 ppm. The appearance of C1 peaks from GAGs at ~ 110 ppm is clear after ~10 hours of dehydration. This observation is understandable as more water is associated with the charged GAG molecules and the dehydration process is expected to have a significant effect on the spectral lines from GAGs.</p><p>The close resemblance of the spectrum of a rehydrated specimen to that of the initial fully-hydrated cartilage specimen (before the dehydration process) is very interesting. In fact, the peaks arising from GAGs also disappear due to rehydration of the specimen due to the increased motion that considerably suppresses the cross-polarization. This observation suggests that the macromolecular assembly enable the dehydration and rehydration processes without resulting in a major damage to their structures or the molecular architecture of cartilage. This dehydration/rehydration behavior of the whole cartilage system can be compared to the action of sponge towards dehydration and absorption of water. Our results also suggest that the dynamics of collagen and GAGs can be controlled by dehydration and rehydration processes. Therefore, the dynamics of collagen and GAGs play a major role in the function of cartilage.</p><!><p>As mentioned above, the 1H/13C RINEPT sequence was used to detect mobile carbon structures, with the main contributions arising from chondroitin sulfate, the most abundant GAG in cartilage, with minor contributions from keratin sulfate and hyaluronan. Since GAGs are associated with a large amount of water molecules, the removal of water molecules through dehydration decreased the overall intensity of spectral lines and increased the line width as shown in Figure 4. There was no observable 13C signal after 30 hours of dehydration (~87% water loss estimated from 1H spectra). These data further confirm the increasing rigidity of polysaccharide chains of GAGs with dehydration; the rigid molecules do not respond to RINEPT but do respond to Ramp-CP as explained earlier.32,25,39 Interestingly, the 13C RINEPT spectrum of cartilage after rehydration (see Figure 4E) is very similar (if not identical) to the fully-hydrated intact cartilage (Figure 1D). To further confirm this observation, spectra were obtained from a cartilage specimen that was rehydrated using D2O (Figure 5). The spectra of the D2O hydrated specimens resemble that of the native specimen and narrowing of spectral lines were observed as the H/D exchange suppress the residual proton dipolar couplings. This observation suggests that the removal of water molecules does not denature cartilage while the rehydration process (even as short as 2 minutes rehydration) fully restores the architecture of cartilage back to normal. This further suggests that any dehydration-induced changes in the structure and dynamics of molecules and intermolecular interactions are reversible upon hydration. Therefore, we believe that this complete reversibility of the flexibility of GAGs and collagen molecules play important roles in the viscoelasticity and mechanical properties of cartilage.</p><!><p>In order to fully understand the dehydration effect on collagen structure present in cartilage and to probe the interactions between collagen and GAGs, we removed the proteoglycan using a published procedure.4013C Ramp-CP MAS spectra of GAGs-removed cartilage specimens were obtained (Figure 6). Though the NMR spectra of fully-hydrated intact (Figure 3A) and GAGs-free (Figure 6A) cartilage are similar, significant differences can be seen in spectra of corresponding dehydrated specimens (see Figures 3B–E and Figure 6B). For example, the broad peaks from GAGs appearing ~75 ppm and ~185 ppm due to dehydration in an intact cartilage (as seen in Figures 3B–E) are absent in GAGs-removed cartilage (Figure 6B). The relatively narrow spectral lines in the GAGs-free specimen (Figure 6B) suggest that the presence of GAGs introduces additional heterogeneity to the dynamically disordered collagen upon dehydration. On the other hand, the spectra of rehydrated specimens (shown in Figures 3F and 6C) are similar as well. Therefore, the observed similarities and differences between these spectra of specimens with and without GAGs suggest that the removal of GAGs does not change the structure of collagen in cartilage but the change in the flexibility of collagen molecules is influenced by GAGs in cartilage.</p><!><p>Understanding the molecular structure of cartilage extracellular matrix molecules continues to be a significant challenge. We chose bovine articular cartilage as a model system for these NMR experiments because the molecular structure of cartilage extracellular matrix molecules depends on the extensive interactions that occur in situ. High-resolution MAS solid-state NMR experiments in combination with time-resolved hydration/rehydration processes were used to probe the dynamical structures of molecular components of cartilage at atomic-level resolution. As demonstrated by the experimental results in this study, the use of Ramp-CP and RINEPT preparation pulse sequences is a powerful method to differentiate the rigid and mobile molecular components present in intact cartilage, and to selectively detect and characterize the type of GAGs in cartilage. Though it is possible to directly obtain 13C MAS spectra of GAGs from cartilage, the use of the RINEPT pulse sequence under MAS enhances the sensitivity of detection. Results from our study show that the dehydration process affected both rigid and mobile structures of molecular components, with most of the changes appearing from a change in the time scale of motions for collagen and the GAG ring structure. Not only did this study provide insights into the structural role of water in cartilage, but also served as a model for studying dehydration effects in tissues, hydrogels or thin films. Particularly, a combination of HRMAS and RINEPT would be a powerful approach in the high-resolution dynamical structural studies of cartilage, tissues and bone materials.7,41–47 It is worth noting that unlike other techniques that require enzymatic digestion, extraction, fixation, cryopreservation or cryosectioning, the NMR methods described here require little specimen preparation and minimal amounts of tissue.</p>

PubMed Author Manuscript

Medicinal Chemistry Profiling of Monocyclic 1,2-Azaborines

The first examples of biologically active monocyclic 1,2-azaborines have been synthesized and demonstrated to exhibit not only improved in vitro aqueous solubility in comparison to the corresponding carbonaceous analogues, but in the context of a CDK2 inhibitor, also improved biological activity and better in vivo oral bioavailability. This proof-of-concept study establishes the viability of monocyclic 1,2-azaborines as a novel pharmacophore with distinct pharmacological profiles that can help address challenges associated with solubility in drug development research.

medicinal_chemistry_profiling_of_monocyclic_1,2-azaborines

<p>One of the main goals of synthetic chemistry is to create structural diversity – and as a consequence produce new functions and properties – beyond what Nature can achieve. For instance, a key impetus behind the laboratory syntheses of bioactive natural products is to diversify the original portfolio of structures to systematically investigate structure-function relationships and elucidate mechanism of action.1 This exploration of new chemical space facilitates the development of reagents that illuminate new biology and the development of therapeutics that can benefit society. BN/CC isosterism2 (i.e., the replacement of a carbon-carbon unit with a boron-nitrogen (BN) unit) has recently emerged as a strategy to increase the chemical space of compounds relevant to biomedical research.3 When applied to a "privileged" structural motif in medicinal chemistry,4 this approach can produce a new versatile pharmacophore. Aromatic rings are ubiquitous in medicinal chemistry, and arene-containing compounds prevail among topselling small-molecule drugs.5 BN/CC isosterism of arenes results in the so-called azaborine heterocycles where specifically 1,2-azaborines are designated as compounds with the boron and nitrogen atoms adjacent to each other (Scheme 1).6 It has been demonstrated that 1,2-azaborines can bind to aryl recognition pockets7 in biological targets and engage in hydrogen bonding inside those binding pockets.8 Furthermore, it has been shown that both the B- and N-Et BN isosteres of ethylbenzene are inhibitors of ethylbenzene dehydrogenase (EbDH), in contrast to ethylbenzene itself, which is the naturally evolved substrate for the EbDH.9 Despite the recent advances made in the area of azaborine chemistry,10 the progress toward evaluating these heterocycles in the context of medicinal chemistry has remained underexplored. BN isosteres of naphthalene have recently been profiled in vitro and in vivo in terms of biological activity and ADMET (absorption, distribution, metabolism, excretion, toxicity) properties.11,12 However, to the best of our knowledge, profiling of the arguably more versatile monocyclic 1,2-azaborine motif has not been reported. Thus, essential questions such as stability, biological activity, pharmacological properties of monocyclic 1,2-azaborines have remained unanswered. In our initial exploration in this area, we sought to investigate 1,2-azaborine isosteres of biologically active biphenyl carboxamides, the biphenyl motif being a "privileged" sub-motif of the arene family in drug discovery research.13,14 In this communication, we establish that 1,2- azaborine-based biphenyl carboxylic acids are compatible with the CDMT/NMM amide coupling conditions, and that the resulting amides 1) are air and water stable, 2) are more soluble in water than their carbonaceous counterparts, 3) exhibit better in vivo oral availability, and 4) can exhibit stronger biological activity due to hydrogen bonding.</p><p>In 2013, we reported a functional-group tolerant Rh-catalyzed B-arylation of B-Cl-substituted 1,2-azaborines and as a demonstration synthesized the BN isostere of Felbinac, a nonsteroidal anti-inflammatory drug.15 Recognizing the versatility of the carboxylic acid functional group present in Felbinac, we sought to develop amide-coupling conditions to access BN isosteres of the ubiquitous biphenyl carboxamide family of biologically active compounds. Gratifyingly, the use of the 2-chloro-4,6-dimethoxy-1,3,5-triazine/N-methylmorpholine (CDMT/NMM) conditions16 furnished the desired amide coupling products in moderate to good yield (Table 1). We specifically chose three biphenyl carboxamides that inhibit a distinct set of biological targets (dopamine D3 (BN-1),17 PPAR γ and δ (BN-2),18 and CDK2 (BN-3)19 to evaluate the effects of BN/CC isosterism on their pharmacological properties. It is worth noting that the amide coupling can be conducted in air. Furthermore, stability studies reveal no decomposition when BN-1, BN-2, and BN-3 are exposed to air and water at 50 °C for 24 hours, demonstrating the viability of these BN heterocycles in medicinal chemistry applications.20</p><p>Table 2 shows the ADMET behavior of BN-1, BN-2, and BN-3 in direct comparison to their carbonaceous analogues CC-1, CC-2, and CC-3.21 A general trend can be observed in terms of the effect of BN/CC isosterism on aqueous solubility properties: the BN isosteres are more soluble both under buffered and FASSIF conditions. As a result, they have decreased membrane permeability (more negative PAMPA value) than their carbonaceous analogues. The better aqueous solubility behavior of 1,2-azaborine derivatives is consistent with reported electronic structure analysis that revealed a 2.1 D dipole moment for 1,2-dihydro-1,2-azaborine in contrast to benzene's dipole moment of 0 D.22 Thus, the incorporation of the 1,2-azaborine motif renders the relatively hydrophobic biphenyl motif more hydrophilic. A majority of currently marketed drugs are poorly soluble.23 Thus, BN/CC isosterism can potentially be used as a design strategy to produce more soluble active pharmaceutical ingredients. No general trend can be discerned from the RLM Cl, CYP3A4, and hERG data. It appears that functional groups unrelated to BN/CC isosterism may be more responsible for the observed data. Overall, our ADMET data indicate that there is no particular red flag associated with the use of 1,2-azaborines as a pharmacophore in medicinal chemistry.</p><p>We then turned our attention to evaluating the biological activity of our BN isosteres in comparison to their all-carbon derivatives. Compound CC-1 was reported as a selective dopamine D3 antagonist,17 and in our analysis CC-1 exhibited an IC50 value of 1 nM (Scheme 2). Its BN isostere BN-1 is also biologically active although the activity is slightly attenuated with an IC50 value of 3 nM. CC-2 has been investigated as antagonists of PPAR γ and δ18 and in our assay we have determined IC50 values of 1 and 2 µM, respectively. Similarly, the corresponding BN isostere BN-2 also exhibits low micromolar activity against PPAR γ and δ (Scheme 2). Compound CC-3 was reported as a potent nanomolar antiproliferative agent in a CDK2 kinase assay.19 In our CDK2 assay CC-3 showed an IC50 of 320 nM. Interestingly, the BN derivative BN-3 (IC50 = 87 nM) showed improved potency than CC-3. Compound BN-3 is selective for CDK2. When tested against a panel of 29 kinases, BN-3 was found to be a more selective inhbitor of CDK2 than CDK1 (IC50 = 460 nM).</p><p>The improved biological activity of BN-3 vs. CC-3 was intriguing. To understand this improvement in potency, BN-3 and CC-3 were analyzed by docking24,25 in a high resolution crystal structure of CDK2/cyclin A (PDB entry 1VYW).19 Shown in Figure 1 is one of the three docking poses obtained for BN-3 in the active site of CDK2. In addition to the hydrogen bonding interaction between the pyrazole amide fragment and hinge residues Leu83, Glu81, an additional hydrogen bonding interaction was observed between the NH of the azaborine and the backbone carbonyl of Ile10. The 3–4 fold improvement in binding of BN-3 vs. CC-3 may be attributed to this NH…O=C(amide) hydrogen bonding which we have recently quantified to be ~ 1 kcal/mol in strength (in the context of binding to T4 Lysozymes).8</p><p>Finally, we asked the question whether the observed improved in vitro solubility for BN-3 vs. CC-3 would translate into in vivo pharmacokinetic behavior. Gratifyingly, we determined that BN-3 exhibits pharmacokinetic properties that are superior to CC-3 in male Sprague Dawley Rat models (Table 3). When dosed intravenously, BN-3 showed lower clearance and a longer terminal half-life (t1/2) than CC-3. Additionally, BN-3 gave a two-fold increase in AUCpo (area under the curve per oral administration) relative to CC-3. This results from a combination of lower clearance and greater bioavailability. The maximum concentration (Cmax) of CC-3, 692 nM, is observed at 0.5 hour after oral dosing. BN-3 on the other hand, has maximum concentration of 746 nM at 1.5 hours after dosing, probably due to the increased solubility prolonging the precipitation time and allowing BN-3 to be absorbed further down the intestine than CC-3. Despite the slightly lower permeability of BN-3 relative to CC-3 in vitro, the improved solubility and lower clearance of BN-3 in vivo enabled an increase in oral exposure for BN-3 compared to CC-3.</p><p>In summary, we have synthesized the first examples of biologically active monocyclic 1,2-azaborines and demonstrated that BN/CC isosterism in the context of biphenyl carboxamides leads to improvement in vitro aqueous solubility and better in vivo oral availability. The BN isosteres of biologically active biphenyl carboxamides are air and moisture stable, and they exhibit biological activity that is comparable to their carbonaceous counterparts. Furthermore, in the context of a CDK2 inhibitor, we have demonstrated that the presence of a 1,2-azaborine motif can lead to improved biological activity likely from an additional hydrogen bonding interaction associated with the NH of the 1,2-azaborine moiety. Overall, we have demonstrated the viability of the monocyclic 1,2-azaborine motif serving as a novel pharmacophore with a distinct pharmacological profile. In view of the solubility challenges associated with many aryl-based drug candidates, BN/CC isosterism may represent a new design principle in medicinal chemistry to address this challenge.</p><p>Supporting information for this article is given via a link at the end of the document.</p>

PubMed Author Manuscript

Synthesis and Pharmacological Evaluation of Nucleoside Prodrugs Designed to Target Siderophore Biosynthesis in Mycobacterium tuberculosis

The nucleoside antibiotic, 5\xe2\x80\xb2-O-[N-(salicyl)sulfamoyl]adenosine (1), possesses potent whole-cell activity against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). This compound is also active in vivo, but suffers from poor drug disposition properties that result in poor bioavailability and rapid clearance. The synthesis and evaluation of a systematic series of lipophilic ester prodrugs containing linear and \xce\xb1-branched alkanoyl groups from two to twelve carbons at the 3\xe2\x80\xb2-position of a 2\xe2\x80\xb2-fluorinated analogue of 1 is reported with the goal to improve oral bioavailability. The prodrugs were stable in simulated gastric fluid (pH 1.2) and under physiological conditions (pH 7.4). The prodrugs were also remarkably stable in mouse, rat, and human serum (relative serum stability: human~rat>>mouse) displaying a parabolic trend in the SAR with hydrolysis rates increasing with chain length up to eight carbons (t1/2 = 1.6 h for octanoyl prodrug 7 in mouse serum) and then decreasing again with higher chain lengths. The permeability of the prodrugs was also assessed in a Caco-2 cell transwell model. All of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction and enhanced permeation in the basolateral-to-apical direction relative to the parent compound 2, resulting in efflux ratios 5\xe2\x80\x9328 times greater than 2. Additionally, Caco-2 cells were found to hydrolyze the prodrugs with SAR mirroring the serum stability results and a preference for hydrolysis on the apical side. Taken together, these results suggest that the described prodrug strategy will lead to lower than expected oral bioavailability of 2 and highlight the contribution of intestinal esterases for prodrug hydrolysis.

synthesis_and_pharmacological_evaluation_of_nucleoside_prodrugs_designed_to_target_siderophore_biosy

1. Introduction<!>2.1. Chemistry<!>2.2. Aqueous and plasma stability<!>2.3 Caco-2 permeability<!>3. Conclusion<!>4.1.1 General Chemistry Methods<!>4.1.2. 9-[5-Azido-2,5-dideoxy-2-fluoro-\xce\xb2-d-ribofuranosyl]adenine (12)<!>4.1.3. 9-[5-Azido-3-O-(tert-butyldimethylsilyl)-2,5-dideoxy-2-fluoro-\xce\xb2-d-rib` ofuranosyl]adenine (13)<!>4.1.4. 9-[3-O-(tert-Butyldimethylsilyl)-2,5-dideoxy-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-arabinofuranosyl] adenine (14)<!>4.1.5. 9-[2,5-Dideoxy-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (15)<!>Method A<!>Method B<!>4.1.6. General procedure for the preparation of compounds 16\xe2\x80\x9323<!>4.1.6.1. 9-[3-O-(Acetyl)-2,5-dideoxy-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (16)<!>4.1.6.2. 9-[2,5-Dideoxy-2-fluoro-3-O-(propionyl)-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (17)<!>4.1.6.3. 9-[3-O-(Butyryl)-2,5-dideoxy-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (18)<!>4.1.6.4. 9-[2,5-Dideoxy-2-fluoro-3-O-(pentanoyl)-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (19)<!>4.1.6.5. 9-[2,5-Dideoxy-2-fluoro-3-O-(octanoyl)-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (20)<!>4.1.6.6. 9-[2,5-Dideoxy-3-O-(dodecanoyl)-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (21)<!>4.1.6.7. 9-[2,5-Dideoxy-2-fluoro-3-O-(isobutyryl)-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (22)<!>4.1.6.8. 9-[2,5-Dideoxy-3-O-(2-ethylbutyryl)-2-fluoro-5-(N-sulfamoyl)amino-\xce\xb2-d-ribofuranosyl]adenine (23)<!>4.1.7. General procedure for the preparation of prodrugs 3\xe2\x80\x9310<!>4.1.7.1. 9-[3-O-(Acetyl)-2,5-dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (3)<!>4.1.7.2. 9-[2,5-Dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-3-O-(propionyl)-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (4)<!>4.1.7.3. 9-[3-O-(Butyryl)-2,5-dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (5)<!>4.1.7.4. 9-[2,5-Dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-3-O-(pentanoyl)-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (6)<!>4.1.7.5. 9-[2,5-Dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-3-O-(octanoyl)-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (7)<!>4.1.7.6. 9-[2,5-Dideoxy-3-O-(dodecanoyl)-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (8)<!>4.1.7.7. 9-[2,5-Dideoxy-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-3-O-(isobutyryl)-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (9)<!>4.1.7.8. 9-[2,5-Dideoxy-3-O-(2-ethylbutyryl)-2-fluoro-5-{N-(N-2-hydroxybenzoyl)sulfamoyl}amino-\xce\xb2-d-ribofuranosyl]adenine Triethylammonium Salt (10)<!>4.2. HPLC method<!>4.3. Aqueous stability<!>4.4. Plasma stability<!>4.5. Caco-2 permeability<!>

<p>Tuberculosis (TB), one of the oldest recorded diseases of humankind, is caused by the slowgrowing bacterium Mycobacterium tuberculosis (Mtb) as well as several closely related mycobacterial species. TB is a devastating disease clinically manifested as a persistent cough followed by hemoptysis, general malaise and fatigue, and severe weight loss as the disease progresses. TB is extremely difficult to treat relative to other bacterial infections due to a number of unique factors in the pathology and metabolism of Mtb.1–4 Thus, in the case of the simplest drug-sensitive TB, one must employ a four-drug regimen comprised of isoniazid, rifampicin, ethambutol, and pyrazinamide for the first two months followed by 4–7 months of isoniazid and rifampicin. Drug-resistant strains are even more challenging to treat with corresponding lower cure rates. As a result, TB has now overtaken malaria and HIV as the leading cause of infectious disease mortality.5 The development of new antitubercular agents that are effective against drug-resistant strains and reduce the duration of treatment will be necessary to bring TB back under control.</p><p>The nucleoside antibiotic 5′-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS (1), Figure 1) originally described by Quadri, Tan and co-workers was rationally designed to inhibit siderophore biosynthesis in Mtb, an essential process under iron-deficient conditions found in the host.6–9 Sal-AMS (1) possesses nanomolar enzyme inhibition of MbtA, which catalyzes the first committed step of mycobactin biosynthesis, and potent on-target whole-cell activity. Proof-of-concept in vivo efficacy was also demonstrated; however, 1 suffers from poor physicochemical properties that result in high clearance and low oral bioavailability.10 To further advance this new class of antibiotics, we previously explored a large number of modifications to 1.8,11–16 The 2′-Fluoro analogue 2 emerged as a lead compound with improved in vitro antitubercular activity (2-fold more potent than 1) while displaying enhanced in vitro (Caco-2 permeability of 4.2 × 10−6 cm/s or 3.5-fold greater than 1, indicative of a medium-permeable compound) and in vivo drug disposition properties (3-fold reduced clearance resulting in a commensurate 3-fold improved oral exposure relative to 1), but no improvement in bioavailability.15</p><p>A common strategy to improve oral bioavailability is to synthesize an ester prodrug that increases the lipophilicity and thereby enhances gastrointestinal absorption.17,18 The ester prodrug is then cleaved by serum or tissue esterases to release the parent drug. Herein we report the synthesis, chemical and enzymatic stability, as well as the in vitro membrane permeability of a systematic series of eight prodrugs (3–10) of the 2′-fluoro analogue 2 through esterification at the 3′-OH group (Figure 1). Prodrugs 3–8 contain a linear alkyl chain from two to twelve carbons while 9 and 10 are branched at the α-position to explore the importance of steric hindrance of the promoiety. The pivaloyl ester, which contains a tertiary carbon at the α-position, was not prepared due to the chronic toxicity of pivalate.19 The phenolic hydroxyl group was left unmasked because the formation of an intramolecular hydrogen bond with the charged acylsulfamide moiety was expected to shield the polarity through charge delocalization.14</p><!><p>Synthesis of the key intermediate 15 for preparation of the prodrugs began with commercially available 2′-deoxy-2′-fluoroadenosine 11 (Scheme 1). Regioselective 5′-azidation of 11 with NaN3 using the classic Appel conditions (CBr4, PPh3, DMF) afforded 12 in 78% yield. The 3′-OH in 12 was then protected as the TBS ether 13 in 80% yield. Catalytic hydrogenation of 13 employing wet Pd/C led to reduction of the azide to the corresponding amine in quantitative yield. The crude aminonucleoside intermediate was refluxed with sulfamide (NH2SO2NH2) in 1,4-dioxane to provide 14 in 85% yield over two steps. Deprotection of the TBS group with HCl furnished the desired sulfamide 15 in quantitative yield. The conversion of azide 12 to sulfamide 15 was accomplished in four steps with a 68% overall yield by this optimized synthetic route. We also demonstrated that azide 12 could be directly converted to sulfamide 15 in 58% overall yield in two steps circumventing the TBS protection-deprotection sequence utilizing an analogous series of reactions for the conversion of 13 to 14. However, the former route was preferred due to its higher overall yield and avoidance of chromatographic purification of the polar nucleoside 15.</p><p>With an efficient route to the common intermediate 15 in place, we then developed a reliable procedure for introduction of the promoieties onto the 3′-OH of the nucleoside. Although selective acylation of the 3′-OH over the sulfamide could be accomplished at −5 °C; in practice the extended reaction times made this experimentally impractical. Thus, the reactions were conducted at 0 °C leading to dual acylation. The acyl group on the sulfamide moiety was subsequently cleaved in situ by treatment with formic acid in methanol. By this method, the 3′-O-acylated sulfamides 16–23 were obtained in 61–88% yield. The salicyl group was introduced by Cs2CO3 mediated coupling with N-hydroxysuccinimdyl ester 24 followed by hydrogenolysis of the benzyl protected phenol and purification by column chromatography (coelution with 2% Et3N) afforded prodrugs 3–10 as triethylammonium salts in 37–57% yield over these final two steps.</p><!><p>The ideal prodrug for our application should be stable in aqueous solutions (for 1–2 hours to enable oral absorption), but rapidly hydrolyzed to the parent drug in plasma. The aqueous stability of the prodrugs 3–10 was thus studied in simulated gastric fluid (SGF, pH 1.2) and HEPES buffer (pH 7.4), which mimic gastric and physiological pH, respectively. The prodrugs 3–10 (100 µM) were incubated at 37 °C in the indicated buffers containing 2% DMSO and the amount of both hydrolyzed product (i.e. parent drug 2) and the prodrug remaining in the solution were monitored by HPLC. Most of the prodrugs were extremely stable and showed little degradation or hydrolysis at 2 hours in both buffers (Table 1). Surprisingly, in the case of most lipophilic dodecanoyl prodrug 8 at pH 1.2, only 76% of the prodrug remained in solution after 2 hours at 37 °C. Chemical hydrolysis to release the parent drug 2 was not observed. Instead we noticed that the prodrug began to slowly precipitate over time, due to the low solubility at pH 1.2. This was also observed with octanoyl prodrug 7 to a minor extent (93% remaining in solution at 2 hours). Only the acetate prodrug 3 was slightly hydrolyzed to parent drug 2 (8% hydrolyzed at pH 7.4 at 2 h). These results confirm that the prodrugs are stable under aqueous conditions.</p><p>Next, the stability of the prodrugs in mouse, rat, and human plasma at 37 °C was investigated. The prodrugs were hydrolyzed very slowly in rat and human plasma. On the other hand, most of the prodrugs were hydrolyzed more rapidly by mouse plasma and followed first order kinetics allowing determination of their half-lives. For prodrugs that showed less than 50% hydrolysis at 6 hours, the percentage of prodrug remaining at this terminal time point was measured. The half-lives and percent remaining at 6 hours are listed in Table 1. In mouse plasma, the fastest rate of hydrolysis was observed with octanoyl prodrug 7 with a half-life (t1/2) of 0.7 hours. The hydrolysis rate was slightly slower for prodrugs with larger alkyl groups such as dodecanoyl 8 (t1/2 = 1.6 h) and much slower for branched alkyl chains such as 9 and 10. It was also slower for prodrugs 3, 4, 5, and 6 containing shorter ester promoieties with a clear trend paralleling chain length. In rat and human plasma, the hydrolysis was slow for all prodrugs with a trend of slower hydrolysis for longer and branched promoiet ies. Nonetheless, the prodrugs studied were far more susceptible to enzymatic hydrolysis than chemical hydrolysis in aqueous buffers. We also confirmed that the parent compound 2 was 100% stable at the last incubation time in both the aqueous and plasma stability experiments.</p><!><p>A representative set of prodrugs (acetyl 3, butanoyl 5, octanoyl 7, isobutyryl 9, and 2-ethylbutyryl 10) along with the parent drug 2 were then evaluated in the bidirectional Caco-2 cell transwell model to measure their permeability and potential for improved oral absorption. Because ester prodrugs can be hydrolyzed by esterases produced by Caco-2 cells, the concentration of both the parent compound and the prodrug were monitored. The permeability coefficients Papp of each prodrug for transport from the apical-to-basolateral (AP-BL) and the converse direction (BL-AP) were determined under initial velocity conditions (<10% transport of prodrug at the last time point) by measuring the transport of compound across the Caco-2 monolayer as a function of time. The permeability coefficients reported herein represent the sum of each prodrug and the parent drug 2 (produced by prodrug hydrolysis before, during, or after transport) detected in the acceptor compartment. The efflux ratio is the ratio of Papp values in the BL-AP and AP-BL direction. The results show the all of the prodrugs evaluated, except 7, had substantially lower permeation in AP-BL direction compared to that of parent drug 2 (Table 2). The prodrugs all had higher permeation in BL-AP direction resulting in high efflux ratios ranging from 1.5 to 8.5 compared to a value of just 0.3 for 2 (Table 2). These high efflux ratios suggest that these prodrugs will almost certainly have poor oral bioavailability.</p><p>Since Caco-2 cells contain esterases, we measured the residual amount of each prodrug remaining intact at 2 hours in both the apical and basolateral side. The amount of prodrug remaining in the apical side is represented by RAAP. Similarly, the amount of prodrug remaining in the basolateral side is represented as RABL (Table 2). The RAAP and RABL values also depend on which side of the monolayer, the prodrug is introduced. Thus, RAAP for AP-BL signifies the relative residual amount of prodrug in the apical solution when the donor solution is in the apical side. This value correlates the amount of hydrolyzed prodrug released by Caco-2 cells into the apical side. On the other hand, RAAP for BL-AP is the relative amount of prodrug in the apical side when the donor solution is in the basolateral side and corresponds to the amount of hydrolysis occurring while the prodrug is passing through the cell monolayer as well as after permeation in the apical side. Similarly, the relative amount of prodrug in the basolateral solution (RABL) was calculated for both conditions where the donor solution is in either the apical (AP-BL) or basolateral (BL-AP) side. As an example, octanoyl prodrug 7 was almost completely hydrolyzed during transport across the Caco-2 cells, with only 1 and 3% remaining at 2 hours in the apical and basolateral sides, respectively, after BL-AP and AP-BL permeation. The result also showed that the prodrugs are hydrolyzed not only during the permeation across the monolayer by cytosolic esterases, but also on both sides of the monolayer, presumably by secreted esterases.20,21 Additionally, Caco-2 cells contain more esterases on the apical side than on the basolateral side because the prodrugs were hydrolyzed to a greater extent on the apical side than on the basolateral side. Prodrugs with short promieties such as acetyl 3 or branched chain promoieties such as isobutyryl 9 and 2-ethylbutyryl 10 were hydrolyzed to a lesser extent in these experiments. The higher stability of these prodrugs was also observed in aqueous and plasma stability studies.</p><!><p>A systematic series of prodrugs of the 2′-fluoro nucleoside 2 with linear and α-branched alkanoyl groups from two to twelve carbons were synthesized through esterification at the 3′-OH position of the nucleoside in order to improve membrane permeability and oral bioavailability. We developed a scalable synthesis of the key intermediate 9-[2,5-dideoxy-2-fluoro-5-(N-sulfamoyl) amino-β-d-arabinofuranosyl]adenine 15 and reliable conditions for direct installation of the ester promoieties on the unprotected nucleoside. The ester prodrugs 3–10 were stable in aqueous conditions at pH 1.2 and 7.4 and surprisingly showed high serum stability in rat and human serum with most prodrugs showing less than 50% hydrolysis at 6 hours. By contrast, the prodrugs were hydrolyzed more rapidly in mouse serum with half-lives ranging from 0.7 to 4.6 hours for 3–9 showing a parabolic relationship with maximal cleavage rates for the octanoyl prodrug 7 (t1/2 = 0.7 hours). The cleavage rates decreased proportionately with alkanoyl chain-length from eight to two carbons and also decreased for longer and α-branched alkanoyl promoieties. The relative hydrolysis in rat and human serum, although substantially lower in magnitude, also paralleled this trend. Selected prodrugs were then evaluated in the Caco-2 transwell permeability assay to evaluate membrane permeability and potential for enhanced oral bioavailability relative to 2. Surprisingly, all of the prodrugs were found to have reduced permeation in the apical-to-basolateral direction with permeability coefficients (Papp) ranging from 0.4 to 3.9 × 10−6 cm/s, compared to 2, whose Papp was 4.2 × 10−6 cm/s. More significantly, all of the prodrugs were found to have enhanced permeation in the basolateral-to-apical direction with Papp's ranging from 1.6 to 6.9 × 10−6 cm/s, compared to 2, whose Papp was 1.2 × 10−6 cm/s. As a result the efflux ratios of 3–10 were found to be 5 to 28-fold greater than 2. These data indicate that despite greater cLogP values (Table 1), the prodrugs are substrates for efflux pumps and are also hydrolyzed intracellularly. As expected, the more sterically hindered isobutyryl prodrug 9 was hydrolyzed to a lesser extent than the butyryl prodrug 5, but the overall permeability was lower likely due to efflux pumps on the apical membrane. Further examination showed that the Caco-2 cells were able to hydrolyze the prodrugs with the same trend observed with the serum stability studies. Thus, octanoyl prodrug 7 was largely hydrolyzed by esterases on the apical side and only 29% percent remained in the apical compartment at 2 hours. The amount of intact prodrug 7 that was successfully transported across the Caco-2 monolayer during AP-BL experiment was only 1% of the total amount (2 + 7) indicating rapid hydrolysis by cellular esterases or efflux of 7 back into the apical compartment. These results collectively suggest that the described prodrug strategy using simple alkanoyl esters will not be successful to improve the oral bioavailability of 2. While this approach was ultimately unsuccessful, it has provided a useful benchmark to guide future studies and also highlights the potential contribution of intestinal epithelial cells for release of ester prodrugs, which in some cases may be faster than serum esterases.</p><!><p>All commercial reagents were used as provided unless otherwise indicated. 2′-Deoxy-2′-fluoroadenosine was purchased from Metkinen Chemistry, Finland. ACS and HPLC grade solvents were purchased from Fischer Scientific. An anhydrous solvent dispensing system using packed columns of neutral alumina was used for drying THF and CH2Cl2, while packed columns of 4 Å molecular sieves were used to dry DMF, and the solvents were dispensed under nitrogen. All reactions were performed under an inert atmosphere of argon in oven dried (130–150 °C) glassware. Thin-layer chromatography (TLC plate, Merck) was performed on a pre-coated silica gel 60 F254 plates. The detection of compounds was carried out with UV light. Flash chromatography was performed with silica gel P60 (Silicycle) with the indicated solvent system. All NMR spectra were recorded on Varian 400 or 600 MHz spectrometers. 1H NMR spectra were referenced to residual CDCl3 (7.27 ppm), DMSO-d6 (2.50 ppm), or CD3OD (3.31 ppm); 13C NMR spectra were referenced to CDCl3 (77.23 ppm) DMSO-d6 (39.51 ppm), or CD3OD (49.15 ppm). NMR chemical shift data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant, integration. Coupling constants are given in Hertz (Hz). High-resolution mass spectra (HRMS) were obtained on a Bruker BioTOF II instrument.</p><!><p>To a solution of 2′-deoxy-2′-fluoroadenosine 11 (1.70 g, 6.37 mmol) in DMF (60 mL) was added triphenylphosphine (4.96 g, 18.91 mmol, 3.0 equiv) and NaN3 (2.05 g, 31.54 mmol, 5.0 equiv). After 5 min at rt, the reaction mixture was cooled to 0 °C, CBr4 (6.27 g, 18.91 mmol, 3.0 equiv) was added, and the reaction mixture was gradually warmed to rt. After 24 h, the reaction was quenched with methanol and the resulting mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2, hexanes–EtOAc–EtOH, 1:3:0–0:100:5) to yield the title compound (1.44 g, 78%) as a white amorphous solid: Rf = 0.35 (5:95 EtOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 8.21 (s, 1H), 6.29 (dd, J = 19.4, 1.8 Hz, 1H), 5.53 (ddd, J = 52.8, 4.5, 1.8 Hz, 1H), 4.81 (ddd, J = 20.7, 12.5, 4.5 Hz, 1H), 4.21–4.14 (m, 1H), 3.74 (dd, J = 13.5, 2.8 Hz, 1H), 3.56 (dd, J = 13.5, 2.8 Hz, 1H); 13C NMR (100 MHz, CD3OD) δ 157.5, 154.2, 150.4, 141.5, 120.7, 94.8 (d, J = 185 Hz), 88.8 (d, J = 31 Hz), 83.0, 71.2 (d, J = 17 Hz), 52.4; HRMS (ESI+) m/z calcd for C10H11FN8O2Na+ [M + Na]+ 317.0881, found 317.0880 (Δ 0.3 ppm).</p><!><p>To a solution of azide 12 (3.17 g, 10.8 mmol, 1.0 equiv) in DMF (110 mL) was added imidazole (4.40 g, 64.6 mmol, 6.0 equiv), DMAP (1.32 g, 10.8 mmol, 1.0 equiv) and TBSCl (4.87 g, 32.3 mmol, 3.0 equiv) and the reaction mixture was stirred for 6 h at rt. At 6 h, more TBSCl (1.62 g, 10.8 mmol, 1.0 equiv) was added. After an additional 1 h at rt, the reaction was quenched with methanol and the resulting mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2, hexanes–EtOAc, 1:1–1:3) to yield the title compound (3.51 g, 80%) as a white amorphous solid: Rf = 0.40 (1:2 EtOAc/hexanes); 1H NMR (400 MHz, CD3OD) δ 8.25 (s, 1H), 8.21 (s, 1H), 6.26 (dd, J = 17.4, 1.6 Hz, 1H), 5.64 (ddd, J = 53.1, 4.6, 1.6 Hz, 1H), 5.11 (ddd, J = 18.9, 7.5, 4.6 Hz, 1H), 4.20–4.13 (m, 1H), 3.71 (dd, J = 13.7, 2.9 Hz, 1H), 3.44 (dd, J = 13.7, 4.2 Hz, 1H), 0.96 (s, 9H), 0.21 (s, 6H); 13C NMR (100 MHz, CD3OD) δ 157.6, 154.1, 150.4, 142.2, 120.8, 94.0 (d, J = 189 Hz), 89.0 (d, J = 35 Hz), 83.6, 72.2 (d, J = 16 Hz), 51.8, 26.3, 19.1, −4.5, −4.8; HRMS (ESI+) m/z calcd for C16H25FN8O2SiNa+ [M + Na]+ 481.1746, found 431.1758 (Δ 2.8 ppm).</p><!><p>To a solution of compound 13 (2.97 g, 7.28 mmol, 1.0 equiv) in methanol (70 mL) was added 10% Pd/C wetted with 55% water (2.00 g, TCI America). The flask was evacuated, purged with hydrogen (balloon pressure) and the reaction was stirred under an atmosphere of hydrogen for 4 h at rt. The resulting mixture was filtered through a short pad of Celite and the filtrate was concentrated under reduced pressure to give the corresponding amine as a white solid. To a solution of the solid in 1,4-dioxane (70 mL) was added sulfamide (2.80 g, 29.1 mmol, 4.0 equiv) and the resulting mixture was refluxed for 13 h. The reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column chromatography (SiO2, hexanes–EtOAc–EtOH, 1:3:0–0:100:15) to yield the title compound (2.73 g, 81% over 2 steps) as a white amorphous solid: Rf = 0.58 (5:95 EtOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.26 (s, 1H), 8.25 (s, 1H), 6.22 (dd, J = 14.3, 4.9 Hz, 1H), 5.63 (dt, J = 52.2, 4.9 Hz, 1H), 4.83 (ddd, J = 17.3, 8.6, 4.9 Hz, 1H), 4.32–4.25 (m, 1H), 3.44 (dd, J = 13.7, 2.8 Hz, 1H), 3.35 (dd, J = 13.7, 3.5 Hz, 1H), 0.96 (s, 9H), 0.19 (s, 3H), 0.17 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 157.7, 154.2, 150.1, 142.4, 121.1, 92.6 (d, J = 194 Hz), 89.1 (d, J = 32 Hz), 85.9, 72.9 (d, J = 15 Hz), 45.4, 26.4, 19.2, −4.48, −4.71; HRMS (ESI+) m/z calcd for C16H28FN7O4SSiNa+ [M + Na]+ 484.1569, found 484.1566 (Δ 0.6 ppm).</p><!><p>This was prepared using two different methods.</p><!><p>To a solution of sulfamide 14 (210 mg, 0.454 mmol) in methanol (12 mL) was added 4 M HCl in 1,4-dioxane (4.0 mL). After 3 h, the reaction mixture was concentrated under reduced pressure to yield the title compound as the hydrochloride salt (182 mg, quant.) as a white amorphous solid, which was used in the next step without further purification. Characterization data is shown below in Method B.</p><!><p>To a solution of azide 12 (21.2 mg, 0.0721 mmol) in methanol (1.0 mL) was added 10% Pd/C wetted with 55% water (20 mg). The flask was evacuated, back-filled with hydrogen and the reaction mixture was stirred under an atmosphere of hydrogen for 6 h at rt. The reaction mixture was filtered through a short pad of Celite and the filtrate was concentrated under reduced pressure to give the corresponding amine as a white solid. To a solution of the solid aminonucleoside in 1,4-dioxane (1.0 mL) was added sulfamide (27.7 mg, 0.288 mmol, 4.0 equiv) and the resulting mixture was refluxed for 17 h. The reaction mixture was concentrated under reduced pressure and the crude product was purified by flash column chromatography (SiO2, EtOAc–MeOH, 100:0–100:5–75:25) to yield the title compound (14.5 mg, 58% over 2 steps) as a white amorphous solid: Rf = 0.23 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 8.43 (s, 1H), 6.36 (dd, J = 16.9, 2.6 Hz, 1H), 5.49 (ddd, J = 52.4, 4.4, 2.6 Hz, 1H), 4.67 (ddd, J = 16.9, 6.7, 4.4 Hz, 1H), 4.28–4.21 (m, 1H), 3.49 (dd, J = 14.1, 3.2 Hz, 1H), 3.39 (dd, J = 14.1, 4.7 Hz, 1H); 1H NMR (400 MHz, DMSO-d6) δ 8.35 (s, 1H), 8.16 (s, 1H), 7.41 (s, 2H), 7.22 (dd, J = 7.1, 4.9 Hz, 1H), 6.60 (s, 2H), 6.24 (dd, J = 17.1, 3.7 Hz, 1H), 5.82 (d, J = 5.6 Hz, 1H), 5.54 (ddd, J = 52.8, 5.7, 3.7 Hz, 1H), 4.52 (ddd, J = 14.3, 11.0, 5.7 Hz, 1H), 4.17–4.07 (m, 1H), 3.36–3.26 (m, 1H), 3.21–3.09 (m, 1H); 13C NMR (100 MHz, CD3OD) δ 151.9, 149.6, 145.6, 144.7, 121.1, 94.6 (d, J = 189 Hz), 89.3 (d, J = 34 Hz), 84.1, 71.3 (d, J = 16 Hz), 45.0; 13C NMR (100 MHz, DMSO-d6) δ 156.3, 152.7, 148.6, 140.1, 119.4, 92.4 (d, J = 189 Hz), 86.2 (d, J = 32 Hz), 82.2, 69.6 (d, J = 16 Hz), 44.3; HRMS (ESI+) m/z calcd for C10H14FN7O4SNa+ [M + Na]+ 370.0704, found 370.0691 (Δ 3.5 ppm).</p><!><p>To a solution of alcohol 15 (1.0 mmol, 1.0 equiv) in pyridine (6.0 mL) was added acid chloride (3.0 equiv) or anhydride (6.0 equiv) at 0 °C and the reaction mixture was stirred until the starting material disappeared as monitored by TLC. The reaction was quenched with methanol and the resulting mixture was concentrated under reduced pressure. To a solution of the residue in methanol (15 mL) was added formic acid (1.5 mL). After 16 h at rt, the reaction mixture was concentrated under reduced pressure. The crude product was purified by flash column chromatography (SiO2, hexanes–EtOAc–MeOH, 1:1:0–0:100:0–0:100:10) to yield the corresponding compound.</p><!><p>Ester 16 (318 mg, 0.816 mmol, 88% in 2 steps) was obtained from alcohol 15 (356 mg, 0.928 mmol) and acetic anhydride as a pale yellow amorphous solid: Rf = 0.33 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.28 (s, 1H), 6.29 (dd, J = 15.9, 4.0 Hz, 1H), 5.94–5.71 (m, 1H), 5.70–5.58 (m, 1H), 4.48 (br s, 1H), 3.45 (br s, 2H), 2.17 (s, 3H); 13C NMR (100 MHz, CD3OD) δ 171.6, 157.5, 154.0, 150.1, 142.2, 121.1, 91.5 (d, J = 194 Hz), 89.2 (d, J = 33 Hz), 82.7, 72.8 (d, J = 14 Hz), 45.2, 20.6; HRMS (ESI+) m/z calcd for C12H16FN7O5SNa+ [M + Na]+ 412.0810, found 412.0806 (Δ 1.0 ppm).</p><!><p>Ester 17 (274 mg, 0.679 mmol, 78% in 2 steps) was obtained from alcohol 15 (336 mg, 0.874 mmol) and propionic anhydride as a white amorphous solid: Rf = 0.30 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.27 (s, 1H), 6.29 (dd, J = 15.6, 4.7 Hz, 1H), 5.82 (ddd, J = 51.4, 4.9, 4.7 Hz, 1H), 5.65 (ddd, J = 10.1, 4.9, 4.3 Hz, 1H), 4.52–4.43 (m, 1H), 3.51–3.39 (m, 2H), 2.49 (q, J = 7.4 Hz, 2H), 1.18 (t, J = 7.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 175.0, 157.6, 154.1, 150.1, 142.2, 121.1, 91.6 (d, J = 195 Hz), 89.2 (d, J = 32 Hz), 82.7, 72.7 (d, J = 14 Hz), 45.3, 28.1, 9.5; HRMS (ESI+) m/z calcd for C13H18FN7O5SNa+ [M + Na]+ 426.0966, found 426.0964 (Δ 0.5 ppm).</p><!><p>Ester 18 (317 mg, 0.759 mmol, 83% in 2 steps) was obtained from alcohol 15 (350 mg, 0.913 mmol) and butyric anhydride as a pale yellow amorphous solid: Rf = 0.35 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.27 (s, 1H), 6.29 (dd, J = 15.6, 4.7 Hz, 1H), 5.83 (ddd, J = 51.6, 5.1, 4.7 Hz, 1H), 5.66 (ddd, J = 10.1, 5.1, 4.7, 1H), 4.51–4.44 (m, 1H), 3.52–3.39 (m, 2H), 2.45 (t, J = 7.3 Hz, 2H), 1.76–1.64 (m, 2H), 0.99 (t, J = 7.3 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 174.1, 157.6, 154.2, 150.1, 142.2, 121.1, 91.6 (d, J = 194 Hz), 91.1 (d, J = 33 Hz), 82.7, 72.6 (d, J = 14 Hz), 42.3, 36.7, 19.5, 14.0; HRMS (ESI+) m/z calcd for C14H20FN7O5SNa+ [M + Na]+ 440.1123, found 440.1151 (Δ 6.3 ppm).</p><!><p>Ester 19 (322 mg, 0.746 mmol, 81% in 2 steps) was obtained from alcohol 15 (354 mg, 0.922 mmol) and pentanoic anhydride as a pale yellow solid: Rf = 0.40 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.28 (s, 1H), 6.38–6.21 (m, 1H), 5.95–5.70 (m, 1H), 5.72–5.58 (m, 1H), 4.54–4.39 (m, 1H), 3.57–3.37 (m, 2H), 2.58–2.35 (m, 2H), 1.78–1.52 (m, 2H), 1.52–1.31 (m, 2H), 1.06–0.82 (m, 3H); 13C NMR (100 MHz, CD3OD) δ 174.3, 157.3, 153.7, 150.1, 142.3, 121.1, 91.6 (d, J = 195 Hz), 89.3 (d, J = 33 Hz), 82.7, 72.6 (d, J = 14 Hz), 45.2, 34.5, 28.2, 23.3, 14.2; HRMS (ESI+) m/z calcd for C15H22FN7O5SNa+ [M + Na]+ 454.1279, found 454.1275 (Δ 0.9 ppm).</p><!><p>Ester 20 (340 mg, 0.719 mmol, 79% in 2 steps) was obtained from alcohol 15 (351 mg, 0.915 mmol) and octanoic anhydride as a white amorphous solid: Rf = 0.43 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.27 (s, 1H), 6.29 (dd, J = 15.4, 4.6 Hz, 1H), 5.83 (ddd, J = 51.5, 5.3, 4.6 Hz, 1H), 5.66 (ddd, J = 10.1, 5.3, 4.8 Hz, 1H), 4.52–4.45 (m, 1H), 3.52–3.41 (m, 2H), 2.47 (t, J = 7.3 Hz, 2H), 1.73–1.62 (m, 2H), 1.43–1.26 (m, 8H), 0.91 (t, J = 6.8 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 174.3, 157.7, 154.4, 150.1, 142.1, 121.1, 91.6 (d, J = 196 Hz), 89.2 (d, J = 32 Hz), 82.7, 72.6 (d, J = 14 Hz), 45.3, 34.8, 33.0, 30.2, 30.2, 26.1, 23.8, 14.5; HRMS (ESI+) m/z calcd for C18H28FN7O5SNa+ [M + Na]+ 496.1749, found 496.1736 (Δ 2.6 ppm).</p><!><p>Ester 21 (328 mg, 0.619 mmol, 68% in 2 steps) was obtained from alcohol 15 (350 mg, 0.913 mmol) and dodecanoic anhydride as a white amorphous solid: Rf = 0.48 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.28 (s, 1H), 8.27 (s, 1H), 6.29 (dd, J = 15.5, 4.7 Hz, 1H), 5.83 (ddd, J = 51.4, 5.1, 4.7 Hz, 1H), 5.66 (ddd, J = 10.0, 5.1, 4.7 Hz), 4.51–4.44 (m, 1H), 3.52–3.40 (m, 2H), 2.46 (t, J = 7.1 Hz, 2H), 1.72–1.62 (m, 2H), 1.42–1.23 (m, 16H), 0.89 (t, J = 6.5 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 174.3, 157.7, 154.4, 150.1, 142.1, 121.1, 91.5 (d, J = 196 Hz), 89.2 (d, J = 33 Hz), 82.7, 72.6 (d, J = 14 Hz), 45.3, 34.8, 33.2, 30.9, 30.9, 30.7, 30.6, 30.5, 30.3, 26.1, 23.9, 14.6; HRMS (ESI+) m/z calcd for C22H36FN7O5SNa+ [M + Na]+ 552.2375, found 552.2347 (Δ 5.0 ppm).</p><!><p>Ester 22 (324 mg, 0.777 mmol, 85% in 2 steps) was obtained from alcohol 15 (350 mg, 0.913 mmol) and isobutyric anhydride as a pale yellow solid: Rf = 0.38 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.29 (s, 1H), 8.29 (s, 1H), 6.40–6.19 (m, 1H), 5.97–5.67 (m, 1H), 5.73–5.55 (m, 1H), 4.55–4.37 (m, 1H), 3.59–3.36 (m, 2H), 2.81–2.59 (m, 1H), 1.23 (br s, 6H); 13C NMR (100 MHz, CD3OD) δ 177.5, 157.0, 153.3, 150.0, 142.5, 121.1, 91.7 (d, J = 194 Hz), 89.3 (d, J = 34 Hz), 82.6, 72.5 (d, J = 14 Hz), 45.2, 35.1, 19.5, 19.3; HRMS (ESI+) m/z calcd for C14H20FN7O5SNa+ [M + Na]+ 440.1123, found 440.1129 (Δ 1.4 ppm).</p><!><p>Ester 23 (251 mg, 0.563 mmol, 61% in 2 steps) was obtained from alcohol 15 (354 mg, 0.923 mmol) and 2-ethylbutyryl chloride as a pale yellow solid: Rf = 0.43 (5:95 MeOH/EtOAc); 1H NMR (400 MHz, CD3OD) δ 8.33 (s, 1H), 8.31 (s, 1H), 6.37–6.24 (m, 1H), 5.94–5.72 (m, 1H), 5.73–5.60 (m, 1H), 4.54–4.40 (m, 1H), 3.55–3.36 (m, 2H), 2.42–2.28 (m, 1H), 1.87–1.47 (m, 4H), 1.05–0.81 (m, 6H); 13C NMR (100 MHz, CD3OD) δ 176.6, 156.2, 152.2, 150.0, 142.8, 121.1, 91.9 (d, J = 194 Hz), 89.4 (d, J = 32 Hz), 82.5, 72.4 (d, J = 15 Hz), 50.2, 45.0, 26.2, 26.2, 12.3, 12.1; HRMS (ESI+) m/z calcd for C16H24FN7O5SNa+ [M + Na]+ 468.1436, found 468.1424 (Δ 2.6 ppm).</p><!><p>To a solution of the 3′-esterified nucleoside (1.0 equiv) in DMF (5.0 mL, 0.2 M in limiting reagent) was added 248 (1.0 equiv) and Cs2CO3 (2.5 equiv) at 0 °C and the reaction mixture was gradually warmed to rt. After 16 h, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2Cl2 and washed with 1 M HCl and brine. The organic layer was dried over MgSO4, filtered, and the solvent was removed under reduced pressure to give the corresponding coupled product. To a solution of the coupled product in CH2Cl2 (10 mL) was added methanol (5.0 mL) and 10% Pd/C wetted with 55% water (450 mg). The flask was evacuated, back-filled with hydrogen gas and the reaction mixture was stirred under an atmosphere of hydrogen for 16 h. The reaction mixture was filtered through a short pad of Celite and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (CH2Cl2–MeOH–Et3N, 100:0:2–100:10:2) to yield the corresponding title compound as the triethylammonium salt.</p><!><p>The title compound 3 (195 mg, 41% in 2 steps) was obtained from 16 (307 mg, 0.788 mmol) as a white amorphous solid: Rf = 0.20 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.33 (s, 1H), 8.22 (s, 1H), 7.94–7.91 (m, 1H), 7.32–7.27 (m, 1H), 6.86–6.82 (m, 1H), 6.82–6.78 (m, 1H), 6.21 (dd, J = 15.3, 4.5 Hz, 1H), 5.80 (dt, J = 51.6, 4.5 Hz, 1H), 5.61 (ddd, J = 10.5, 5.4, 4.5 Hz, 1H), 4.48–4.44 (m, 1H), 3.47 (dd, J = 13.6, 2.8 Hz, 1H), 3.42 (dd, J = 13.6, 4.0 Hz, 1H), 2.92 (q, J = 7.5 Hz, 6H), 2.17 (s, 3H), 1.20 (t, J = 7.4 Hz, 9H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 172.9, 169.8, 159.9, 155.4, 152.7, 148.5, 139.4, 132.6, 129.4, 119.1, 119.1, 117.8, 116.3, 90.0 (d, J = 195 Hz), 86.7 (d, J = 32 Hz), 80.6, 71.0 (d, J = 15 Hz), 45.9, 43.8, 19.6, 8.81; HRMS (ESI−) m/z calcd for C19H19FN7O7S [M − H]− 508.1056, found 508.1079 (Δ 4.5 ppm).</p><!><p>The title compound 4 (194 mg, 47% over 2 steps) was obtained from 17 (270 mg, 0.668 mmol) as a pale yellow amorphous solid: Rf = 0.23 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.33 (s, 1H), 8.22 (s, 1H), 7.94–7.90 (m, 1H), 7.32–7.26 (m, 1H), 6.86–6.83 (m, 1H), 6.83–6.78 (m, 1H), 6.21 (dd, J = 15.5, 4.3 Hz, 1H), 5.81 (ddd, J = 51.5, 4.6, 4.3 Hz, 1H), 5.61 (ddd, J = 10.1, 4.9, 4.6 Hz, 1H), 4.49–4.44 (m, 1H), 3.47 (dd, J = 13.8, 2.9 Hz, 1H), 3.43 (dd, J = 13.8, 4.0 Hz, 1H), 3.14 (q, J = 7.4 Hz, 6H), 2.46 (q, J = 7.2 Hz, 2H), 1.29 (t, J = 7.4 Hz, 9H), 1.18 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 173.2, 172.8, 159.9, 155.4, 152.7, 148.5, 139.4, 132.6, 129.4, 119.1, 119.1, 117.8, 116.3, 90.0 (d, J = 196 Hz), 86.8 (d, J = 33 Hz), 80.6, 70.9 (d, J = 14 Hz), 46.2, 43.9, 26.6, 8.2, 8.1; HRMS (ESI−) m/z calcd for C20H21FN7O7S [M − H]− 522.1213, found 522.1302 (Δ 17.0 ppm, slightly displaced by nearby internal standard peak).</p><!><p>The title compound 5 (260 mg, 57% over 2 steps) was obtained from 18 (300 mg, 0.719 mmol) as a white amorphous solid: Rf = 0.24 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.33 (s, 1H), 8.21 (s, 1H), 7.94–7.90 (m, 1H), 7.32–7.26 (m, 1H), 6.87–6.83 (m, 1H), 6.82–6.78 (m, 1H), 6.20 (dd, J = 15.6, 4.3 Hz, 1H), 5.81 (ddd, J = 51.4, 5.3, 4.3 Hz, 1H), 5.61 (dt, J = 10.6, 5.3 Hz, 1H), 4.49–4.44 (m, 1H), 3.47 (dd, J = 13.6, 3.0 Hz, 1H), 3.43 (dd, J = 13.6, 4.3 Hz, 1H), 3.09 (q, J = 7.3 Hz, 6H), 2.44–2.38 (m, 2H), 1.74–1.65 (m, 2H), 1.27 (t, J = 7.6 Hz, 9H), 0.98 (t, J = 7.6 Hz, 3H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 172.8, 172.4, 159.9, 155.4, 152.7, 148.5, 139.4, 132.6, 129.4, 129.4, 119.1, 117.8, 116.3, 90.0 (d, J = 196 Hz), 86.8 (d, J = 33 Hz), 80.7, 70.8 (d, J = 16 Hz), 46.2, 43.9, 35.2, 17.8, 12.8, 8.3; HRMS (ESI−) m/z calcd for C21H23FN7O7S [M − H]− 536.1369, found 536.1344 (Δ 4.7 ppm).</p><!><p>The title compound 6 (173 mg, 37% over 2 steps) was obtained from 19 (310 mg, 0.718 mmol) as a white amorphous solid: Rf = 0.26 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.33 (s, 1H), 8.20 (s, 1H), 7.94–7.90 (m, 1H), 7.32–7.27 (m, 1H), 6.86–6.83 (m, 1H), 6.83–6.78 (m, 1H), 6.20 (dd, J = 15.6, 4.4 Hz, 1H), 5.81 (ddd, J = 51.7, 4.9, 4.4 Hz, 1H), 5.60 (dt, J = 10.3, 4.9 Hz, 1H), 4.49–4.41 (m, 1H), 3.48 (dd, J = 13.8, 3.1 Hz, 1H), 3.43 (dd, J = 13.8, 4.1 Hz, 1H), 3.10 (q, J = 7.2 Hz, 6H), 2.46–2.41 (m, 2H), 1.68–1.61 (m, 2H), 1.43–1.35 (m, 2H), 1.27 (t, J = 7.2 Hz, 9H), 0.94 (t, J = 7.1 Hz, 3H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 172.9, 172.6, 160.0, 155.4, 152.8, 148.6, 139.4, 132.7, 129.4, 119.2, 119.2, 117.9, 116.4, 90.0 (d, J = 196 Hz), 86.8 (d, J = 32 Hz), 80.7, 70.9 (d, J = 15 Hz), 46.2, 43.9, 33.1, 26.4, 21.7, 13.0, 8.3; HRMS (ESI−) m/z calcd for C22H25FN7O7S [M − H]− 550.1526, found 550.1543 (Δ 3.1 ppm).</p><!><p>The title compound 7 (250 mg, 52% over 2 steps) was obtained from 20 (329 mg, 0.695 mmol) as a white amorphous solid: Rf = 0.30 (10:90 MeOH/CH2Cl2); 1H NMR (400 MHz, CD3OD) δ 8.31 (s, 1H), 8.30 (s, 1H), 7.90–7.83 (m, 1H), 7.30–7.22 (m, 1H), 6.82–6.72 (m, 2H), 6.26 (dd, J = 15.7, 4.2 Hz, 1H), 5.85 (ddd, J = 51.7, 5.1, 4.2 Hz, 1H), 5.67 (dt, J = 10.6, 5.1 Hz, 1H), 4.47–4.39 (m, 1H), 3.43–3.35 (m, 2H), 3.16 (q, J = 7.4 Hz, 6H), 2.45–2.35 (m, 2H), 1.69–1.57 (m, 2H), 1.39–1.23 (m, 8H), 1.26 (t, J = 7.4 Hz, 9H), 0.90 (t, J = 6.4 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 174.4, 174.2, 162.0, 157.6, 154.5, 150.4, 141.8, 134.1, 131.1, 121.1, 120.9, 119.3, 118.0, 92.0 (d, J = 195 Hz), 88.8 (d, J = 32 Hz), 82.5, 72.8 (d, J = 15 Hz), 48.0, 45.7, 34.7, 33.0, 30.2 (×2), 26.1, 23.8, 14.6, 9.5; HRMS (ESI−) m/z calcd for C25H31FN7O7S [M − H]− 592.1995, found 592.1967 (Δ 4.7 ppm).</p><!><p>The title compound 8 (175 mg, 39% over 2 steps) was obtained from 21 (317 mg, 0.598 mmol) as a white amorphous solid: Rf = 0.36 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.34 (s, 1H), 8.20 (s, 1H), 7.94–7.90 (m, 1H), 7.33–7.27 (m, 1H), 6.87–6.83 (m, 1H), 6.83–6.78 (m, 1H), 6.20 (dd, J = 15.5, 4.1 Hz, 1H), 5.81 (ddd, J = 51.6, 5.1, 4.1 Hz, 1H), 5.60 (dt, J = 10.1, 5.1 Hz, 1H), 4.50–4.42 (m, 1H), 3.47 (dd, J = 13.9, 3.2 Hz, 1H), 3.43 (dd, J = 13.9, 4.1 Hz, 1H), 3.16 (q, J = 7.6 Hz, 6H), 2.48–2.38 (m, 2H), 1.70–1.62 (m, 2H), 1.38–1.22 (m, 16H), 1.30 (t, J = 7.6 Hz, 9H), 0.88 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 172.6 (2C), 172.6, 159.9, 155.4, 152.8, 148.6, 139.4, 132.7, 129.4, 119.2, 119.0, 117.9, 116.4, 90.0 (d, J = 197 Hz), 86.8 (d, J = 32 Hz), 80.7, 70.9 (d, J = 14 Hz), 46.3, 43.9, 33.3, 31.4, 29.1 (2C), 29.0, 28.9, 28.8, 28.6, 24.3, 22.2, 13.4, 8.1; HRMS (ESI−) m/z calcd for C29H39FN7O7S [M − H]− 648.2621, found 648.2592 (Δ 4.5 ppm).</p><!><p>The title compound 9 (193 mg, 41% over 2 steps) was obtained from 22 (309 mg, 0.741 mmol) as a pale yellow amorphous solid: Rf = 0.24 (10:90 MeOH/CH2Cl2); 1H NMR (600 MHz, 2:1 CDCl3–CD3OD) δ 8.33 (s, 1H), 8.21 (s, 1H), 7.95–7.90 (m, 1H), 7.32–7.27 (m, 1H), 6.86–6.83 (m, 1H), 6.83–6.78 (m, 1H), 6.20 (dd, J = 15.7, 4.6 Hz, 1H), 5.81 (dt, J = 51.2, 4.6 Hz, 1H), 5.60 (ddd, J = 10.0, 5.2, 4.6 Hz, 1H), 4.50–4.42 (m, 1H), 3.47 (dd, J = 13.7, 3.2 Hz, 1H), 3.43 (dd, J = 13.7, 4.3 Hz, 1H), 3.15 (q, J = 7.2 Hz, 6H), 2.73–2.65 (m, 1H), 1.29 (t, J = 7.2 Hz, 9H), 1.24 (d, J = 5.7 Hz, 3H), 1.22 (d, J = 5.7 Hz, 3H); 13C NMR (150 MHz, 2:1 CDCl3–CD3OD) δ 175.9, 172.8, 159.9, 155.4, 152.8, 148.6, 139.5, 132.7, 129.4, 119.2, 119.1, 117.9, 116.4, 90.0 (d, J = 196 Hz), 86.9 (d, J = 33 Hz), 80.7, 70.8 (d, J = 16 Hz), 46.3, 44.0, 33.4, 18.3, 18.1, 8.2; HRMS (ESI−) m/z calcd for C21H23FN7O7S [M − H]− 536.1369, found 536.1373 (Δ 0.7 ppm).</p><!><p>The title compound 10 (185 mg, 51% over 2 steps) was obtained from 23 (241 mg, 0.542 mmol) as a white amorphous solid: Rf = 0.33 (10:90 MeOH/CH2Cl2); 1H NMR (400 MHz, CD3OD) δ 8.32 (s, 1H), 8.31 (s, 1H), 7.89–7.83 (m, 1H), 7.30–7.22 (m, 1H), 6.82–6.72 (m, 2H), 6.26 (dd, J = 16.1, 4.0 Hz, 1H), 5.88 (ddd, J = 51.6, 5.1, 4.0 Hz, 1H), 5.69 (dt, J = 10.7, 5.1 Hz, 1H), 4.47–4.40 (m, 1H), 3.41–3.35 (m, 2H), 3.11 (q, J = 7.4 Hz, 6H), 2.38–2.27 (m, 1H), 1.73–1.50 (m, 4H), 1.24 (t, J = 7.4 Hz, 9H), 0.97–0.88 (m, 6H); 13C NMR (100 MHz, CD3OD) δ 176.5, 174.5, 162.0, 157.6, 154.5, 150.4, 141.9, 134.1, 131.0, 121.1, 120.9, 119.3, 118.0, 92.0 (d, J = 195 Hz), 88.9 (d, J = 32 Hz), 82.6, 72.8 (d, J = 15 Hz), 50.2, 48.0, 45.8, 26.2 (2C), 12.3, 12.1, 9.6; HRMS (ESI−) m/z calcd for C23H27FN7O7S [M − H]− 564.1682, found 564.1703 (Δ 3.7 ppm).</p><!><p>Reversed-phase HPLC analysis was performed on an Agilent 1260 instrument (Agilent Technologies, Santa Clara, CA) equipped with a Phenomenex Gemini C18, 5 micron, 4.6 × 250 mm column (Phenomenex, Torrence, CA). The mobile phase consisted of 20 mM triethylammonium bicarbonate (TEAB) buffer, pH 7.5 as aqueous (A) and acetonitrile as organic (B) components. The 20 mM TEAB buffer was prepared by freshly diluting a 1 M stock which, in turn, was prepared by bubbling CO2 into 1 M aqueous trimethylamine at 4 °C until the pH reached 7.5. Elution was performed with the following gradient: 5 to 70% B over 10 min, 90% B from 10 to 12 min, isocratic at 90% B from 12 to 16 min, and re-equilibration at 5% B for 3 min as a postrun before the next injection. The retention times and purity of the parent drug 2 and prodrugs 3–10 are listed in the supporting information (Table S1) along with HPLC traces. The detection wavelength was set at 254 nm and the flow rate was 1.0 mL/min. The precision and accuracy of the analytical methods was ≤ 10% at all concentrations. Standard curves were processed in duplicate with the samples.</p><!><p>A previously described method was followed to determine the stability of prodrugs in aqueous buffers.22 The parent drug 2 and prodrugs 3–10 (100 µM) were incubated at 37 °C in simulated gastric fluid (pH 1.2) and 100 mM HEPES (pH 7.4). In triplicate, buffers (980 µL) were pre-incubated for 5 min at 37 °C, 20 µL of a 20.0 mM DMSO stock solution of parent drug 2 or prodrugs 3–10 were added, the mixtures were briefly vortexed (3 sec), and incubated at 37 °C. Aliquots (100 µL) were removed from the incubation solution at 0, 5, 10, 15, 30, 45, 60, and 120 min, and immediately placed on ice and injected (50 µL) onto the HPLC. Once we realized that the prodrugs are quite stable in these buffers, only 0 and 120 min time points were used for subsequent HPLC analysis. Stability was determined by calculating the percentage of prodrug remaining after the 120 min incubation time.</p><!><p>The plasma stability of prodrugs was investigated with CD-1 female pooled mouse plasma, Sprague-Dawley female pooled rat plasma, and female pooled human plasma (BioChemed, Winchester, VA). In triplicate, plasma (980 µL) was pre-incubated for 5 min at 37 °C followed by the addition of 20 µL of a 10.0 mM DMSO stock solution of 2 and prodrugs 3–10. Aliquots (100 µL) were withdrawn at 0, 10, 30, 60, 120, 240, and 360 min and immediately quenched with an equal volume of 10% TCA to precipitate the proteins. The samples were vortexed, centrifuged (30 sec at 15,000 × g), and 140 µL of the supernatant were collected in HPLC vials and kept in ice. Samples were analyzed by injecting 100 µL of samples onto the HPLC. The stability of the prodrugs was determined by calculating the half-lives of the parent compounds.</p><!><p>Studies to determine permeability of prodrugs in Caco-2 monolayers followed an adapted protocol described by Hubatsch and co-workers23 and a method previously published from our group.16 Caco-2 cells (ATCC® HTB-37™) from the stock were thawed and cultivated in Dulbecco's modified Eagle's medium (DMEM) media containing 17% fetal calf serum and 1% PEST (penicillin 10,000 U mL−1, streptomycin 10,000 µg mL−1). Cells of desired passage number (50–70) were seeded on transwell membrane plate (12-well, 12 mm insert, 0.4 µm pore, Corning No. 3401) and grown for 21 days (37 °C, CO2 incubator, 5% CO2, water saturated atmosphere). The medium was changed every other day to support the required amount of growth. On the day of experiment, the media in the plates was aspirated and the apical side washed twice with cell assay buffer (CAB) pH 6.0 (3.56 g NaCl, 1.05 g NaHCO3, 0.9 g glucose, 0.975 g MES, 0.112 g KCl, 0.147 g MgSO4, 0.10 g CaCl2, 0.035 g K2HPO4 in 500 mL of water). The basolateral side was washed with CAB pH 7.4 (Same composition as CAB 6.0, except 1.19 g HEPES instead of MES). The apical side was filled with 0.5 mL of CAB, pH 6.0, and the basolateral side was filled with 1.5 mL of CAB, pH 7.4. With an EVOM epithelial voltammeter, electrical resistance across the differentiated Caco-2 cells were measured in triplicate. Only the wells with transepithelial electrical resistance (TER) of more than 300 Ωcm2 were used for the studies. The plate with the CAB was preincubated in an incubator shaker for 60 min, 65 rpm. Donor solution (2.5 mL) for the apical side was made with the test compound (12.5 µL, 20 mM DMSO stock), luciferin yellow (12.5 µL, 20 mM in water) and CAB pH 6 (2.475 mL). Donor solution (6 mL) for the basolateral side was made with the test compound (30 µL, 20 mM in DMSO), luciferin yellow (30 µL, 20 mM in water) and CAB pH 7.4 (5.97 mL). The concentration of test compound in both donor solutions was 100 µM. Luciferin yellow was added to assess the intactness of tight junctions in the donor solutions. The CAB solutions were aspirated and the experiment was initiated by filling the apical side with 0.5 mL of donor solution and the basolateral side with 1.5 mL of donor solution. Samples (100 µL) were taken out at 0, 30, 60 and 120 minutes in triplicates from both the apical and basolateral sides and were analyzed by HPLC. Papp AP-BL and Papp BL-AP, and efflux ratio were calculated based on the cumulative sampling, correcting for the successive dilutions. The Papp values were calculated using equation (1): (eq 1)Papp=(dQdt)(1AC0) where (dQdt) is the steady state flux, A is the surface area of the filter (cm2) and C0 is the initial concentration in the donor chamber.</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> Supplementary data </p><p>Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.xxxx.xx.xxx.</p>

PubMed Author Manuscript

The following article has been submitted to J. Chem Phys (2021) Communication: Effect of Oxidation on Excited State Dynamics of Neutral TinO2n-x (n<10, x<4) Clusters

Excited state lifetimes of neutral titanium oxide clusters (TinO2n-x, n < 10, x < 4) were measured using a sequence of 400 nm pump and 800 nm probe femtosecond laser pulses. Despite large differences in electronic properties between the closed shell stoichiometric TinO2n clusters and the suboxide TinO2n-x (x = 1-3) clusters, the transient responses for all clusters contain a fast response of 35 fs followed by a sub-picosecond excited state lifetime. In this non-scalable size regime, subtle changes in the sub-ps lifetimes are attributed to variations in the coordination of Ti atoms and localization of charge carriers following UV photoexcitation. In general, clusters exhibit longer lifetimes with increased size and also with addition of O atoms. This suggests that removal of O atoms develops stronger Ti-Ti interactions as the system transitions from a semiconducting character into a fast metallic electronic relaxation mechanism.

the_following_article_has_been_submitted_to_j._chem_phys_(2021)_communication:_effect_of_oxidation_o

INTRODUCTION<!>EXPERIMENTAL METHODS<!>TITANIUM OXIDE CLUSTER DISTRIBUTION<!>SIZE EFFECT ON SUBOXIDE CLUSTER LIFETIMES<!>OXIDATION EFFECT ON SUBOXIDE CLUSTER LIFETIME<!>CONCLUSION<!>Supporting Information<!>Corresponding Author

<p>Bulk titania (TiO2) materials are the subject of numerous experimental and theoretical investigations 1,2 due to their wide application in water splitting, [3][4][5] white pigments, 6 and photocatalysis. 7 The most important aspect of photoactivity is the production of charge carriers with sufficient lifetimes to participate in chemical reactions. Absorption of a photon exceeding the optical gap results in the creation of an exciton, or electron-hole pair, that can recombine through non-radiative processes such as internal conversion or relax through electron scattering. In strongly correlated materials, such as titania, the charge carriers are coupled to lattice vibrations to form polarons, which serve to trap mobile carriers by reducing their mobility and photoconversion yields. Polaron formation is affected by the local geometry of the material, where despite identical chemical compositions, recombination is two orders of magnitude faster in the rutile phase than the anatase phase of TiO2. 8,9 TiO2 nanoparticles follow two relaxation channels, 10 with holes and electrons relaxing separately over ns to μs dependent upon the particle size and structure. [11][12][13] However, the factors affecting these separate lifetimes and how to control these mechanisms remains poorly understood.</p><p>A major limitation for bulk titania is the large bandgap which limits electron transport. Defect engineering has become a major focus for titanium oxides, where oxygen deficient materials contain a smaller bandgap, increasingly delocalized density of states (DoS) 14,15 and therefore can utilize more of the sun's visible spectrum. Suboxides of titania, referred to as TinO2n-x (x > 0), are easily produced from bulk TiO2 materials, [16][17][18] are non-toxic, 19 possess unique optical properties, 18,20 and may have increased catalytic activity over their stoichiometric counterpart. 17,21 In particular, Magnéli phase titanium oxides (TinO2n-1, n = 4-9) display enhanced electrical conductivity 22 and increased stability over stoichiometric titania. 23 The overall reactivity of bulk titanium oxides is thought to be heavily dependent on O vacancies and associated Ti 3+ sites, 24,25 yet a precise understanding of their influence on the behavior of excitons, polarons, and free charge carriers in titania is needed.</p><p>Clusters serve as atomically precise models that enable fundamental studies on the factors that affect charge carrier recombination in bulk materials. Similar to the band structure and photoabsorption in bulk titania, stoichiometric (TinO2n) clusters are closed shell systems with low energy photoexcitation, described as an electron transfer from the O 2p-orbitals to the Ti 3d-orbitals or ligand to metal charge transfer (LMCT). Our measurements on the excited state lifetimes of sub-nanometer TinO2n (n < 10) clusters found their large optical gap acts as a relaxation bottleneck, making the properties of the S1 state strongly correlated with the measured lifetimes. 26 The variation in sub-picosecond relaxation dynamics revealed the local cluster geometry and charge carrier separation to be important in adjusting excited state lifetimes. 26 Similarly, suboxide clusters act as models for the distortions caused by O vacancies on the surfaces of titania materials. However, the electronic properties of suboxide clusters are quite different due to the presence of partially filled 3d-orbitals, dramatically changing the DoS below the photoexcitation energy and are therefore expected to possess different excited state dynamics. Here, fs pump-probe spectroscopy is employed to measure the excited state lifetimes of suboxide TinO2n-x clusters (n < 10, x < 4). This study supplies a foundation for understanding molecular-scale titania, which will ultimately lead to the production of materials with improved reactivity.</p><!><p>The experimental methods and instrumental setup were described previously. 26,27 Briefly, neutral clusters were produced through ablation of a pure 0.25" diameter Ti rod by the second harmonic of a pulsed Nd:YAG laser in the presence of a seeded He gas pulse (1% O2). The ablation plume was confined to a 1 x 60 mm collision region and reduced to a molecular beam diameter of 2 mm by a charged skimmer, deflecting all but the neutral clusters. Neutral clusters were ionized by a sequence of 35 fs laser pulses from a Ti:Sapphire laser and analyzed using a home-built Wiley-McLaren 28 type time-of-flight mass spectrometer. The ionized clusters separated in arrival time due to their m/z ratio within a fieldfree region and were subsequently recorded using a microchannel plate (MCP) detector. The 400 nm (3.1 eV) pulse was used to excite (pump) the clusters to an intermediate state and the 800 nm (1.55 eV) probe pulse was sent through a programmed delay-stage before recombining with the 400 nm beam for ionization. A delay to the 800 nm laser pulse was scanned by 10 fs steps and the change of intensity with delay was recorded. An average of 200 shots per time step as the probe was delayed from -1.6 -6.8 ps. The pump-probe instrumental response function (IRF) of 35 fs was measured using non-resonant excitation of Ar gas. Mass spectra were recorded using 400 nm pump and 800 nm probe pulses of 9.9 x 10 14 W/cm 2 and 3.1 x 10 15 W/cm 2 intensity, respectively. All transient signals were fit using a combination of two Gaussian functions convoluted with an exponential decay to account for the relaxation lifetimes. 27,29 3. RESULTS AND DISCUSSION</p><!><p>Ionization of the neutral titanium oxide molecular beam by the 400 nm pump and 800 nm probe lasers at temporal overlap produced a mass spectrum of Ti2O to (TiO2)11 (Fig. 1). The primary clusters recorded follow the series of TinO2n-x (x = 0-3) and grow through addition of TiO2 units. The cluster distribution is consistent with previous studies, 30 with the highest intensity peaks generally composed of TinO2n-1 or TinO2n-2, and is in agreement with their stabilities. 15,31 Neutral stoichiometric (TinO2n) clusters have previously only been recorded experimentally using single photon (VUV) ionization thought to be void of fragmentation, 32 supporting that fragmentation is not significant in our experiment. Thus, the formation of O deficient clusters is due to kinetic limitations during the growth of clusters in the ablation plasma.</p><p>Sub-nm titanium oxide clusters form hollow cage-like geometries 33,34 that are different from the bulk lattice structure. The cluster geometries and electronic structures were calculated using time-dependent density functional theory and CAM-B3LYP functional as described in detail in other publications. 26,31 The two terminal O atoms in TinO2n clusters have the lowest binding energy, making the geometry of TinO2n-1 clusters similar to TinO2n but lacking one dangling O atom. The lack of one dangling O atom causes the HOMO to shift from an O 2p-orbital onto a Ti 3d-orbital making both the HOMO and LUMO primarily localized on Ti atoms. 31,35 Therefore, photoexcitation in suboxides is a d-d transition and occurs with a much smaller optical gap. 35 The TinO2n-</p><!><p>Surprisingly, despite the large changes to the electronic and structural characteristics of the clusters as they gain and lose O atoms, 26,31 their excited state dynamics remain roughly consistent. The transient signals for all clusters contain a fast (35 fs) response and a sub-ps relaxation lifetime (τ). No longlived states are recorded, and the ratios (κ) of fast/sub-ps fitting coefficients are similar (Table 1). These similarities suggest that the sub-nm scale is perhaps the most important feature, and that relaxation is efficient in these sub-nm clusters due to the restricted proximity of their diameter. The fast component of the transient signal is attributed to a rapid relaxation of a nonresonant excited state. The remaining sub-ps transient ion signal is proportional to the neutral cluster's intermediate excited state population as it relaxes to lower energy. Pumpprobe transients of the TinO2n-1 (n < 10) cluster series is presented in Figure 2. Transient signals of the remaining suboxide clusters are presented in the Supplemental Information (Fig. S1 and Fig. S2).</p><p>Clusters represent a size regime of non-scalable properties, where every atom impacts the collective electronic and structural properties. Therefore, subtle differences in the excited state lifetimes highlight the variation of cluster geometries and electronic structures on dynamics and excited state lifetimes. Although there is variation in the lifetimes of all cluster series, each TinO2n-x series (x = 0-3) exhibits a gradual increase in excited state lifetime with the addition of TiO2 units (Table 1), due to a combination of increased charge carrier separation and overall increase in bonding coordination.</p><p>Despite the similarities, each cluster series exhibits a unique trend as they grow in size. A near linear increase in lifetime occurs with size in TinO2n-1 clusters up to n = 7, with the exception of Ti6O11 (Fig. 3b). All clusters in this series (except Ti6O11) contain a mirror plane to stabilize the reduced Ti atoms. 31 Thus, due to a lack of symmetry, the tri-coordinated Ti site of Ti6O11 may retain additional d-electrons that facilitate a faster decay, deviating from the trend. The TinO2n-2 clusters are more compact, given the absence of any terminal O atoms, and universally have shorter lifetimes. The TinO2n-2 cluster series of n ≥ 4 shows an oscillatory nature (Fig. 3c), where oddnumbered clusters have a longer lifetime over even-numbered clusters due to a higher localization of charge carriers. The lifetimes of the TinO2n-3 series also alternate with increasing cluster size and is most pronounced for the smallest cluster sizes (Fig. 3d). Both oxygen-deficient series exhibit the opposite behavior from the TinO2n series, where even-numbered clusters exhibited longer lifetimes than odd-numbered clusters. 26</p><!><p>The oxidation states of the Ti atoms are commonly assumed to involve complete electron transfer, where each O atom removes 2 electrons from the Ti atoms. Removal of each O atom from the stoichiometric cluster returns two d-electrons to the Ti atoms. Therefore, the suboxides contain many delocalized d-electrons that should influence the metallicity and consequently the relaxation dynamics of the cluster. Unfortunately, measurements of metallic behavior, such as conductivity, are not possible for clusters of just a few atoms. Another indicator for metallicity is short excited state lifetimes [36][37][38] and has been well established for small metal clusters. [39][40][41][42][43] Metallic and nonmetallic properties can be identified by the different relaxation behaviors of optically excited states. Strong interactions between delocalized valence d-electrons causes relaxation in metallic species on the fs timescale via Auger-like electron-electron scattering, whereas a weak coupling between electronic excitation and nuclear motion facilitates long (picosecond or longer) lifetimes of electron-hole characteristics in non-metallic semiconductors. Metallic scattering processes dominate if there are many delocalized electrons and a sufficiently high DoS, such as is the case even in small clusters. Thus, the relaxation by electron scattering processes results in many electrons occupying lowlying excited states, similar to the bulk.</p><p>However, internal conversion is an alternative possible pathway of relaxation and cannot be ruled out as contributing here. Small molecules can exhibit excited state lifetimes on the order of 10s of fs, particularly when there is passage through a conical intersection between two potential energy surfaces. Despite the well-known role of dangling O atoms in facilitating energy relaxation through conical intersections, 44 clusters void of terminal O atoms exhibit similar lifetimes to those with them. This suggests that internal conversion is not driving the relaxation. Further, the clusters are sufficiently small such that electrostatic interactions between the hole and electron are efficient for relaxation. The transient signals of neutral Ti2O4-x (x < 4) clusters reveal an increase in excited state lifetimes with oxidation (Fig. 4). Here, each O atom changes the oxidation state of the Ti atoms linearly, from a formal oxidation state of +3 (Ti2O3) and +2 (Ti2O2) which decreases the lifetime by 15% and 26% from the stoichiometric cluster, respectively. Geometries of Ti2O4-x (x < 4) clusters are well established 31,45 (Fig. 4). Ti2O4 is the least rigid cluster (containing two terminal O) and therefore should be the easiest to traverse a conical intersection since internal conversion is less effective in rigid clusters. Yet, it contains the longest lifetime of the series. In contrast, the more rigid ring structure of Ti2O2 contains no dangling O atoms and has a faster relaxation, suggesting that internal conversion is not the dominant relaxation mechanism occurring here. Although the number of relaxation pathways decrease with the removal of O atoms, the bond distances shorten 31 and d-orbital occupancy increases, resulting in a faster relaxation. This influence of O content on lifetime aligns with a metallic to insulator transition occurring with oxidation. Thus, our data supports that relaxation in clusters occurs via Auger-like electron scattering processes similar to bulk metals.</p><p>The transient response in O deficient Ti3O6-x (x < 4) clusters is different than the other cluster series (Fig. 5). The fully oxidized cluster, Ti3O6, has the shortest excited state lifetime in the Ti3On series, while Ti3O3 shows a longer lifetime. However, the Ti3O3 transient contains an unreliable sub-ps component with high experimental noise. Therefore, the high variation in κ is not expected to indicate any valuable scientific trend. Ti3O6 is unique among the stoichiometric clusters in that it contains a tri-coordinated Ti atom and tri-coordinated O atom, which are not present in other stoichiometric (n < 6) structures. 15,26,35 The tri-coordinated Ti atom sites are common in clusters exhibiting suppressed lifetimes. Ti3O5 exhibits a slightly longer lifetime, even though it is further undercoordinated, due to presence of partially filled d orbitals which are delocalized across two Ti atoms. In general, the delocalization of the d orbitals is correlated with extended lifetimes in suboxides. Similar to the Ti2O4-x clusters, Ti4O8-x (x < 4) clusters also highlight an increased lifetime with oxidation (Fig. 3a), supporting a metallic to semiconducting transition. Ti4O8-x (x < 4) clusters have 3D structures with the Ti atoms forming a tetrahedron core and O atoms bridging the Ti atoms or as terminal groups for Ti4O7 and Ti4O8. The Td symmetry of Ti4O6 lacks terminal O atoms and also contains an increased number of d-electrons which manifest in a large decrease in lifetime. Ti4O5 is similar to Ti4O6 but contains one less bridging O atom that reduces the Ti-O coordination and decreases the Ti-Ti bond length, resulting in a slightly shorter lifetime. Although there is minimal change in geometry in Ti4O8-x (x < 4) clusters, a higher d-orbital occupancy occurs with decreased O atoms from Ti4O8, 35 resulting in a linear decrease in the excited state lifetime.</p><p>Lifetimes of Ti5O10-x (x < 4) clusters do not change significantly with O (Fig. 3a). The similar lifetimes are due to a similar Cs symmetry and align with the size transition between local and global excitations, where charge carrier delocalization no longer fills the cluster diameter. Further, Ti5O10 exhibits a slightly reduced lifetime in the stoichiometric series, bringing the dynamics closer to Ti5O9. Interestingly, several stoichiometric clusters exhibit shortened lifetimes that deviate from the proposed metallic trend. The TinO2n clusters, where n = (3,5,6), contain a tri-coordinated Ti atom instead of the fully tetra-coordinated Ti atoms of the other clusters, and therefore may retain d-electrons that reduce their lifetimes. Such incomplete electron transfer leading to an atypical 3+ oxidation state is proposed for clusters as small as TiO2. 46 Larger clusters (TinO2n-x, n = 6-9) follow similar trends in excited state behavior (Fig. 3a). Generally, the TinO2n-1 and TinO2n clusters show longer lifetimes, and exhibit similar lifetimes due to related structures and possibly incomplete electron transfer, leading to retention of d-electrons on the stoichiometric cluster. 15 Further, in TinO2n clusters, the excited state avoids localization on the Ti atoms with terminal Ti-O bonds, 26 which ensures that the excited states behave similarly in the various O deficient clusters and accounts for the minimum influence of lifetime with oxidation. Clusters without terminal O atoms (TinO2n-2 and TinO2n-3) show shorter lifetimes and increased d-electron occupancy, indicating that the delectron scattering is a dominant mechanism affecting dynamics and that bridging O atoms have a minor effect on excited state lifetimes.</p><p>A particular outlier to the described trends is Ti7O13, which exhibits a significantly longer lifetime than Ti7O14 (Fig. 3a). This switched behavior is attributed to its unique structural features, where removal of an O atom from Ti7O14 drives a significant compression of the local Ti-O bonds and creates a new bond forming a tetra-coordinated Ti site adjacent to a tetracoordinated O atom. This high coordination site may account for its exceptionally long lifetime, in opposition to tricoordinated Ti sites facilitating fast relaxation. Further, Ti7O13 exhibits the largest separation of charge carriers and electron delocalization, supporting that this delocalization of is correlated with lifetime.</p><p>Clusters generally exhibit longer lifetimes with higher oxidation and shorter lifetimes upon removal of O atoms. Although the optical gap of suboxides decreases by ~3 eV from the stoichiometric cluster, 15 it does not have a significant influence on the excited state lifetime. This suggests that removal of O atoms develops metallic Ti-Ti bonds of lower coordination, causing the system to transition into a fast scattering-type electronic relaxation mechanism. This is consistent with the idea that as the clusters become more metallic, the lifetimes decrease. Excited state lifetimes are modified by electron-hole interactions which are influenced by Ti bond coordination and cluster size. These results suggest that enhanced excited state lifetimes in bulk titania materials may be achieved through manufacturing structures similar to the Ti7O13 cluster that contain delocalized d-electrons and higher Ti coordination.</p><!><p>The low-lying excited state lifetimes of neutral TinO2n-x (n = 1-9 and x < 4) clusters were measured using fs pump-probe spectroscopy, and trends in their transient signals related to the size and oxidation are presented. An oscillation in lifetimes as clusters grow in size is attributed to structural differences between the clusters that control charge localization and polaron-like formation. The signal returns to baseline for all clusters, suggesting that relaxation is efficient for these sub-nm materials. We show that the level of coordination increases with cluster size, related to a longer lifetime. The excited state lifetimes of titanium oxide clusters change with oxidation, which affect the Ti coordination and charge carrier localization. The lifetimes show a behavior consistent with a metallic to semiconducting transition with oxidation and related removal of d-electrons from the system. The fundamental information provided herein leads to a deeper understanding of the factors affecting O vacancies in bulk-scale titania materials and will assist in the production of future catalysts of increased reactivity.</p><!><p>See supplemental material for experimental measurements and fitting of larger TinO2n-x (n = 4-9, x < 4) clusters.</p><!><p>*Scott.Sayres@asu.edu</p>

ChemRxiv

Breaching the Axial Limits in Ln(III) Single-Ion Magnets Using External Electric Field

Single-Molecule Magnets have potential applications in several nano-technology applications including in high-dense information storage devices and realization of this potential application lies in enhancing the barrier height for magnetization reversal (Ueff).Recent literature examples suggest that the maximum values that one can obtain using a ligand field are already accomplished. Here we have explored using a combination of DFT and ab initio CASSCF calculations, the way to enhance the barrier height using an oriented external electric field for top three Single-ion Magnets ([Dy(Py) 2) and [Dy(Cp Me3 )Cl] ( 3)). For the first time our study reveals that, for apt molecules, if appropriate direction and value of electric fields are chosen, the barrier height could be enhanced twice that of the limit set by the ligand field. This novel non-chemical-fine tuning approach to modulate the magnetic anisotropy is expected to yield new generation SIMs.

breaching_the_axial_limits_in_ln(iii)_single-ion_magnets_using_external_electric_field

<p>There is a great interest in the area of single-molecule magnets (SMMs) as they are reported to have potential applications in information storage devices, cryogenic refrigeration, quantum computing and spintronics devices to name a few. 1 SMMs containing Lanthanide(III) ions are gaining interest in recent years as they possess huge barrier height for magnetization reversal (Ueff) and at the same time possess record high blocking temperatures (TB). While there are various classes of molecules exhibit blocking temperatures in the range of 4-15 K, 2 higher blocking temperatures are found for organometallic Dy(III) single-ion magnets (SIMs) containing substituted cyclopentadienyl ligands (TB in the range of 48 K to 80 K). 3 It is well-known that the shape of the electron density of the ground state mJ levels of the Lanthanide ion is critical in dictating their magnetic properties. The Ln(III) ions are classified as those possessing oblate density require strong axial with no/weak equatorial ligation and those with prolate density demands strong equatorial ligand with weak/no axial ligation. Synthetic chemists have been utilizing these ideas to develop novel molecules with attractive Ueff and TB values. 4 While most of the molecules possessing very high-blocking temperature also possess substantial Ueff values, often the TB is only a fraction of the Ueff values reported. While establishing the relationship between the Ueff and TB and mechanism beyond the singleion relaxation has gained attention, 5 it is also equally important to realize large Ueff values to move forward.</p><p>To obtain a larger Ueff value for lanthanide complexes, various chemical fine-tuning methods such as (i)designer ligands that control the ligand field around the Ln(III) ion in an anticipated fashion, 6 (ii) maintaining symmetry around the metal centre, 2, 7 (iii) incorporating diamagnetic elements in the cluster aggregation to enhance the axiality 8 or (iv) incorporate transition metal or radicals to induce exchange interaction as a way to suppress tunneling have been explored. 4,9 With numerous Dy(III) mononuclear complexes reported in the literature, it has been stated that the axial limit that controls the overall Ueff value has been reached. 2a While increasing TB value being the focus at present, other avenues to enhance the Ueff values have not been explored. As chemical fine-tuning of the ligand field has already reached its potential, we aim to search an alternative route to enhance the Ueff values in Ln(III) SIMs. In this context, using various computational tools, here we set out to explore the role of an applied electric field in the magnetization reversal of Ln(III) SIMs. Recent examples in this area where electric field has been utilized to modulate the magnetic properties offers strong motivation for this work. 10 To enumerate the effected of an oriented external electric field (OEEF) on Lanthanide SIMs, we have chosen three examples [Dy(Py)5 11 (2) and [Dy(Cp Me3 )Cl] 3a (3) complexes. All three complexes are characterized well and among the best-known SIMs in their family. Particularly complex 1 found to exhibit a Ueff value of 1815 K with a blocking temperature of 14 K while complex 2 found to have Ueff value of 63 K with a TB of 3 K. Complex 3, on the other hand, do not exhibit any out-of-phase signals and hence is not a Single-ion magnet. 3c Computing the magnetic anisotropy of the Ln(III) SIMs in the presence of electric field has not been attempted, and there are multiple challenges present to account for such effects. The application of oriented electric fields is expected to distort the geometry and capturing this effect is crucial in understanding the magnetic anisotropy. As Ln(III) SIMs are known to be extremely sensitive to small structural changes, static OEEF on Xray structure unlikely to reveal the real scenario. As structure optimization with the ab initio CASSCF calculations is not practical at present, here we have chosen a combination of methodology wherein DFT calculations in the presence of electric field were performed to obtain reasonable structures. These structures were then subject to ab initio CASSCF/RASSI-SO/SINGLE_ANISO calculations in the presence of same electric field to capture both the structural distortion and also the electric field effect on the magnetic anisotropy (see computational details for more information). Ab initio calculations were performed on the crystal structures of complexes (or models derived from the X-ray structure) of 1 -3 in the absence of any external perturbation (see Table S1-S3 in ESI). Complex 1 and 2 are well-known examples, exhibiting strong axiality in the estimated gz values with the computed barrier height of 1183 cm -1 and 181 cm -1 , respectively (relaxation via 4 th excited Kramers doublet). 2a, 12 .</p><p>As geometries of 1 and 2 are relaxed in the presence of an electric field, it is imperative to understand how the optimized geometry in the gas phase fair to the X-ray structure. The optimized geometry of complexes (1opt and 2opt) reveal elongation of all the bonds within the molecule as intermolecular interactions in the crystal lattice are removed. The axial Dy-O(1) bond length increases from 2.110 Å in the X-ray structure to 2.142 Å in 1opt, and the average Dy-N bond also increases ~0.05 Å in 1opt geometry (see Table 1). Similar elongation has been witnessed in Er-N/Cl bond lengths in complex 2. The CASSCF calculations on 1opt and 2opt yield Ucal value of 1118 cm -1 and 144 cm -1 , respectively, assuming relaxation via 4 th excited state (See Fig. 1) and these values are marginally smaller than the X-ray geometry due to relatively weaker axial ligand field (LF) in optimized geometries (see Table S4-S5).</p><p>In the next step, we have attempted to optimize the geometry in the presence of oriented external electric field (OEEF) starting from 0.004 au (atomic unit and equivalent to 0.2 V/Å). 10c, 13 The electric field applied here varies from 0.004 au to 0.026 au and lies within the limit of ionization energies, bond dissociation energies and is accessible for most of the STM tips. [13][14] While the electric field induced spectroscopic techniques uses smaller field, organic reactions performed using OEFF are comparable to the electric field utilized here. [13][14] Applying the electric field along the +z-axis in 1opt (See Fig. 1a and Fig. S1 in ESI), elongates the Dy-O(1) bond and at the same time shortens the Dy-O(2) bond and therefore breaks the pseudo D5h symmetry of the molecule. We have performed ab initio CASSCF calculations on this optimized geometry 4z 1opt (here superscript denotes the amount of OEEF applied x 10 -3 au along +z direction) in the presence of electric field (EF) wherein a reduction in the barrier height was witnessed. This is due to the fact that Dy-O(1) elongation cause the weakening of the axial LF and hence reduces the axial anisotropy for the oblate Dy(III) ion. Although there is a simultaneous shortening of Dy-O(2) is noticed, 4z 1opt geometry reveal that elongation is greater than the shortening (see Fig. S1) and hence this is not fully symmetric leading to a smaller Ucal value of 1108 cm -1 for 4z 1opt. In the next step, we increase the OEEF value in a stepwise manner to 0.012 au and see clearly that increase in the electric field increase the Dy-O(1) bond further and at the same time shortens the Dy-O(2) bond albeit asymmetrically. This led to a further reduction in the barrier height with a value of 1040 cm -1 noted for 12z 1opt structure (see Table S6 and S9-S11 in ESI). This reduction in barrier height can be rationalized by analyzing Lo-Prop charges at the spin-free ground state. By increasing OEEF, the LoProp charge on O(1) gradually decreases while it increases in O(2) (see Table S8 and S16). Perceiving this effect, we switched the OEEF direction along the x/y direction of complex 1opt (see Fig. S1 in ESI). The optimized structure of 4x 1opt (here superscript denoted the amount of OEEF applied x 10 -3 au along +x direction). Here Dy-N(1) bond length found to increase sharply from 2.62 Å to 2.80 Å vis a vis 1opt vs 12x 1opt (see Table 1) geometry and at the same time two of the Dy-N (along the -x direction) found to shorten asymmetrically. Also, the effect of applying OEEF along Dy-N(1) direction can be seen in a substantial decrease in the LoProp charge of N(1) atom while the charges on the oxygen atoms remain unaltered (see Table S8 in ESI). As three Dy-N bonds are significantly elongated at 12x 1opt geometry, one could expect a large barrier height, however, ab initio calculations reveal a contrary with a barrier height diminishing with an increase in OEEF value yielding a Ucal value of 939 cm -1 for 12x 1opt which relaxes via 3 rd excited KDs. (see Table S6 and S12-S14 in ESI). This is due to alteration of Dy-N distances that are accompanied by a variation in the O-Dy-O angle, which is reduced to 157 in 12x 1opt from 178 in 1opt geometry (see Table 1). Thus, the application of the electric field along the perpendicular or gx-direction worsens the barrier height for complex 1. To prove that the reduction is solely due to the O-Dy-O bending, we have performed one additional set of calculation on 12x 1opt geometry wherein the O-Dy-O angle is fictitiously set at 178 and this structure yield a barrier height of 1162 cm -1 (See Fig. S2 and Table S15 in ESI). This value estimated is ~50 cm -1 higher compared to optimized geometry offering a possibility, however small, to enhance Ucal value in 1 using an applied electric field. Furthermore, increasing the OEEF at 0.016 au results in dissociation of the Dy-N bond, which sets the electric field limit at x/y direction of the molecule.</p><p>To further understand how the structure alteration occurs due to OEEF, it is important to understand the nature of dipoles and their behavior in the applied electric field conditions. Application of OEEF expected to polarize a non-polar bond and enhance the ionic character of a polar bond. 13 For a Ln-L bond, the application of OEEF will stretch it further if the dipolar field creates opposite dipole with respect to the Ln-L dipole and will help to shorten it, if the dipolar field is in the same direction as the Ln-L dipole (see Fig. 2b). Therefore, the molecule has to be chosen in such a way that an increase in the Ln-L bond length will enhance the magnetic anisotropy and will subsequently increase barrier height (Ueff). Applying the OEEF along an equatorial Ln-L bond in oblate ions such as Dy 3+ or along an axial Ln-L bond in prolate such as Er 3+ or Yb 3+ thus likely to increase the Ueff value beyond the X-ray structure reported values. However, if the OEEF is applied along the opposite directions, this is expected to decrease the Ueff value further.</p><p>Based on the knowledge gained, we intuitively expand the study to a prolate Er(III) ion using complex 2. We have narrow down to this example for two reasons (i) to choose a well-studied prolate Er(III) SIM with a significant barrier height (ii) to choose an Er(III) SIM with a strong equatorial ligand and a weak axial ligand along only one direction as this would be expected to facilitate the enhancement of the Ucal value upon application of OEEF. Upon applying the OEEF along the Er-Cl direction (gz axis, see Fig. 2), with the same step size as before, the Er-Cl bond length found to increase significantly (see Fig. S3 in ESI and Table 1) reaching a maximum value of 3.04 Å at 0.026 au EZ ( 26z 2Opt). The application of OEEF beyond this value found to cleave Er-Cl bond suggesting possible ionization/decomposition limit.</p><p>Additionally, the {N3Er} out-of-plane pyramidal shift (parameter  see Fig. 2 and S3 in ESI) also found to alter upon application of OEEF. As OEEF is applied along the Er-Cl bond, this bond elongates and also pushes the Er 3+ ion down and hence decreases the  value. The  value decreases from 0.5 Å at the 2opt to 0.3 Å at 26z 2opt. If the OEEF is applied along the -z direction (Cl-Er direction), this tends to enhance the pyramidalization (see Fig. S3 in ESI) and thus,  value increases to 0.62 Å at 26-z 2opt. Theoretical studies performed earlier on complex 2 reveal that this is an important parameter that enhances the barrier height. 15 Application of OEEF along the gz in 2 (.i.e along Er-Cl bond) axis enhances the Ucal 163 cm -1 at 4z 2opt to a remarkable 317 cm -1 at 26z 2opt. This estimate is one of the highest obtained for any Er(III) SIMs. 16 Computed QTM (and TA-QTM) values reveal a smooth decrease of these values from 2.2 at 4z 2opt to 1.3 at 26z 2opt. (see Table S17-S24 in ESI). Also, a smooth linear increase of the negative B2 0 parameter was observed for complex 2 under the applied electric field range along the +z direction (see Fig. S4 and Table S27 in ESI). If OEEF applied on the reverse direction on complex 2, i.e., along the -z direction, a reverse trend was visible with a gradual decrease in Ucal value. As expected, here the Er(III)-Cl bond length decreases and a  value were noticed upon applying an electric field in the -z direction. The Ucal value decreases from 131 cm -1 at 4-z 2opt to a much smaller value of 52 cm -1 (via 3 rd excites state) at 24-z 2opt structure (see Table S25-S27 at ESI). Further, the Ucal vanishes to zero at 26-z 2opt with a notable ground state QTM. We have also plotted the β-electron density of the Er(III) under the applied electric field conditions, and this nicely reflects the changes observed (see Fig. S5 for plot corresponding to 26-z 2opt, 2opt and 26z 2opt).</p><p>After achieving such a large Ucal value for complex 2, we extended the study further to another Dy(III) example namely [Dy(Cp Me3 )2Cl] (complex 3) (Cp Me3 = trimethylcyclopentadienyl) (see Figure S6 top) which is a model complex derived from X-ray structure of the famous precursor [Dy(Cp ttt )2Cl]. 3a Our calculations on the optimized structure reveals a very small Ucal value of 144 cm -1 relaxing via first excited state due to high QTM being operation due to the coordination of -Cl along the equatorial direction (see Table S28 in ESI). To quench this QTM, we have applied the OEEF along the Dy-Cl bond direction and this leads to weakening of Dy-Cl bond and gradually the Ucal value increases from 160 cm -1 at 4z 3opt to 519 cm -1 at 22z 3opt structure (see Table S28 and Figure S7). The Dy-Cl bond length increases from 2.59 Å at 4z 3opt to 2.94 Å at 22z 3opt. As the Dy-Cl bond increases with the electric field, a simultaneous increase of Cp-Dy-Cp angle was observed. Application of the electric field beyond 0.022 au results in rupture of the Dy-Cl bond. Thus, the Ucal value increases by three times more than that of the optimized geometry.</p><p>To this end, here we have explored the possibility of finetuning the barrier height for magnetization reversal using oriented external electric fields in Ln(III) SIMs. Enhancement in Ucal value twice that of the X-ray structures offers a viable nonchemical fine-tuning way to enhance the barrier height beyond the limit set by the ligand fields. This novel approach expected to trigger substantial interests to obtain new generation SIMs unveiling its potential applications.</p>

ChemRxiv

Kinetic Studies of Inhibition of the A\xce\xb2(1\xe2\x80\x9342) Aggregation Using a Ferrocene-tagged \xce\xb2-Sheet Breaker Peptide

The aggregation of amyloidogenic proteins/peptides has been closely linked to the neuropathology of several important neurological disorders. In Alzheimer\'s disease (AD), amyloid beta (A\xce\xb2) peptides and their aggregation are believed to be at least partially responsible for the etiology of AD. The aggregate-inflicted cellular toxicity can be inhibited by short peptides whose sequence are homologous to segments of the A\xce\xb2(1\xe2\x80\x9342) peptide responsible for \xce\xb2-sheet stacking (referred to as the \xce\xb2-sheet breaker peptides). Herein a water-soluble ferrocene (Fc)-tagged \xce\xb2-sheet breaker peptide (Fc-KLVFFK6) is used as an electrochemical probe for kinetic studies of the inhibition of the A\xce\xb2(1\xe2\x80\x9342) fibrillation process and for determination of the optimal concentration of \xce\xb2-sheet breaker peptide for efficient inhibition. Our results demonstrated that Fc-KLVFFK6 interacts with the A\xce\xb2 aggregates instantaneously in solution, and sub-stoichiometric amount of Fc-KLVFFK6 is sufficient to inhibit the formation of the A\xce\xb2 oligomers and fibrils and to reduce the toxicity of A\xce\xb2(1\xe2\x80\x9342). The interaction between Fc-KLVFFK6 and A\xce\xb2(1\xe2\x80\x9342) follows a pseudo-first-order reaction, with a rate constant of 1.89 \xc2\xb1 0.05 \xc3\x97 10\xe2\x88\x924 s\xe2\x88\x921. Tagging \xce\xb2-sheet breaker peptides with a redox label facilitates design, screening, and rational use of peptidic inhibitors for impeding/altering A\xce\xb2 aggregation.

kinetic_studies_of_inhibition_of_the_a\xce\xb2(1\xe2\x80\x9342)_aggregation_using_a_ferrocene-tagged

Introduction<!>Materials<!>Peptide Synthesis<!>Electrochemical Measurements<!>Size-Exclusion Chromatography<!>Atomic Force Microscopic Measurements<!>Cell Cytotoxicity Assay<!>Results and Discussion<!>Conclusions

<p>The pathology of several neurological disorders has been attributed to one or several of the following processes: misfolding of amyloidogenic peptides/proteins, staking of the β-sheet-rich oligomers, further aggregation of oligomers into larger and higher-ordered aggregates, and the ultimate deposition of filamentous aggregates onto neuronal cells. For Parkinson's disease and prion diseases, the culprit proteins are believed to be the α-synuclein protein and the prion protein, respectively [1-4]. Alzheimer's disease, on the other hand, is manifested by aggregated amyloid beta (Aβ) peptides in senile plaques and the intracellular aggregates of the τ protein [5, 6]. The Aβ peptides include variants containing 39–43 amino acid residues, with Aβ(1–42) as the major amyloidogenic component. In general, the aggregation pathway of these proteins/peptides is believed to proceed with the transformation of monomers into β-sheet-containing intermediates or nucleating units, which is followed by stacking of these units to form higher-ordered aggregates (e.g., protofibrils and fibrils) [7-9]. Addition of monomers is presumed to be responsible for the elongation and smoothening of the surface of the fibrils [9-11]. By monitoring the aggregation of fluorophore-labelled Aβ(17–22), Lynn and co-workers recently showed that fibril-like amyloid assemblies could emerge from early, micrometer-sized aggregates [12]. The observation suggests that the early Aβ aggregates might be viable targets for therapeutic intervention.</p><p>Peptidic inhibitors (also referred to as the β-sheet breaker peptides [13]) are a class of compounds known to be highly potent in ameliorating Aβ(1–42)- or α-synuclein-inflicted cell toxicity [14-22]. Typically the sequences of such peptides are homologous to parts of the hydrophobic segments of the amyloidogenic molecule responsible for the β-sheet formation. To inhibit the Aβ aggregation process, Tjernberg et al. designed an inhibitor of the KLVFF sequence (residues 16-20 of Aβ(1–42)) [23] and Soto and co-workers have shown that the LPFFD peptide is highly effective in inhibiting Aβ amyloidogenesis [24]. Murphy and co-workers found that linking a lysine hexamer to the C-terminus of KLVFF improves both the solubility and inhibitory efficacy [25]. In a later work, they investigated the interaction between KLVFFK6 and Aβ(1–40) and posited that the mode of inhibition was an accelerated assembly of filamentous Aβ intermediates into short fibrils [26]. As a result, the amount of the more neurotoxic Aβ oligomers is greatly reduced in solution.</p><p>Despite an extensive effort in developing peptidic inhibitors and analogs, in many cases it is not clear how a given peptidic inhibitor interacts the various protein/peptide forms (e.g., monomers, oligomers, large filaments, and amorphous aggregates) to reduce the Aβ-elicited cytotoxicity [23-26]. These uncertainties are partly caused by a lack of sensitive methods for real-time monitoring of the entire inhibition process. Atomic force microscopy (AFM) and electron microscopy are typically used to image aggregates formed after a relatively long incubation. Other real-time techniques, such as dynamic light scattering or fluorescence spectrometry of β-sheet-intercalating dye molecules (e.g., Thioflavin T) are also more suitable to studies of similarly late events wherein aggregates of certain sizes (a few nanometers or greater) and shapes (e.g., rod-like protofibrils and fibrils) are formed [27, 28]. While circular dichroism (CD) spectroscopy can detect the transition of natively unstructured peptides/proteins into oligomers rich in the β-sheet conformation (the very first step in the aggregation process), the different stages of the aggregation cannot be differentiated. Moreover, some β-sheet breaker peptides themselves are capable of forming β-sheet-containing structures. Consequently, these β-sheet breaker peptides could obscure CD signals given rise by β-sheet formation (or lack of) in the amyloidogenic peptides/proteins. Recently label-free electrochemical methods were explored for studying the aggregation of Aβ(1–42). It was found that the redox activity of the tyrosine-10 (Tyr-10) residue of Aβ(1–42) changes during the Aβ(1–42) aggregation process [29]. However, due to the fact that the Tyr-10-encompassing hydrophilic termini of Aβ(1–42) molecules emanate into solution from the core of the aggregates [30], the difference in the tyrosine redox currents is not significant between unstructured and aggregated Aβ(1–42) species. In other words, the oligomerization and fibrillation (aggregation) processes produce gradually changed Tyr-10 oxidation currents and the electrochemical method consequently cannot differentiate oligomers of different sizes or even oligomers from protofibrils, fibrils or amorphous aggregates. This is in contrast to the commonly used Thioflavin T (ThT) assay in which the presence of a lag phase is indicative of a nucleation-elongation mechanism [9, 11] and the induced ThT fluorescence signals the formation of fibrils. As for interrogating the inhibition of aggregation by a β-sheet breaker, any uncertainty in recording small current changes can affect the accuracy of the kinetics of the inhibition reaction [31]. Furthermore, β-sheet breakers disrupt Aβ(1–42) aggregation/fibrillation via interacting with the hydrophobic domain of Aβ(1–42), which does not significantly block/affect the electrochemical oxidation reaction of Tyr-10, which resides in the hydrophilic domain of Aβ(1–42). Due to the aforementioned issues, it has been difficult to gauge the effective breaker dosage for inhibiting/disrupting the aggregation/fibrillation. Furthermore, the paucity of knowledge about the kinetics of the inhibitory process makes the investigation on the time-dependent aggregation disruption a largely trial-and-error practice. Typically multiple aliquots of solutions incubated for different times are incrementally sampled and studied. Such a practice is both laborious and sample-consuming.</p><p>Tagging various biomolecules and small organic compounds with the ferrocene (Fc) moiety has been widely used for electrochemical studies of a wide range of biomolecular interactions [32-36]. We previously synthesized a ferrocenoyl pentapeptide (Fc-KLVFF) and found it to possess some inhibitory effect on the Aβ fibril formation [37]. But its solubility in water is rather poor. As a consequence of the poor water solubility, a high Fc-KLVFF concentration in a mixed organic/water solvent had to be used to elicit inhibition of the Aβ(1–42) aggregation. As a result, kinetic information about the β-sheet breaking and inhibition of aggregation could not be obtained and the optimal dosage for the aggregation inhibition in a physiologically relevant medium could not be determined. It was also not clear whether tagging KLVFF with Fc would compromise the ability of KLVFF in attenuating the cell toxicity of Aβ(1–42) aggregates. KLVFF is also known to self-aggregate [38, 39] and therefore is not considered as an ideal inhibitor (especially for cytotoxicity or animal model studies). In an attempt to gain insight into the kinetics of the impediment engendered by a β-sheet breaker peptide to the Aβ(1–42) aggregation, we attached the Fc moiety to the N-terminus of the water-soluble KLVFFK6 breaker. By monitoring changes in the electrochemical current of the Fc tag, real-time electrochemical monitoring of the inhibition of the Aβ(1–42) aggregation was accomplished. Inhibition of the Aβ(1–42) aggregation was found to follow a pseudo-first-order reaction and effective inhibition can be obtained at a breaker concentration that is much smaller than that of Aβ(1–42). Fc-KLVFFK6 was found to not self-aggregate, eliminating ambiguities in our previous studies [37]. Remarkably, we found that Fc-KLVFFK6 completely halts the Aβ(1–42) fibrillation and its inhibition of the Aβ(1–42)-inflicted cytotoxicity compares well to that of KLVFFK6.</p><!><p>Aβ(1–42) was purchased from American Peptide Co. Inc. (Sunnyvale, CA), and pretreated by the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) method [40-42]. The treatment of HFIP follows a centrifugation of the solution for 30 min at 13800 rps and the supernatant solution was lyophilized for further sample preparation. Stock solution of Aβ (1–42) (0.5 mM) was prepared as previously reported [43]. Ferrocene monocarboxylic acid (Fc-COOH) was obtained from Sigma-Aldrich (Milwaukee, WI). Wang resin, Fmoc-protected amino acids, diisopropylcarbodiimide (DIC), pyridine and piperidine were purchased from Anaspec, Inc. (San Jose, CA). SH-SY5Y cell (human neuroblastoma) was obtained from American Type Culture Collection, Inc. (Manassas, VA) and fetal bovine serum (FBS) was from Omega (Tarzana, CA). Both Dulbecco's Modified Eagle Medium (DMEM) and Ham's F12 Medium were acquired from Mediatech Inc. (Manassas, VA). The mixture of penicillin and streptomycin for cell culture and cytotoxicity study was purchased from Millipore (Billerica, MA). The other chemicals were obtained from Thermo-Fisher Scientific Inc. (Pittsburgh, PA).</p><!><p>Fmoc-K(Boc)LVFFK(Boc)6 attached to the Wang resin was synthesized through the solid-phase Fmoc chemistry on a Symphony Quartet peptide synthesizer (Protein Technologies, Tucson, AZ). To remove the Fmoc group, the resin was allowed to react with 20% piperidine in 20 mL dimethylformamide (DMF) for 45 min. Filtering the resin and washing it sequentially with DMF, CH2Cl2 and CH3OH produced NH2–K(Boc)LVFFK(Boc)6 on the resin. To couple the Fc tag to the peptide, Fc-COOH (0.1 mol) in 20 mL DMF was mixed with DIC, N-hydroxybenzotriazole and 4-dimethylaminopyridine (0.1 mole each) for 30 min and the resultant mixture was mixed with the resin covered with NH2–K(Boc)LVFFK(Boc)6. The solution was then shaken at room temperature for 6 h. The resin was filtered and washed with DMF and CH3OH to produce Fc-K(Boc)LVFFK(Boc)6. Finally, a mixture containing 95% trifluoroacetic acid (TFA) and 5% triisopropylsilane was used to remove the Boc group and to cleave the peptide off the resin. The filtrate was recrystallized with cold ether to yield the crude product of Fc-KLVFFK6, which was then purified by semi-preparative reversed-phase HPLC (Shimadzu AD, Columbia, MO) using a Jupiter-10-C18-300 column (10 mm i.d. × 250 mm; Phenomenex Inc., Torrance, CA). The eluting solutions were 0.1% TFA in water (mobile phase A) and 0.1% TFA in acetonitrile (mobile phase B). At a flow rate of 4.75 mL/min, an elution gradient of 20–45% phase B lasted for 12 min. The as-purified peptide was characterized by electrospray-mass spectrometry (ES–MS), which showed a single peak at m/z = 1635.6 (theoretical m/z = 1636.9). Fc-K6 was synthesized and purified similarly.</p><!><p>All electrochemical measurements were carried out with a CHI660B electrochemical workstation (CH Instruments, Austin, TX). The working electrode was a glassy carbon disk with a diameter of 3 mm (Bioanalytical System Inc., West Lafayette, IN). A platinum wire and a Ag/AgCl electrode were used as the auxiliary and the reference electrodes, respectively. Aβ(1–42) and Fc-KLVFFK6 were dissolved in 100 mM phosphate buffer/50 mM KClO4 (pH 7.4). The entire experimental setup was lowered into a water bath maintained at 37 °C. For differential pulse voltammetric measurements, the following parameters were used: sample width = 17 ms, pulse amplitude = 50 mV, pulse width = 50 ms, and pulse period = 200 ms.</p><!><p>Blue dextran (2000 kD), bovine serum albumin (66 kD), chymotrypsinogen A (25 kD), aprotinin (6.7 kD), and vitamin B12 (1.35 kD) were used to calibrate the retention time of the size exclusion chromatographic columns (GFC 2000 from Phenomenex Inc). Two columns were connected in series and the separation/detection was carried out on a Waters 600 HPLC system (Milford, MA) that is equipped with a photodiode array detector (Model 2996). Phosphate buffer (100 mM, pH 7.4) was utilized as the mobile phase and the flow rate was 0.2 mL/min. Elutions of Aβ species, Fc-KLVFFK6, and other peptides were monitored at 220 nm. For each assay, a 20-μL aliquot taken from a solution incubated in a 37 °C water bath was injected into the columns.</p><!><p>Freshly cleaved mica sheets were pretreated with Ni(II) in 10 mM NiCl2 for 15 min. Prior to imaging, aliquots were taken from incubated solutions containing Aβ(1–42), Fc-KLVFFK6/Aβ(1–42), KLVFFK6/Aβ(1–42), or Fc-KLVFFK6 and cast onto these treated mica sheets. The mica sheets were then rinsed with water to remove salt residues, and dried with nitrogen prior to imaging. The morphology of the various Aβ aggregates was characterized with an MFP-3D-SA microscope (Asylum Research, Santa Barbara, CA) using the tapping mode.</p><!><p>SH-SY5Y cells were cultured in a medium of 44.5% DMEM comprising L-glutamine (4 mM), Ham's F12, FBS, and a mixture of penicillin and streptomycin (V:V:V:V = 44.5%:44.5%:10%:1%). The cultured cells were then transferred to a sterile 96-well plate with approximately 20000 cells per well. These cells were allowed to acclimatize overnight in the DMEM/F12 media containing 5% FBS in a humidified incubator under 5% CO2 at 37 °C. Solutions of Fc-KLVFFK6, KLVFFK6, Aβ(1–42), Fc-KLVFFK6/Aβ(1–42) and KLVFFK6/Aβ(1–42) were pre-incubated at 37 °C for 24 h and were allowed to react with the SH-SY5Y cells for 24 h. Viability of cells exposed to each solution was determined based on the 3,[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (EMD Inc., Gibbstown, NJ) assay, as described by others and our previous work [44, 45].</p><!><p>KLVFF-containing short peptides have been demonstrated to be effective in inhibiting Aβ aggregation through hydrophobic interaction and salt bridge formation with residues 18 – 23 of Aβ(1–42), though truncated or shortened fibrils were observed as some of the end products [25]. As reported by Murphy and co-workers, attachment of a hexalysine segment to the C-terminus of KLVFF not only increases the breaker's solubility, but also reduces cell toxicity of the Aβ aggregates [46]. We envision that, when Fc is attached to the water-soluble KLVFFK6 peptide and the resultant molecule is allowed to interact with the Aβ(1–42) oligomers and larger aggregates (e.g., protofibrils and fibrils), diffusion of the soluble oligomers with Fc-KLVFFK6 incorporated should be retarded. Incorporation of Fc-KLVFFK6 into and co-precipitation with insoluble and higher-ordered Aβ(1–42) aggregates would also decrease the Fc-KLVFFK6 bulk concentration. Both of the above processes will decrease the electrochemical signal, through which the kinetics of the inhibition reaction can be interrogated (cf schematics in Figure 1). As shown in Figure 2, Fc-KLVFFK6 displays a cyclic voltammogram (CV) with an oxidation peak at 499.2 mV and a reduction peak at 430.6 mV (inset). The oxidation potential is much less positive than that of the tyrosine oxidation, which has been shown by us [43, 47] and others [29, 31] (see also Figure S1 in Supporting Information for comparison) to be around 0.72 V vs. Ag/AgCl. On the basis that the peak potential splitting (ΔEp) = 68.6 mV and the current ratio between the anodic and cathodic peaks (ipa/ipc) = 1.03 are close to the theoretical values (59.6 mV and 1.00, respectively) and the fact that the oxidation peak current (ipa) is proportional to the scan rate [48], we conclude that the Fc tag undergoes facile electron transfer with the electrode. Figure 2A is an overlay of a series of differential pulse voltammograms (DPVs) of 20 μM Fc-KLVFFK6 in the presence of equimolar Aβ(1–42) collected at different incubation times. The DPV oxidation peak decreases with incubation time, confirming that the Fc tag can serve as a sensitive probe for studying the interaction between a potential β-sheet breaker peptide and Aβ(1–42). Our final consideration of using Fc as a tag is that Fc derivatives have been shown to be innocuous to many cell lines [49] and consequently the Fc tag would not compromise the ability of KLVFFK6 of alleviating the cell toxicity inflicted by Aβ(1–42) aggregates.</p><p>By plotting the electrochemical currents of Fc-KLVFFK6 as a function of time and overlaying the curves obtained in solutions of different amounts of Aβ(1–42), kinetic information about the interaction between Fc-KLVFFK6 and Aβ(1–42) can be garnered. As shown in Figure 2B, the precipitous drop in the black curves at the beginning suggests that Fc-KLVFFK6 binds to Aβ(1–42) instantaneously. In the absence of Aβ(1–42), a steady DPV current was maintained in an Fc-KLVFFK6 solution for 24 h (red curve), suggesting that Fc-KLVFFK6 does not undergo self-aggregation (unlike the water-insoluble KLVFF [38, 39]). We also monitored the current of Fc-K6, an Fc-tagged hexalysine peptide, at various incubation times in the presence of Aβ(1–42). The essentially constant current (blue curve) suggests that Fc-K6, which lacks the KLVFF domain, does not interact with Aβ(1–42).</p><p>Continuous electrochemical monitoring of the interaction between Fc-KLVFFK6 and Aβ(1–42) at different stoichiometric ratios all produced plateaus at about 6 h and beyond. We also centrifuged samples incubated for at least 6 h and recorded DPV currents in both the supernatants and the residual solutions. The currents from the supernatants (data not shown) are essentially congruent with the respective curves in Figure 2B, whereas those in the residual solutions did not produce noticeable Fc oxidation currents. Thus, the plateaus in Figure 2B correspond to excess Fc-KLVFFK6 remaining in solution. We found that all the decay curves in Figure 2B can be fitted with a pseudo-first-order reaction rate equation:</p><p>where i0 and i represent the current values at the beginning and time t of an incubation, respectively. A is a constant and ka is the forward reaction rate constant in the following reaction:</p><p>The rate constants deduced from these simulations (black curves in Figure 2B) are within a relatively narrow range of 1.26-1.89 × 10−4 s−1 (Table 1), and the half-life of the conjugate formed between Fc-KLVFFK6 and the Aβ(1–42) aggregates is 4547.4 ± 705.8 s. Notice that the fits are excellent even at [Fc-KLVFFK6]/[Aβ(1–42)] < 1, indicating that a significant amount of free Fc-KLVFFK6 has remained in solution. This observation suggests that a single Fc-KLVFFK6 molecule is associated with aggregates comprising multiple Aβ(1–42) molecules. Another possible explanation is that the binding affinity between Fc-KLVFFK6 and the Aβ(1–42) aggregates is in the μM range, which establishes an equilibrium between free Fc-KLVFFK6 in solution and Fc-KLVFFK6 bound to the aggregates.</p><p>As mentioned above, the decrease in the DPV peak currents in Figure 2B could be ascribed to twopossible processes—(1) a decrease of the bulk Fc-KLVFFK6 concentration resulted from the Fc- KLVFFK6 incorporation in the precipitates of large and insoluble Aβ(1–42) aggregates and (2) a slower diffusion dueito its association with soluble Aβ(1–42) oligomers. To pinpoint the specific form of Aβ(1–42) (soluble oligomers or large aggregates) to which Fc-KLVFFK6 is bound, we conducted size exclusion chromatography (SEC) of mixtures of Aβ(1–42) and Fc-KLVFFK6 incubated for various times. As shown in Figure 3A, under our chromatographic condition, the Aβ(1–42) monomer elutes at ca. 32 min. During the Aβ(1–42) sample injection and its migration across the column (ca. 50 min), the freshly prepared Aβ(1–42) sample plug has already produced a discernible amount of dimers and a small amount of soluble oligomers (cf. the pentamer peak at 25 kD). The time for forming dimers and pentamers is consistent with those reported in some studies [50, 51]. Aβ pentamers and hexamers are the predominant oligomeric species, consistent with findings from previous reports [52]. Incubation of an Aβ(1–42) solution for 2–3 h converts monomers and small oligomers into larger oligomers (cf. the peak identified as a 160 mer at 740 kD). Very few monomers and oligomers remained in Aβ(1–42) solutions that had been incubated for 6 h or longer, as evidenced by the disappearance of the Aβ(1–42) monomer and dimer peaks. The decrease in the concentrations of monomers and soluble oligomers is due to the formation of insoluble, higher-ordered aggregates, which are not amenable to the SEC separation. We should note that the small peak at ca. 40 min with a size of 1.3–2 kD was verified by electrospray mass spectrometry to be an impurity in the synthetic Aβ(1–42) sample.</p><p>The SEC separation of an Fc-KLVFFK6-only (20 μM) solution did not show any elution peaks (cf. Figure S2A in Supporting Information), because of the small molar extinction coefficient of Fc-KLVFFK6. We also confirmed with SEC that Fc-KLVFFK6 alone does not aggregate throughout the incubation process (Figure S2B). When Fc-KLVFFK6 was present in the Aβ(1–42) solution, no soluble oligomers larger than the Aβ(1–42) dimer were observed (Figure 3B). In addition, the monomer concentration at the inception of the incubation is significantly greater (two times as high as the combined area of the monomer and dimer peaks in Figure 3A). Unlike in Figure 3A, it is more difficult to resolve the monomer and dimer peaks in Figure 3B, though peaks in the latter chromatogram appear to be of higher abundance than those in the former. Since Murphy and coworkers have shown that KLVFFK6 does not directly interact with the Aβ(1–40) monomer [26], we believe that the large and broad peak in Figure 3B does not contain Aβ(1–42) monomers conjugated with Fc-KLVFFK6. Therefore, the much enhanced monomer/dimer peak must be resulted from the alteration of the Aβ(1–42) aggregation process, which stabilizes both the monomer and dimer at the initial stage of the incubation. We posit that, instead of directly interacting with Aβ(1–42) monomers and soluble oligomers, Fc-KLVFFK6 alters the Aβ(1–42) aggregation pathway by interacting with larger, intermediate Aβ(1–42) aggregates, thereby preventing further attachment of Aβ(1–42) monomers to such aggregates. To observe insoluble aggregates, we conducted the following atomic force microscopic measurements.</p><p>Figure 4 are atomic force microscopy (AFM) images obtained from Aβ(1–42) solutions in the absence (row A) and presence of Fc-KLVFFK6 (row B). In the absence of Fc-KLVFFK6, few insoluble globular aggregates were observed at the beginning of the incubation. The abundance of the globular aggregates became much greater at 3 h. At 6 h and later times, the predominant aggregate is the Aβ(1–42) fibril. In contrast, in the presence of Fc-KLVFFK6, large aggregates with heights around 20 nm and diameters in sub-micrometers were observed in the first 3 h and are predominately of the amorphous morphology (cf. the cross-sectional contours). The abundance of the amorphous aggregates became greater at 6 h, and after 24 h, the amorphous aggregates were the predominant end product. We also collected AFM images from a mixture of KLVFFK6 (the soluble β-sheet breaker peptide without the Fc tag) and Aβ(1–42) at various incubation times. As shown in row C of Figure 4, the aggregates observed at 3 h and 6 h have essentially the same morphology (amorphous aggregates) as those shown in row B. The slight difference is that after extensive incubation (24 h), the mixture of Aβ(1–42) and KLVFFK6 produced a mixture of short fibrils and amorphous aggregates, whereas in row B, the solution was populated with amorphous aggregates. We conclude that the mode of inhibition by Fc-KLVFFK6 and KLVFFK6 should be quite similar. The small difference in the final product(s) between the Fc-KLVFFK6- and KLVFFK6-containing solutions (largely amorphous aggregates vs. a mixture of short fibrils and amorphous aggregates) can be rationalized by the presence of the Fc moiety in the former. The additional steric hindrance rendered by the Fc tag perhaps contributes to the overall breakage of the stacked, β-sheet-rich Aβ(1–42) oligomers. Consequently, the more disordered amorphous aggregates, instead of shortened fibrils, are produced.</p><p>AFM also showed that Fc-KLVFFK6 does not self-aggregate when incubated alone (row D). This is consistent with our electrochemical results (cf. red curve in Figure 2B). This again demonstrates the advantage and greater biochemical relevance of using a water-soluble p-sheet breaker peptide over its aggregation-prone counterpart (i.e., Fc-KLVFF [37]). Therefore, the amorphous aggregates shown in rows B and C of Figure 4 must be produced from altered Aβ(1–42) aggregation pathway resulted from the interaction between Aβ(1–42) aggregates and the β-sheet breaker peptides. Taken together, the mode of inhibition by KLFVVK6 (or its Fc-tagged analog) is to interact with the aggregation intermediates, thereby diverting the Aβ(1–42) aggregation away from the main aggregation (i.e., fibril-producing) pathway. This point is in line with the finding by Murphy and co-workers who showed that KLVFFK6 can interact with linear aggregate of Aβ(1–42) or protofibril-like aggregate [26].</p><p>It appears that Fc-KLVFFK6, analogous to KLVFFK6, completely eliminates soluble Aβ(1–42) oligomers and fibrils in solution. The soluble oligomers (sometimes referred to as amyloid-derived diffusible ligand [53]) are known to be the most cytotoxic among all of the Aβ(1–42) aggregates [54]. To assess whether the amorphous aggregates formed in the Fc-KLVFFK6/Aβ(1–42) mixture are less pernicious to cells, SH-SY5Y human neuroblastoma cells were exposed to various solutions. As shown in Figure 5, both Fc-KLVFFK6 and KLVFFK6 are largely benign to the SH-SY5Y cells (viability values are 91.4 and 96.7%, respectively). However, the cell viability upon exposure to a pre-incubated Aβ(1–42) solution dropped to 53.7%. As shown by the AFM images in row A of Figure 4, an Aβ(1–42) solution incubated for 24 h produced a mixture of fibrils, protofibrils, and globular aggregates, with mature fibrils being the most abundant. Thus, these aggregates have collectively exerted toxicity to the SH-SY5Y cells. However, when Aβ(1–42) was pre-incubated with Fc-KLVFFK6 and then added into the SH-SY5Y cell media, the cell viability was rescued to 92.4%. When a pre-incubated KLVFFK6/Aβ(1–42) mixture was added into the SH-SY5Y cell media, the final cell viability was determined to be 82.9%. Because the error associated with the former is 5% and that with the latter is 7%, the difference between the two cases is statistically insignificant. Any slight difference is most likely contributed by the existence of a small number of short fibrils present in the KLVFFK6/Aβ(1–42) mixture (cf. AFM image at 24 h in row C). Thus, tagging an Fc moiety to the N-terminus of the KLVFFK6 peptide does not alter the inhibitory effect of KLVFFK6 but facilitates kinetic studies of the breakage/disruption of the stacking of β-sheet-containing Aβ(1–42) aggregates.</p><!><p>By tagging a water-soluble β-sheet breaker (KLVFFK6) with an Fc moiety, we demonstrate that disruption of the Aβ(1–42) aggregation/fibrillation process can be monitored in real time by electrochemical methods. We found that a sub-stoichiometric amount of Fc-KLVFFK6 is sufficient to completely prevent the Aβ(1–42) oligomers and fibrils from being formed, an observation confirmed by size exclusion chromatographic and atomic force microscopic experiments. For the first time, we show that the aggregation inhibition is a pseudo-first-order reaction. Our study confirms that the inhibitory effect is realized through a strong interaction between Fc-KLVFFK6 and the Aβ(1–42) aggregation intermediates, similar to that for KLVFFK6. As a consequence, cytotoxicity caused by Aβ(1–42) aggregates is abolished extensively. The method described herein is of general appeal, as β-sheet breaker peptides have been touted as a potential therapeutic remedy to treat AD, PD, and other related neurological disorders. Kinetic information about the inhibition of the aggregation of amyloidogenic proteins/peptides and knowledge of the effective dosage of β-sheet breaker molecule are essential for rational design and high-throughput screening of potential therapeutic drugs for treating these neurological disorders.</p>

PubMed Author Manuscript

Expression and roles of Slit/Robo in human ovarian cancer

The Slit glycoproteins and their Roundabout (Robo) receptors regulate migration and growth of many types of cells including human cancer cells. However, little is known about the expression and roles of Slit/Robo in human ovarian cancer. Herein, we examined the expression of Slit/Robo in human normal and malignant ovarian tissues and its potential participation in regulating migration and proliferation of human ovarian cancer cells using two ovarian cancer cell lines, OVCAR-3 and SKOV-3. We demonstrated that Slit2/3 and Robo1 were immunolocalized primarily in stromal cells in human normal ovaries and in cancer cells in many histotypes of ovarian cancer tissues. Protein expression of Slit2/3 and Robo1/4 was also identified in OVCAR-3 and SKOV-3 cells. However, recombinant human Slit2 did not significantly affect SKOV-3 cell migration, and OVCAR-3 and SKOV-3 cell proliferation. Slit2 also did not induce ERK1/2 and AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells. The current findings indicate that three major members (Slit2/3 and Robo1) of Slit/Robo family are widely expressed in the human normal and malignant ovarian tissues and in OVCAR-3 and SKOV-3 cells. However, Slit/Robo signaling may not play an important role in regulating human ovarian cancer cell proliferation and migration.

expression_and_roles_of_slit/robo_in_human_ovarian_cancer

Introduction<!>Human ovarian tissue microarray and human ovarian cancer cell lines<!>Immunohistochemistry<!>Western blot analysis for Slit2/3, Robo1/4, ERK1/2, and AKT1<!>Cell migration<!>Cell proliferation<!>Wound healing assay<!>Statistics procedures<!>Immunolocalization of Slit/Robo in human ovarian cancer tissues<!>Expression of Slit2/3 and Robo1/4 in OVCAR-3 and SKOV-3 cells<!>Activation of ERK1/2 and AKT1<!>Effects of Slit2 on cell migration, proliferation, and wound healing<!>Discussion

<p>Although there have been great advances in our understanding of ovarian cancer biology and significant improvement in surgical technology and therapy regimens over the last three decades, the mobility and mortality of ovarian cancer remain high with an estimated 21,880 new cases and 13,850 deaths in the USA alone in 2010 (American Cancer Society at http://www.cancer.org 2010). This high mortality is largely due to the fact that the majority (~70%) of ovarian cancer is diagnosed at an advanced, chemoresistant stage (Wang et al. 2010). To date, the cellular and molecular pathogenesis of human ovarian cancers is poorly understood (Bast et al. 2009), even though it is believed that approximately 90% of primary malignant ovarian tumors arise from the ovarian surface epithelium (Bell 2005). Therefore, a better understanding of the cellular and molecular pathogenesis of ovarian cancer will assist the development of novel therapeutic interventions for this deadly gynecological disease.</p><p>The secreted Slit glycoproteins and their Roundabout (Robo) receptors, consisting of three Slit (Slit1–3) and four Robo (Robo1–4) members in vertebrates, were originally identified in the nervous system as a repulsive cue to prevent axons from migrating to inappropriate locations during the assembly of the nervous system (Andrews et al. 2007; Brose et al. 1999). Each mammalian Slit contains four leucine-rich regions (Robo-binding domains), nine epidermal growth factor repeats, a laminin G domain, and a C-terminal cysteine-rich domain (Hohenester 2008). Slits can be proteolytically cleaved into the N- and C-terminal fragments in vivo and in vitro (Ba-Charvet et al. 2001; Brose et al. 1999; Little et al. 2001; Patel et al. 2001). It is believed that only full length and the N-terminus, both of which are predominantly membrane bound, of the Slits can bind and activate Robos (Ba-Charvet et al. 2001; Brose et al. 1999; Little et al. 2001; Patel et al. 2001). Robos belong to the Ig superfamily of transmembrane signaling molecules (Hohenester 2008; Morlot et al. 2007). Robo1–3 consist of five Ig-like and three type III fibronectin domains in the ectodomain, whereas Robo4 has only two Ig domains. To date, the specific high-affinity Slit binding has been demonstrated biochemically for all Robos, except for Robo4 (Hohenester 2008; Morlot et al. 2007).</p><p>As many Slit/Robo family members are detected in a variety of non-nervous tissues, critical roles of the Slit/Robo signal in regulating growth, development, and remodeling of these non-nervous tissues have been recognized (Dickinson and Duncan 2010; Hinck 2004; Ypsilanti et al. 2010). This notion is firmly supported by the observation that homozygous knockout of Slit1/2/3 (Liu et al. 2003; Plump et al. 2002; Yuan et al. 2003) and Robo1/2 (Grieshammer et al. 2004; Xian et al. 2001) often results in impaired development of lungs, hearts, and kidneys, leading to newborn death shortly after birth.</p><p>Recently, Slit/Robo has also been identified in major organs of the female reproductive system including fetal and adult ovaries, endometria, and placentas (Dickinson et al. 2008; Dickinson and Duncan 2010; Dickinson et al. 2010; Duncan et al. 2010; Ma et al. 2010). In sheep fetal ovary, Slit/Robo is present primarily in oocytes of the primordial follicles, and its increased expression is accompanied by a reduction in the number of proliferating oocytes in the developing ovary (Dickinson et al. 2010). The Slit/Robo family members have also been detected in luteal cells and stromal cells of the human adult ovary, in which blockade of Slit/Robo activity promotes cell migration and decreases apoptosis in primary cultured luteal cells, suggesting that Slit/Robo may regulate lute-olysis in women (Dickinson et al. 2008).</p><p>Slit/Robo is expressed in many types of human cancers (Legg et al. 2008; Liao et al. 2010b). Nonetheless, reports on the roles of Slit/Robo in human cancers are controversial. It has been postulated that Slit/Robo may function as a tumor suppressor (Legg et al. 2008; Liao et al. 2010b; Narayan et al. 2006). This is supported by several lines of evidence. First, homozygous deletion of ROBO1 gene is detected in lung and breast carcinomas (Sundaresan et al. 1998). Second, Slit2 is able to suppress the growth of breast and colorectal tumor cells (Dallol et al. 2002a, b, 2003), and the Slit/Robo signal is often inactivated in breast, lung, colorectal, and brain cancers by hypermethylation of the Slit and Robo promoters (Dallol et al. 2002a, b, 2003; Dickinson et al. 2004; Morris et al. 2003). Third, the expression of certain Slit/Robo members is decreased in many cancer tissues, including lung cancer, breast cancer, adult renal cell carcinoma, cervical cancer, and prostate cancer (Dallol et al. 2002a, b; Latil et al. 2003; Ma et al. 2010; Tanno et al. 2006), as compared to normal tissues or less advanced cancer tissues. In contrast, increased expression of certain Slit/Robo members has also been reported in prostate cancer (Latil et al. 2003), breast cancer (Tanno et al. 2006), colorectal cancer (Grone et al. 2006), hepatocellular carcinoma (Ito et al. 2006), lung, kidney, liver, and metastatic melanoma (Seth et al. 2005). Furthermore, a recent report has shown that neutralization of Robo1 reduces the cancer mass and the microvascular density of human malignant melanoma in vivo, suggesting that Slit/Robo may promote cancer growth and development, plausibly via promoting cancer angiogenesis (Wang et al. 2003). Thus, Slit/Robo might either promote or inhibit cancer development, depending on individual type of cancers.</p><p>Little is known about the expression and roles of Slit/Robo in human ovarian cancer. Herein, we examined the expression of Slit2/3 and Robo1/4, four major members of Slit/Robo in human normal and malignant ovary tissues, as well as SKOV-3 and OVCAR-3 cells, two human ovarian cancer cell lines. We also examined the potential participation of the Slit/Robo signal in regulating migration and proliferation of SKOV-3 and OVCAR-3 cells.</p><!><p>Human ovarian cancer tissue microarrays (cat # OV2084) were purchased from Biomax, Rockville, MD, USA. This tissue microarray contains 16 normal ovarian tissue samples (NORM; including eight adjacent normal ovarian and eight normal ovarian tissues) and 192 cases of ovarian cancer. Cancer histotypes include adult granular cell tumor (AGCT; n = 4), disgerminoma (DISG; n = 5), adenocarcinoma (ADEN; n = 8), teratoma malignant change (TMC; n = 5), yolk sac tumor (YST; n = 6), mucinous adenocarcinoma (Mu-ADEN; n = 20), serous adenocarcinoma (Se-ADEN; n = 136, in which 21 were classified as low grade (L-Se-ADEN) and 115 as high grade (H-Se-ADEN) based on the 2-tie grading system) (Ayhan et al. 2009; Malpica et al. 2004).</p><p>Human ovarian adenocarcinoma cell lines, OVCAR-3 and SKOV-3, were obtained from the American Type Culture Collection (Manassas, VA, USA). OVCAR-3 cells were expanded in RPMI1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% FBS (HyClone, Logan, UT, USA), 1% penicillin/streptomycin (HyClone), and 10 µg/ml insulin (Sigma, St. Louis, MO, USA). SKOV-3 cells were expanded in RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin. Each cell line was maintained in a humidified atmosphere containing 5% CO2 at 37°C.</p><!><p>Immunolocalization of Slit2/3 and Robo1/4 in the human ovarian cancer tissue microarray was carried out as described (Jiang et al. 2010; Zheng et al. 1999). Immunoreactivity of Slit2/3 and Robo1/4 was visualized by indirect detection with the avidin–biotin complex kit (Vector Laboratories, Burlingame, CA, USA) using 3-amino-9-ethylcarbazole (Vector Laboratories) as a chromogen. After deparaffinization, the tissue microarray underwent antigen retrieval in a 10-mM citrate buffer solution (pH 6.0) in a microwave. Endogenous peroxidase activity was quenched in 0.3% H2O2. The tissue microarray was lightly counterstained with hematoxylin (Fisher Scientific, Pittsburg, PA, USA). After blocking the nonspecific binding with 1% horse serum albumin, the tissue microarray was probed with a goat antibody against an internal region of human Slit2 (cat # sc-16619), the C-terminus of human Slit3 (cat # sc-31597) (4 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or a rabbit antibody against human Robo1 (cat # ab7279), or Robo4 (cat # ab10547) (4 µg/ml; Abcam, Cambridge, MA, USA). After washing, the tissue microarray was incubated with a biotinylated secondary antibody. Two more tissue microarrays were probed with preimmune goat and rabbit IgG, respectively, at 4 µg/ml as negative controls. The staining was examined and recorded under a Nikon microscope equipped with a Spot Insight QE CCD camera.</p><p>Staining intensity of Slit2/3 and Robo1/4 was semi-quantitatively analyzed as described (Jiang et al. 2010). The images from the cancer tissues with the sample size per cancer histotype ≥4 were taken under a 10× objective, which represented ~83% of area of each tissue core. The images were uniformly converted to 8-bit grayscale pictures. The integrated optical density (OD) value was measured by the MetaMorph analysis software (Molecular Devices, Sunnyvale, CA, USA). All data reported were corrected by subtracting the OD value of the IgG control in each corresponding section. Since no significant difference in Slit2/3 and Robo1/4 staining intensity was observed between adjacent normal ovarian tissues and normal ovarian tissues, data from these two types of tissues were pooled.</p><!><p>To determine the expression of Slit2/3 and Robo1/4 in OVCAR-3 and SKOV-3 cells, Western blot analysis was performed as described (Jiang et al. 2010; Wang et al. 2008; Zheng et al. 1999). OVCAR-3 and SKOV-3 cells were cultured as above. Cells were lysed by sonication in buffer [50 mM HEPES, 0.1 M NaCl, 10 mM EDTA, 4 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM sodium orthovanadate (pH 7.5), 1 mM PMSF, 1% Triton X-100, 5 µg/ml leupeptin, 5 µg/ml aprotinin]. After centrifugation, protein concentrations of the supernatant were determined with BSA (fraction V; Sigma, St. Louis, MO, USA) as standards. The protein samples (50 µg) were separated on 8% or 4–15% gradient SDS-PAGE gels and electrically transferred to PVDF membranes. Human hepatoblastoma cell lysate (50 µg, cat # sc2227; Santa Cruz Biotechnology) and mouse thyroid extract (50 µg, cat # sc2407; Santa Cruz Biotechnology) were run in parallel as positive controls for Slit2 and Slit3, respectively, as recommended by the manufacturer. Human umbilical cord vein endothelial (HUVE) cells were used as the positive control for Robo1/4 (Wang et al. 2003). The membranes were probed with a rabbit antibody against the C-terminus of human Slit2 (1: 200; cat # sc-28945), the N-terminus of human Slit3 (1: 200; cat # sc-28946; Santa Cruz Biotechnology), or Robo1 (1:1,000; cat # Ab7279) or Robo4 (1:1,000; cat # Ab10547; Abcam), followed by reprobing with a mouse GAPDH (1:20,000; cat # H00002597-M01; Abnova, Walnut, CA) or β-actin (1:4,000; Ambion, Austin, TX, USA) as a loading control. Proteins were visualized using enhanced chemiluminescence reagents (Amersham Biosciences, Piscataway, NJ, USA), followed by exposure to chemiluminescence films. The immunoreactive signals were analyzed by a densitometer. HUVE cells were isolated from term placentas of normal pregnant women. Collection of placentas was approved by the Institutional Review Board, Meriter Hospital, and the Health Sciences Institutional Review Boards, the University of Wisconsin- Madison, and followed the recommended guidelines for using human subjects.</p><p>To determine if Slit2 activates ERK1/2 and AKT1, additional OVCAR-3 and SKOV-3 cells were cultured as above. After serum starvation for 16 h, cells were treated without or with 100 ng/ml of recombinant human Slit2 protein (Cat # ab82131, Abcam) for 0, 10, 30 min, 1 h, and 3 h. The protein (15 µg) was subjected to Western blotting as described (Wang et al. 2008; Zheng et al. 1999). For each set of samples, at least two gels were run simultaneously. One membrane was blotted with a phospho-ERK1/2 antibody, followed by reblotting with a total ERK1/2 antibody (both were at 1:2,000). Another membrane was blotted with a phospho-AKT1 antibody (1:500) followed by reblotting with a total AKT1 antibody (1:2,000). All of these four antibodies were purchased from Cell Signaling Technology, Beverly, MA, USA. The membranes were also reprobed with a mouse GAPDH (1:10,000; cat # H00002597-M01; Abnova, Walnut, CA, USA). Proteins were visualized using chemiluminescence reagents (Amersham, Piscataway, NJ, USA). Changes in total and phospho-ERK1/2 and AKT1 protein levels were quantified by a densitometer. Data on phospho-ERK1/2 and AKT1 were normalized to total ERK1/2 and AKT1. Data on total ERK1/2 and AKT1 were normalized to GAPDH.</p><!><p>Cell migration was evaluated using a 24-Multiwell BD Falcon FluoroBlok Insert System (8.0 µm pores; BD Biosciences, San Jose, CA, USA) as described (Liao et al. 2010a). After serum starvation for 24 h, 30,000 cells were seeded in the insert (topside of membrane) and cultured in serum-free media. Slit2 (final concentration 100 ng/ml) in media containing 0.5% heat-inactivated FBS or media containing 0.5% heat-inactivated FBS as a control was added into the bottom wells. After 16 h, cells that migrated to the bottom of the inserts were stained with calcein AM (0.2 µg/ml; Invitrogen) for 30 min, examined, and recorded by an inverted microscope mounted with a CCD camera. The numbers of migrated cells were counted using the MetaMorph image analysis software (Molecular Devices). HUVE cells were run in parallel in a similar fashion as a positive control (Wang et al. 2003).</p><!><p>Cell proliferation assay was carried out as described (Wang et al. 2008; Zheng et al. 1999). Cells seeded in 96-well plates (5,000 cells/well) were cultured in complete growth media. After 16 h of serum starvation, cells were treated with Slit2 at 0, 5, 10, or 100 ng/ml (6 wells/dose). Media without or with Slit2 (100 ng/ml) were changed every 2 days. After 4 days of culture, the number of cells was determined using the crystal violet method. Briefly, cells were rinsed with distilled water and air dried. Once dried, cells were lysed with 2% (w/v) sodium deoxycholate solution with gentle agitation. Absorbance was measured at 570 nm on a microplate reader (BioTek Instrument, Winooski, VT, USA). Wells containing known cell numbers (0, 1,000, 2,000, 5,000, 10,000, 20,000, or 40,000 cells/well; 6 wells/cell density) were used to establish standard curves.</p><!><p>To confirm Slit2's action on ovarian cancer cell migration and proliferation, the wound healing assay was performed as described (Liao et al. 2010a). OVCAR-3 and SKOV-3 cells were cultured on 12-well plates in complete growth media, until reaching confluence, and serum starved overnight. A sterilized 200-µl pipette tip was used to make a straight scratch, simulating a wound. Cells were washed once with serum-free media and then treated without or with recombinant human Slit2 (100 ng/ml; cat # ab82131, Abcam) in serum-free media up to 24 h. Five images per scratch, which represented 60% of the length of each scratch, were photographed under a 4× objective immediately after scratching, and after 16 and 24 h of Slit treatment. Sizes of the wound area (mm2) were calculated using the MetaMorph image analysis software (Molecular Devices).</p><!><p>Data for the Slit/Robo staining intensity in the tissue microarrays and changes in ERK1/2 and AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells were analyzed using one-way ANOVA followed by pairwise comparisons (SigmaStat; Jandel Co., San Rafael, CA, USA). Data for the Slit/Robo protein levels in human ovarian cancer cells were analyzed using the Student's t test. p ≥ 0.05 was considered to be significant.</p><!><p>Positive staining for Slit2/3 and Robo1, but not Robo4 was present in NORM, primarily in stromal cells (Fig. 1A–D). The immunoreactivity of Slit2/3 and Robo1/4 was also found in cancer cells in several histotypes of ovarian cancer tissues (Fig. 1A–D). In Mu-ADEN and Se-ADEN, Slit2/3 and Robo1/4 were localized primarily in epithelial cells (Fig. 1A–D). No staining was observed in the preimmune goat or rabbit IgG control (see the small inserts in h panels of Fig. 1A–D). The semi-quantitative analysis revealed that for each target protein examined, there was no significant difference in staining intensity between the grades, stages, and TNM classifications in each histotype of ovarian cancer, except Se-ADEN (Fig. 1E).</p><p>For Slit2 (Fig. 1A), immunoreactivity was present in AGCT, primarily in granulosa cells, DISG, and YST, as well as in ADEN, Mu-ADEN, L-Se-ADEN, and H-Se-ADEN, primarily in epithelial cells. The overall staining intensity was relatively high in NORM, AGCT, DISG, YST, Mu-ADNE, L-Se-ADEN, and H-Se-ADEN as compared to that in ADEN and TMC. A semi-quantitative analysis confirmed these observations. However, due to high variation of staining intensity and relatively small sample sizes in certain histotypes of ovarian cancers, significant (p ≥ 0.05) differences in the staining intensity were detected only in YST and L-Se-ADEN as compared to NORM (Fig. 1E).</p><p>For Slit3 (Fig. 1B), the strong staining was seen in DISG and YST, while weak staining was presented in NORM, ADEN, AGCT, Mu-ADEN, TMC, L-Se-ADEN, and H-Se-ADEN. The semi-quantitative analysis also indicated that the staining was much more (p ≥ 0.05) intensive in DISG and YST than in the others (Fig. 1E). These differences in the staining intensity were in agreement with data from the semi-quantitative analysis (Fig. 1E).</p><p>For Robo1 (Fig. 1C), moderate and strong staining was observed in NORM and all histotypes of ovarian cancer. The semi-quantification analysis revealed that the staining intensity in DISG, YST, Mu-ADEN, L-Se-ADEN, and H-Se-ADEN was significantly higher (p ≥ 0.05) than that in NORM, AGCT, ADEN, and TMC (Fig. 1E).</p><p>For Robo4 (Fig. 1D), the overall staining was weak in NORM and all histotypes of ovarian cancer tissues except DISG, in which the staining was more intensive than that in all other tissues. These observations were confirmed by the semi-quantitative analysis (Fig. 1E).</p><!><p>The antibody against the C-terminus of human Slit2 detected a band at ~50 kDa in OVCAR-3, SKOV-3, and human hepatoblastoma cells (the positive controls) (Fig. 2). The molecular mass of 50 kDa corresponds to the C-terminus of human Slit2 reported (Ba-Charvet et al. 2001; Brose et al. 1999; Little et al. 2001; Patel et al. 2001). No band was detected at ~200 kDa (the full length Slit2) and ~140 kDa (the N-terminus of Slit2) (Ba-Charvet et al. 2001; Brose et al. 1999; Patel et al. 2001). The Slit2 antibody also detected an additional band right below 50 kDa in SKOV-3 cells (Fig. 2). It is currently unknown if this band represents a unique isoform or a truncated form of Slit2 in SKOV-3 cells.</p><p>The antibody against the N-terminus of human Slit3 detected two strong bands at ~65 and 45 kDa, as well as a weak band at ~100 kDa in OVCAR-3 and SKOV-3 cells, and also in the positive control (mouse thyroid extract) (Fig. 2). These ~65 and 45 kDa bands might represent the C-terminus of Slit3 as previously reported for Slit3 (Little et al. 2001) and Slit1/2 (Ba-Charvet et al. 2001; Brose et al. 1999; Little et al. 2001; Patel et al. 2001). No band was detected for Slit3 at ~200 kDa (the full-length Slit3; data not shown) and ~140 kDa (the N-terminus of Slit3; data not shown) (Ba-Charvet et al. 2001; Little et al. 2001; Patel et al. 2001). The Robo1/4 antibodies detected a single band at ~250 and 170 kDa, respectively, in OVCAR-3, SKOV-3, and HUVE cells (the positive control) (Fig. 2). Both of these molecular masses correspond to human Robo1/4 reported (Seki et al. 2010; Wang et al. 2003). These data indicate the expression of Slit2/3 and Robo1/4 in OVCAR-3 and SKOV-3 cells.</p><!><p>As compared to the control, recombinant human Slit2 at 100 ng/ml did not significantly induce ERK1/2 and AKT1 phosphorylation up to 3 h (Fig. 3). It, however, is noted that the basal level (time 0) of total ERK1/2, but not phospho ERK1/2 in SKOV-3 cells, was much higher (p ≥ 0.05) than that in OVCAR-3 cells; however, the basal level of phospho AKT1 but not total AKT1 in SKOV-3 was higher than that in OVCAR-3 cells (Table 1).</p><!><p>Under a serum-free condition, SKOV-3 underwent migration (Fig. 4). However, no cell migration was observed for OVCAR-3 cells under a similar serum-free condition and even after 10% FBS stimulation (data not shown). Slit2 at 100 ng/ml promoted (p ≥ 0.05) HUVE cell migration by 27% over the control in agreement with the previous report (Wang et al. 2003). Treatment of Slit2 did not alter SKOV-3 (Fig. 4) and OVCAR-3 (data not shown) cell migration.</p><p>After 4 days of treatment, the number of SKOV-3 cells (10,703 ± 556.9) in the control was increased approximately by twofold of the initially seeding cells, while no significant change was observed for OVCAR-3 cells (4,585 ± 574.1). As compared to the control, Slit2 slightly increased (~112% of the control) the number of OVCAR-3 cells at 5, 10, and 100 ng/ml, but this stimulatory effect did not reach significance (Fig. 5). Treatment of Slit2 also did not affect SKOV-3 cell numbers (Fig. 5).</p><p>Treatment of Slit2 did not affect wound healing in both OVCAR-3 and SKOV-3 cells (Fig. 6). In OVCAR-3 cells, the scratch areas did not show significant changes either in the control or in Slit2 treatment up to 24 h. In SKOV-3 cells, the scratch areas were similar between the control and Slit treatment at 16 h, whereas these were almost healed and not detectable in both the control and Slit2 treatment at 24 h (Fig. 6). These data confirm that Slit2 has no effect on proliferation and migration of SKOV-3 cells as described in Figs. 4 and 5.</p><!><p>In the current study, we have demonstrated, for the first time as far as we are aware, the protein expression of three members (Slit2/3 and Robo1) of Slit/Robo family in the adult human normal ovary tissue and in a variety of histotypes of human ovarian cancer tissues, suggesting that Slit/Robo might play an important role in both normal and malignant ovarian tissues. However, given that Slit2 did not affect cell proliferation and migration of ovarian cancer cells, the role of the Slit/Robo signaling in regulating human ovarian cancer cell function remains to be identified.</p><p>Among four Slit/Robo family members studied, Slit2/3 and Robo1 were relatively widely present in human normal and malignant ovarian tissues, while Robo4 was localized only in a few histotypes of ovarian cancer tissues (i.e., DISG, YST, and H-Se-ADEN). These data suggest that Slit proteins could function as autocrine or paracrine factors in human ovarian normal and malignant cancer. Moreover, as compared to Robo4, higher levels of Robo1 in normal ovarian tissues and many histotypes of ovarian cancer tissues studied also imply that Robo1 is a major receptor for Slit in both normal and malignant ovarian tissues. This is in agreement with previous reports showing that Robo1 is the major receptor for Slit proteins in the non-nervous system (Mertsch et al. 2008). It is noteworthy that for each Slit/Robo family member, expression levels were highly varied across different histotypes of ovarian cancer, albeit it could be partially attributed to the small sample size in some of these histotypes of cancer. This highly uneven expression of Slit/Robo is not surprising since human ovarian cancer is characterized by its high heterogeneity (Bast et al. 2009). Moreover, our observations that Robo1 levels in DISG, YST, and Se-ADEN were ~2.5 to 3.7-fold higher than those in NORM imply that Robo1 could serve as a diagnostic marker for these major histotypes of human ovarian cancer and/or could potentially be used as a therapeutic target for these ovarian cancers after its role is defined.</p><p>It is believed that Robo4 is exclusively expressed in endothelial cells, particularly in tumors (Neri and Bicknell 2005), and mediates Slit2-inhibited VEGF165-induced in vitro angiogenesis and VEGF165-induced in vivo vascular permeability (Jones et al. 2008). In contrast, blockade of Robo using a Robo neutralizing antibody could suppress the growth of human malignant melanoma cells in vivo, possibly via inhibiting tumor angiogenesis (Wang et al. 2003). Nonetheless, in the current study, regardless of whether Slit/Robo was pro- or anti-angiogenic, no apparent immunoreactivity of Robo4, and also Robo1 was detected in vascular endothelial cells in normal and malignant ovarian tissues. Thus, it is unlikely that Slit2 can directly target endothelial cells via binding to Robo1/4 in these ovarian tissues to affect ovarian angiogenesis.</p><p>The downstream signaling of Slit remains largely unknown. However, previous reports have implied that Slit2 could function via activating the MEK/ERK1/2 and PI3K/AKT1 signaling pathways in human malignant melanoma (Wang et al. 2003) and Xenopus retinal growth cones (Piper et al. 2006). Our current observations that Slit2 did not alter ERK1/2 and/or AKT1 phosphorylation in OVCAR-3 and SKOV-3 cells up to 3 h suggest that both ERK1/2 and AKT1, two key signaling molecules in mediating many essential cell processes (Kolch 2000), do not directly participate in mediating Slit2's actions in both of these cancer cell lines. It is noteworthy that the basal level of total ERK1/2 and phospho AKT1 in SKOV-3 cells was much higher than that in OVCAR-3 cells. It is currently unclear if such differential regulation has impacts on different behaviors (e.g., different growth rates) between these two cell lines.</p><p>In the current study, we detected mainly the C-terminus, but not full length and N-terminus of Slit2/3 in OVCAR-3 and SKOV-3 cells. These data suggest that the proteolytic fragments (Hohenester 2008) are the major forms of Slit2/3 existing in human ovarian cancer cells as in other non-tumor tissues (Hohenester 2008), and the majority of the N-terminus of Slit2/3 is rapidly released out and dissociate from ovarian cancer cells after their cleavage from the full length of Slit2/3. Given that the C-terminus of Slit2 is biologically inactive (Ba-Charvet et al. 2001; Patel et al. 2001), these C-terminal fragments of Slit2/3 should not play an important role in regulating ovarian cancer cell function. In contrast, the N-terminal fragments of Slit2/3 after their releases from the cells might act on ovarian cancer cells.</p><p>In recent years, most members of the Slit/Robo family have been found in many solid tumors, but their roles in the progression of various cancers have not been clearly defined. The association of high Robo1 levels with DISG, YST, and Se-ADEN supports the notion that the Slit/Robo is pro-cancer in human ovarian cancer, as suggested in many other cancers including endometrial cancer (Narayan et al. 2006), prostate cancer (Latil et al. 2003), breast cancer (Tanno et al. 2006), colorectal cancer (Grone et al. 2006), hepatocellular carcinoma (Ito et al. 2006), lung, kidney, liver, and metastatic melanoma (Seth et al. 2005), and malignant melanoma (Wang et al. 2003). However, in the current in vitro study, we observed that Slit2 did not affect cell proliferation and migration in both OVCAR-3 and SKOV-3 cells, although it stimulated migration of HUVE cells. Thus, we speculate that Slit/Robo may participate in regulating other ovarian cancer functions such as cell differentiation and apoptosis (Chédotal et al. 2005). For example, Slit2 might regulate cell apoptosis in human ovarian cancer, particularly since SKOV-3 and OVCAR-3 cells either keep proliferating (SKOV-3) or maintain their cell number (OVCAR-3) at the initial plating level even after 4 days of serum starvation as observed in the current study.</p>

PubMed Author Manuscript

Design of Novel Amphipathic \xce\xb1-Helical Antimicrobial Peptides with No Toxicity as Therapeutics against the Antibiotic-Resistant Gram-Negative Bacterial Pathogen, Acinetobacter Baumannii

We designed de novo and synthesized two series of five 26-residue amphipathic \xce\xb1-helical cationic antimicrobial peptides (AMPs) with five or six positively charged residues (D-Lys, L-Dab (2,4-diaminobutyric acid) or L-Dap (2,3-diaminopropionic acid)) on the polar face where all other residues are in the D-conformation. Hemolytic activity against human red blood cells was determined using the most stringent conditions for the hemolysis assay, 18h at 37\xc2\xb0C, 1% human erythrocytes and peptide concentrations up to 1000 \xce\xbcg/mL (~380 \xce\xbcM). Antimicrobial activity was determined against 7 Acinetobacter baumannii strains, resistant to polymyxin B and colistin (antibiotics of last resort) to show the effect of positively charged residues in two different locations on the polar face (positions 3, 7, 11, 18, 22 and 26 versus positions 3, 7, 14, 15, 22 and 26). All 10 peptides had two D-Lys residues in the center of the non-polar face as \xe2\x80\x9cspecificity determinants\xe2\x80\x9d at positions 13 and 16 which provide specificity for prokaryotic cells over eukaryotic cells. Specificity determinants also maintain excellent antimicrobial activity in the presence of human sera. This study shows that the location and type of positively charged residue (Dab and Dap) on the polar face are critical to obtain the best therapeutic indices.

design_of_novel_amphipathic_\xce\xb1-helical_antimicrobial_peptides_with_no_toxicity_as_therapeutics

Introduction<!>Solid-phase peptide synthesis and reversed-phase purification<!>Characterization of helical structure<!>Amino acid analysis for peptide quantitation<!>Gram-negative bacterial strains used in this study<!>Antimicrobial activity (MIC) determination<!>Hemolytic activity (HC50) determination<!>Therapeutic index (T.I.) determination<!>Peptide design, location and type of positively charged residue on the polar face<!>Antibacterial activity<!>Hemolytic activity and therapeutic indices<!>Peptide hydrophobicity<!>Peptide helicity<!>Conclusion

<p>The growing emergence of pathogenic bacteria with clinically significant resistance to conventional antibiotics is a major public health concern [1-5]. As noted by Falanga, et al. [6] we are facing a worldwide re-emergence of infectious diseases and a rapid increase in multidrug-resistant (MDR) bacteria, threatening the world with a return to the pre-antibiotic era. Indeed, there are now "Superbugs" that are resistant to most or all available antibiotics [7]. The scope of the challenge in tackling drug-resistant infections globally is reported in detail in a 2016 review on antimicrobial resistance [4]. Thus, it was estimated that, by 2050, 10 million lives a year will be at risk due to the rise of drug-resistant infections if proactive solutions are not quickly found to slow the rate of drug resistance. At present, 700,000 people die every year from drug resistant strains of common bacterial infections, HIV, TB and malaria [4]. While the 2016 review [4] offered a plethora of approaches to slowing down or preventing future bacterial resistance to antibiotics (e.g., promoting vaccine use, avoiding unnecessary antibiotic use, better water and sanitation, decrease in environmental pollution), the fact remains that organisms resistant to conventional antibiotics will still be present and must be dealt with. Indeed, it has frequently been asserted that, as part of a global response to MDR bacteria, we must increase the number of effective antimicrobial drugs to defeat infections that have become resistant to existing antibiotics [4]. Unfortunately, antibiotic discovery has stalled just as we need it the most. Between 1929 and the 1970s, more than 20 new classes (not just analogs of an existing class) of antibiotic reached the market [3]. Since then, only two new classes have reached the market, with the worldwide antibiotic pipeline for new antibiotic classes active against highly resistant Gram-negative bacteria being almost non-existent [3]. It is estimated that only 4 new classes of antibiotics can be expected in the next 30 years, while antibiotic resistance to some pathogens may more than double in the same period [4]. Although, in the 1970s and 1980s, the pharmaceutical industry did produce a stream of antibiotics, these were not new classes but analogs of existing classes [3]. The fundamental problem with this approach is that, although analog development is low risk compared to novel class discovery and development, analogs eventually became more difficult to come by and the process hits a dead end.</p><p>A potential solution to the crisis of medically resistant strains of bacteria lies in a ubiquitous response in nature to bacterial infections, namely the production of antimicrobial peptides (AMPs) [6,8-24]. AMPs are produced by a wide variety of organisms, including bacteria, fungi, plants, insects, amphibians, crustaceans, fish and mammals (including humans) [25]. AMPs (specifically, cationic AMPs) are fast-acting bactericides with generally broad spectrum activity [25]. In addition, AMPs in general do not have specific targets (unlike traditional antibiotics), their mode of action generally involving nonspecific interactions with the cytoplasmic membrane of bacteria. This causes peptide accumulation in the membrane, leading to increased permeability and loss of barrier function [8,9,12,13,15-30]. Development of resistance is not expected since this would require substantial changes in the lipid composition of the cell membranes of microorganisms. The majority of AMPs in current clinical development target skin infections caused by Gram-positive bacteria, i.e., topical use only [31]. In addition, within the last 30 years, only four natural AMPs have found their way onto the market and no systemic AMP has been approved by the Federal Drug Administration in the USA [31]. This dearth of clinically approved AMPs despite the past three decades of attempts and the excellent antimicrobial activity of many AMPs lies mainly in their generally high toxicity to normal cells which prevents their use as a systemic drug. Interestingly, cationic AMPs polymyxin B and polymyxin E (colistin) saw widespread use in the 1960s and 1970s. However, their clinical use in the 1970s was scaled back considerably due to serious neurotoxicity and nephrotoxicity issues [32-36]. Despite these toxicity drawbacks, these two peptides returned as antibiotics of last resort with the emergence of prevalent Gram-negative bacteria with multidrug resistance. However, the aforementioned emergence of polymyxin-resistant "Superbugs" [32,33,36], due to the fact that these particular peptide antibiotics (unlike the AMPs presently under consideration) have specific targets and are thus prone to resistance, means that it is now critical to develop antimicrobials effective against both polymyxin B- and colistin-resistant microorganisms. Worldwide research for the past 30 years to remove toxicity from AMPs, thus enabling a shift of focus from development of peptide drugs for topical use towards agents for systemic administration, has been unsuccessful until recent work in our laboratory.</p><p>Numerous structure/activity studies on both natural and synthetic AMPs identified factors important for antimicrobial activity: the presence of both hydrophobic and basic (positively charged) residues, an amphipathic nature, and preformed or inducible secondary structure (α-helix or β-sheet) [16]. We have always postulated that a de novo design synthetic peptide approach to examining the effect of incremental changes in these parameters would enable rapid progress in the rational design of novel peptide AMPs. Thus, from lessons learned about factors important for antimicrobial activity, as noted above, we utilized the structural framework, or template, of a 26-residue amphipathic α-helical AMP with excellent antimicrobial activity but with, initially, strong hemolytic activity [16]. The 26-residue length of the template was designed to be able to accept amino acid substitutions with minimal effects on peptide properties and stability other than the ones under investigation; at the same time, synthesis and purification of analogs remained straightforward. With this template approach, we determined the effect on biological activity of varying the hydrophobicity of the non-polar face [37] or the number of positively charged residues on the polar face [38]. In addition, utilizing D-enantiomers of amino acids led to excellent stability against proteolytic digestion (a key property for AMPs to be useful as injectable AMPs), whilst maintaining excellent antimicrobial activity [39].</p><p>At this point, a major milestone was our discovery of "specificity determinants" allowing selectivity between eukaryotic cells and Gram-negative microorganisms, i.e., producing a major decrease in toxicity as measured by hemolysis of human red blood cells [39-41]. These "specificity determinants" were one, later two, Lys-substitutions in the middle of the non-polar face of the amphipathic model peptide, a peptide long enough to allow such substitutions whilst maintaining sufficient hydrophobicity on the non-polar face. Briefly, we utilized positively charged residues as specificity determinants (Lys residues at positions 13 and 16 of the non-polar face) of the 26-residue peptide. In addition, we manipulated total hydrophobicity, hydrophobe type and location as design parameters. Taken together, these approaches resulted in unprecedented, at that time, improvements in therapeutic indices (hemolytic activity/antimicrobial activity) [40,41]. This discovery hastened another aspect of our template design, namely the requirement for our peptides to lie parallel to the membrane, surface, i.e., promoting the "carpet model" of interaction [24,42,43] while preventing penetration of the membrane as a transmembrane helix in eukaryotic cells via a "barrel stave" mechanism [24,44], thus preventing hemolysis. Further, we demonstrated that modification of native AMPs (the 22-residue Piscidin 1 and 28-residue Dermaseptin S4) with Lys specificity determinants in the non-polar face of these amphipathic α-helical peptides produced similar results of improved antimicrobial activity and dramatically decreased hemolytic activity [45,46]. Such results are of critical importance to the future of AMPs as therapeutic agents.</p><p>With our focus now on developing a better Gram-negative AMP rather than to maintain broad-spectrum activity in a "one size fits all" approach, thus hastening development of such AMPs, we recently turned our attention to the polar face of our peptide template [47]. Namely, we replaced the Lys residues with Arg residues and unusual amino acids: ornithine (Orn) [22,48-53], diaminobutyric acid (Dab) [22,48-53] or diaminopropionic acid (Dap) [22,48-54]. Excitingly, AMPs with specificity determinants and with L-Dab and L-Dap on the polar face have essentially no hemolytic activity at high peptide concentrations (1000 μg/mL), demonstrating for the first time the importance of these unusual amino acid residues in solving long-standing hemolysis issues of AMPs, whilst maintaining excellent antimicrobial activity against seven Acinetobacter baumannii strains, resistant to polymyxin B and colistin, and 20 A. baumannii isolates from 2016 and 2017 with resistance to 18 different antibiotics.</p><p>The present study serves to continue the success of our template-driven de novo design approach by attempting to fine-tune our recent achievement of utilizing unusual amino acids (Dab and Dap) in the polar face of our AMP to eliminate hemolysis [47]. Thus, we have determined the effect of changing locations of positively charged residues on the polar face of the AMP, as well as eliminated a single positively charged residue at the C-terminal which allows future development of Pegylated AMPs on a C-terminal cysteine residue if prolonged half-life is necessary.</p><!><p>The synthesis and purification methods have been described in detail in a previous publication [47].</p><!><p>Circular dichroism (CD) spectroscopy was used to determine the mean residue molar ellipticities of the peptides, using a Jasco J-815 spectropolarimeter (Jasco, Inc., Easton, MD, USA) under two sets of conditions: at pH 7.0 the buffer was 50 mM NaH2PO4/NaHPO4/100 mM KCl and in the presence of an α-helix inducing solvent, 2, 2, 2-trifluoroethanol, TFE, (50 mM NaH2PO4/NaHPO4/100 mM KCl, pH 7.0 buffer/50% TFE). A 10-fold dilution of an approximately 500μM stock solution of the peptides was loaded into a 0.1 cm quartz cell and its ellipticity scanned from 195 to 250 nm. Peptide concentrations were determined by quantitative amino acid analysis.</p><!><p>The method of Cohen and Michaud [55] was used for amino acid analysis. Each peptide sample was aliquoted into glass tubes and lyophilized followed by acid hydrolysis in 6 M HCl with 0.1% phenol for 48 h at 110°C. The resulting solution was allowed to come to room temperature and then vacuum-dried to remove the HCl. Each sample was then resuspended in 10 mM HCl and 20 μL of sample was added to 60 μL of 0.2M sodium borate buffer, pH 8.8. To this mixture, 20 μL of 6-aminoquinoyl-N-hydroxysuccinimidyl carbamate in acetonitrile was added to derivatize the amino acids present in the sample. This sample was then heated to 55°C for 15 min to convert Tyr byproducts to one form. An Agilent 1260 series instrument with a Waters AccQTag column, 3.9 mm I.D. × 150 mm column was used to separate and quantify the derivatized amino acids present in each sample using UV absorbance at 254 nm.</p><!><p>The A. baumannii strains used in this study consisted of seven strains resistant to Polymyxin B and Colistin (antibiotics of last resort) obtained from MERCK (M89941, M89949, M89951, M89952, M89953, M89955 and M89963). The MICGM in this case is the geometric mean MIC from the seven Acinetobacter baumannii strains used in this study.</p><!><p>The minimal inhibitory concentration (MIC) is defined as the lowest peptide concentration that inhibited bacterial growth. MICs were measured by a standard microtiter dilution method in Mueller Hinton (MH) medium. Briefly, cells were grown overnight at 37°C in MH broth and were diluted in the same medium. Serial dilutions of the peptides were added to the microtiter plates in a volume of 50 μL, followed by the addition of 50 μL of bacteria to give a final inoculum of 5 × 105 colony-forming units (CFU)/mL. The plates were incubated at 37°C for 24h, and the MICs were determined. The MICGM is the geometric mean of the number of MIC values.</p><!><p>Peptide samples (concentrations determined by amino acid analysis) were added to 1% human erythrocytes in phosphate-buffered saline (100 mM NaCl, 80 mM Na2HPO4, 20 mM NaH2PO4, pH 7.4) and the reaction mixtures were incubated at 37°C for 18h in microtiter plates. Two-fold serial dilutions of the peptide samples were carried out. This determination was made by withdrawing aliquots from the hemolysis assays and removing unlysed erythrocytes by centrifugation (800 × g). Hemoglobin release was determined spectrophotometrically at 570 nm. The control for 100% hemolysis was a sample of erythrocytes treated with water. The control for no release of hemoglobin was a sample of 1% erythrocytes without any peptide added. Since erythrocytes were in an isotonic medium, no detectable release (<1% of that released upon complete hemolysis) of hemoglobin was observed from this control during the course of the assay. The hemolytic activity HC50 is the peptide concentration that causes 50% hemolysis of erythrocytes after 18h. HC50 was determined from a plot of percent lysis versus peptide concentration (μM) using 12 different concentrations up to 1000 micrograms per ml for 18h at 37°C. The average of 3 replicates is used with an average variance of less than 4%. Fresh human blood was obtained from Vitalant, Denver, CO, USA.</p><!><p>The therapeutic index is a widely accepted parameter to represent the specificity of antimicrobial peptides for prokaryotic versus eukaryotic cells. It is calculated by the ratio of hemolytic activity (HC50) and antimicrobial activity (MICGM). The MICGM in this case is the geometric mean MIC from the seven Acinetobacter baumannii strains used in this study. Thus, larger values of therapeutic index indicate greater specificity for prokaryotic cells. Thus, the therapeutic index is the HC50/MICGM ratio.</p><!><p>In this study, we designed de novo, synthesized, purified and characterized ten potentially amphipathic α-helical antimicrobial peptides (AMPs) where we changed the location of positively charged residues on the polar face of the α-helix. Location 1 consists of five or six positively charged residues at positions 3, 7, 11, 18, 22 and 26 or 3, 7, 11, 18 and 22 (Figure 1). Location 2 has these residues at positions 3, 7, 14, 15, 22 and 26 or 3, 7, 14, 15 and 22 (Figure 1). The C-terminal positively charged residue (position 26) was replaced from both sets of AMPs with Ser 26 (Table 1). All ten AMPs have two "specificity determinants" (D-Lys residues at 13 and 16 in the center of the non-polar face). We have previously shown the critical importance of "specificity determinants" in these AMPs which encoded selectivity for Gram-negative pathogens and removed both Gram-positive activity and hemolytic activity from broad spectrum AMPs [40,41,45-47]. In addition, we have shown that specificity determinants have another important role of preventing high-affinity to human serum proteins [40,41,45,46].</p><p>Figure 1 shows a general amino acid sequence in a helical wheel and helical net representations where X denotes the positions on the polar face of the positively charged residues (colored blue). We have displayed two versions of the helical nets where the polar residues are displayed along the center of the helical net. Panel A shows the polar face residues at positions 3, 7, 11, 18, 22 and 26 and Panel B shows the polar face residues at positions 3, 7, 14, 15, 22 and 26. The major difference between the two relates to positions 11 and 18 in Panel A and positions 14 and 15 in Panel B. When the positively charged residues are at positions 3, 7, 11, 18, 22 and 26, we have denoted this orientation as −1 at the end of the peptide name (Table 1). When the positively charged residues are at positions 3, 7, 14, 15, 22 and 26, we have denoted this orientation as −2 at the end of the peptide name (Table 1). The −2 orientation creates a positively charged cluster in the sequence at positions 13, 14, 15 and 16. Positions 13 and 16 are the D-Lys residues as the specificity determinants on the non-polar face and positions 14 and 15 are on the polar face (D-Lys, L-Dab or L-Dap) residues. One of the most interesting points of this study was the substitutions of Dab and Dap residues in the L-conformation into an otherwise all D-antimicrobial peptide. The substitution of 5 or 6 positively charged residues on the polar face as either D-Lys, L-Dab or L-Dap (Table 1) was not expected to have any undesired effect on the conformation since our objective was to have as little α-helical structure as possible in aqueous conditions but maximum inducible α-helical structure in the presence of the hydrophobicity of the membrane (mimicked here by determining the helical structure by circular dichroism spectroscopy (CD) in the presence of 50% trifluoroethanol). We did not expect the 5 or 6 L-substitutions of Dab or Dap residues to affect the overall structure in any significant way since there are 20 or 21 positions out of 26 to maintain the structure in the presence of the hydrophobicity of the membrane. The use of the L-conformation for Dab and Dap residues was based on the fact that they are significantly less expensive for peptide synthesis and would not introduce any susceptibility to proteases since the Dab and Dap residues are unusual amino acids and are not recognized by proteases. The hydrophobic/non-polar faces of all ten AMPs have eight Leu residues in two clusters of four (colored yellow) separated by two Lys residues (specificity determinants in the center of the non-polar face (colored red)) (Figure 2). Position 1 in all peptides is D-Lys which we consider is on the non-polar face; thus, the non-polar face contains three D-Lys residues at positions 1, 13 and 16 to give a net charge of +3 on the nonpolar face and +5 or +6 on the polar face resulting in an overall net charge on these AMPs of either +9 or +8 depending on whether there are 6 or 5 positively charged residues on the polar face (Table 1).</p><p>In the helical wheels, the non-polar face is indicated as a yellow arc (Leu residues are colored yellow and position Lys 1 and the specificity determinants at positions 13 and 16 are colored pink). The polar face is indicated as a black arc (positively charged residues are colored blue). In the helical nets, the residues on the non-polar face are circled with the Lys residues colored red (Lys 1 and the specificity determinants, Lys 13 and Lys 16) and the Leu residues in two clusters (L2, L5, L6, L9 for the N-terminal cluster and L17, L20, L21 and L24 for the C-terminal cluster). The black open boxes denote the positively charged residues on the polar face at positions 3, 7, 11, 18, 22 and 26 (Panel A) and positions 3, 7, 14, 15, 22 and 26 (Panel B). The potential i to i +3 or i to i + 4 hydrophobic interactions between large hydrophobes are shown as black solid lines.</p><!><p>Table 2 shows the antibacterial activities against 7 different Acinetobacter baumannii strains resistant to polymyxin B and colistin (antibiotics of last resort). The geometric mean MIC values were determined for the ten AMPs, where the positively charged residue was varied from D-Lys, L-Dab and L-Dap on the polar face in two different locations designated −1 or −2 (Table 2). The MICGM values for Lys residues on polar face at positions 3, 7, 11, 18, 22 and 26 was 0.5 μM and for positions 3, 7, 14, 15, 22 and 26 was 0.4 μM. This suggests that antibacterial activity is not significantly affected by the change in location of the positively charged residues on the polar face. Similarly, replacing D-Lys residues with L-Dab residues had very little effect on the MICGM (0.9 μM at positions denoted −1 and 0.7 μM at positions denoted −2). Replacing D-Lys residues with L-Dap residues was more significant (0.5 μM for D-Lys at positions −1 and 1.2 μM for L-Dap residues at positions −1). At the −2 location, D-Lys residues had a MICGM value of 0.4 μM, while L-Dap residues at these same positions resulted in a value of 0.8 μM. Clearly, shortening the side-chain from Lys to Dab and then to Dap has a small but significant effect on antibacterial activity. The MICGM changed from 0.5 μM to 0.9 μM to 1.2 μM for D-Lys, L-Dab and L-Dap, respectively, when in the −1 location (compare D84, D86 and D105, Table 2). The MICGM changed from 0.4 μM to 0.7 μM to 0.8 μM for D-Lys, L-Dab and L-Dap, respectively, when in the −2 location (compare D88, D89 and D106, Table 2). The largest difference in the two locations occurs when Dap residues are used (compare D105(Lys1-6 Dap-1) MICGM of 1.2 μM with D106(Lys1-6 Dap-2) MICGM of 0.8 μM). There seems to be a major advantage to have Dap residues at position 14 and 15 on the polar face rather than positions 11 and 18. Positions 14 and 15 are between the two specificity determinants (D-Lys residues) on the non-polar face at positions 13 and 16. This is creating a positively charged cluster in the sequence (D-Lys13, L-Dap14, L-Dap15 and D-Lys 16) (Table 2). In the big picture, the changes in the geometric mean MIC value are minor compared to the effect observed in hemolytic activity by changing the residues on the polar face from D-Lys to L-Dab and L-Dap residues (4 carbon atoms, 2 carbon atoms and 1 carbon atom in the side-chain, respectively (Table 2). We discovered that we can eliminate the positively charged residue at position 26 with no significant effect on the geometric mean MIC value (compare D86(Lys1-6 Dab-1), MICGM 0.9 μM to D102(Lys1-5 Dab-1), MICGM 0.7 μM and D89(Lys1-6 Dab-2), MICGM 0.7 μM to D104(Lys1-5 Dab-2), MICGM 0.8 μM) (Table 2).</p><!><p>The biological activities of the ten peptide analogs are shown in table 2. The hemolytic activity is expressed as the HC50 value which is the concentration of peptide that results in 50% hemolysis of human red blood cells. In order to determine that we were able to eliminate hemolysis of human red blood cells, we used the most rigorous test of hemolytic activity (18h at 37°C and up to 1000 μg/mL or >350 μM of AMP). This is in stark contrast to other researchers who routinely use incubation times of just 0.5-2h. We have shown that, when the exposure time is increased from less than 2 h to 18h [16,20,39], substantially greater hemolysis is observed. Clearly, hemolysis should be monitored on human red blood cells for an exposure time up to 18h, since anything less will lead to misleading results. From figure 3, which shows the effect of peptide concentration on human red blood cells lysis, the decrease in hemolytic activity resulting from the use of the two unusual amino acid residues Dab and Dap on the polar face is dramatic. The Lys containing peptides (D84(Lys1-6 Lys-1) and D88(Lys1-6 Lys-2) have HC50 values of 54.3 and 80.6 μM and show 100% hemolysis at high peptide concentration (Figure 3 and Table 2). On the other hand, the two peptides containing Dab and Dap residues show essentially no lysis of human red blood cells at 1000 μg/mL as indicated by the linear lines (Figure 3) (D105(Lys1-6 Dap-1) and D89(Lys1-6 Dab-2)). The location of the positively charged residues on the polar face has a major effect on hemolysis and the best location is dependent on whether Dab or Dap residues are used. Using Dap residues in the −2 location (Dap at positions 14 and 15) results in significant more lysis of human red blood cells versus the −1 location (Dap at positions 11 and 18) compare D106(Lys1-6 Dap-2) to D105(Lys1-6 Dap-1 (Figure 3). See figure 1 to observe the difference in location on the polar face between −1 and −2 locations. When using Dab residues, the exact opposite effect is observed. Dab residues in the −2 location (Dab at positions 14 and 15) results in no measurable hemolysis at 1000 μg/mL (D89(Lys1-6 Dab-2), Figure 3). These results suggest that side-chain length, number of carbon atoms and location can affect hemolysis. The HC50 value is estimated when 50% hemolysis is not observed at 1000 μg/mL by extrapolation of the plots observed in figure 3. The therapeutic indices are calculated from the HC50 (μM)/MICGM (μM). We have also shown that we can remove the C-terminal positively charged residue and replace it with Ser26 without any consequence (Table 2). Compare removing the C-terminal Lys residue D84(Lys1-6 Lys-1) (TI=108.6) to D101(Lys1-5 Lys-1) (TI=129.9) and D88(Lys1-6 Lys-2) (TI=201.5) to D013(Lys1-5 Lys-2) (TI=192.7). Similarly, on removing the C-terminal Dab residue, compare D86(Lys1-6 Dab-1) (TI > 824) to D102(Lys1-5 Dab-1) (TI= >1012) and D89(Lys1-6 Dab-2) (TI> 1589) and D104(Lys1-5 Dab-2) (TI= >1863).</p><!><p>Retention behavior in reversed-phase high-performance liquid chromatography (RP-HPLC) is an excellent method to represent overall peptide hydrophobicity. Even though the non-polar face of an amphipathic α-helical peptide represents the preferred binding domain for its interaction with the hydrophobic matrix of the reversed-phase column [56,57]; the overall hydrophobicity is also affected by the composition of residues on the polar face (five or six positively charged residues) (Figure 1). The RP-HPLC results for these two series of peptides are shown in figure 4 and table 3. Panel A shows the separation of five peptides with positively charged residues in the −1 location (positions 3, 7, 11, 18, 22 and 26 or 3, 7, 11, 18 and 22). Panel B shows the separation of the five peptides with positively charged residues in the −2 location (positions 3, 7, 14, 15, 22 and 26 or 3, 7, 14, 15, and 22). The type of positively charged residue on the polar face has a dramatic effect on the overall hydrophobicity, with the Dab residue being more hydrophilic (less hydrophobic) than the Dap residue even though the Dab residues are a carbon atom larger in their side-chain compared to the Dap residue: Dab peptide D86 (6Dab-1) retention time of 113.9 min compared to Dap peptide D105 (6Dap-1) retention time of 126.6.min (Panel A). Similarly, with these peptides in the −2 location, the D89 (6Dab-2) retention time was 115.8 min compared to the D106 (6Dap-2) peptide retention time of 128.8 min (Panel B). This can be explained by the Dab residues stabilizing the α-helical structure considerably more than the Dap residues. This means the polar face of the Dab peptides are interacting more with the hydrophobic matrix than the polar face of the Dap peptides, which results in a large decrease in retention time (tR for Dap peptides is 126.6 min in the −1 location and tR for Dab peptide is 113.9 min, i.e., a decrease of 12.7 min) or tR for Dap peptides is 128.8 min in the −2 location and tR for Dab peptide is 115.8 min, i.e., a decrease of 13.0 min). Compare Panel A and Panel B of figure 4 and table 3. All the +9 peptides shown in table 1 are identical in sequence except for the six polar face substitutions which are either D-Lys, L-Dab or L-Dap residues. Similarly the +8 peptides in table 1 have either 5 Lys or 5 Dab residues in two different locations (−1 or −2). The Lys peptides are always considerably more hydrophobic than the Dab peptides in location −1 or −2. This agrees with Lys peptides containing 4 carbon atoms in their side-chains relative to Dab peptides with 2 carbon atoms in their side-chains.</p><!><p>The biophysical data for our ten peptides are shown in table 3. Circular dichroism (CD) spectroscopy was used to determine the α-helical content in aqueous conditions at pH 7 (50 mM PO4, 100 mM KCl) and in the presence of 50% trifluoroethanol (TFE) to mimic the hydrophobicity and α-helix inducing ability of the hydrophobic membrane, the target of our AMPs. Our strategy was to minimize α-helical structure in aqueous conditions and maximize the inducible α-helical structure in the presence of the hydrophobicity of the membrane. The % helix in aqueous conditions varied from 6 to 29% and the % inducible α-helix varied from 71 to 94% depending on the peptide (Table 3). The specificity determinants (Lys residues at positions 13 and 16 in the center of the non-polar face) were used to disrupt the continuous hydrophobic face of our template, creating two hydrophobic clusters of leucine residues, cluster one consisted of leucine residues at positions 2, 5, 6 and 9 and cluster two consisted of leucine residues at positions 17, 20, 21 and 24 (Figure 2). Though all AMPs met the general requirement of low α-helical content in aqueous conditions and dramatic increases in α-helical content in the presence of 50% TFE, there was no direct correlation with the type of positively charged residue (Lys, Dab or Dap) used on the polar face and helical content. In summary, inducible α-helical structure plays a critical role in providing our AMPs with desired properties.</p><!><p>The goal of the present study was to determine whether our template-driven de novo designed peptide approach which enabled us to fulfill the long-sought goal of eliminating toxicity from AMPs could be further refined to improve therapeutic indices even more, as well as allow pegylation of the peptide model to enhance AMP half-life during therapeutic use, if required. Thus, our original 26-residue amphipathic α-helical AMP template, containing two D-Lys specificity determinants at positions 13 and 16 of the non-polar face and positively charged residues (D-Lys, L-Dab or L-Dap) at positions 3, 7, 11, 18, 22 and 26 of the polar face (Figures 1 and 2) were modified in two ways: (1) changing the positions of positively charged residues on the polar face originally at positions 11 and 18 (−1 orientation) to positions 14 and 15 (−2 orientation), the latter creating a positively charged cluster at positions 13, 14, 15 and 16 (Figures 1 and 2); and (2) eliminating the positively charged residue at position 26 through replacement with serine. Interestingly, the location of positively charged residues on the polar face had a major effect on hemolysis and the best location was dependent on whether Dab or Dap residues were used, i.e., side-chain length, number of carbon atoms and residue location all appear to affect hemolysis. Significantly, the therapeutic index of the 6Dab-containing −1 analog (>1012) rose to >1589 for the 6Dab-2 analog, an impressive increase in efficacy. In addition, the 5Dab-1 analog with a T.I. of >1012 saw an even greater increase in T.I. (>1863) for the 5Dab-2 peptide.</p><p>Comparing the 6Dab and 5Dab peptide series, the T.I. values for 6Dab-1 and 5Dab-1 were >824 and >1012, respectively; for the 6Dab-2 and 5Dab-2 peptides, the T.I. values were >1589 and >1863, respectively. Thus, we have shown that we can remove the C-terminal positively charged residue and replace it with Ser26 without any consequence; indeed, for the Dab analogs, an improvement in T.I. was observed. Such results now allow us to investigate the effectiveness of pegylation to a C-terminal Cys residue, in place of a positively charged residue, in order to prolong peptide half-life when desired.</p><p>Our continuing studies clearly show the potential of our amphipathic AMPs as potential therapeutics to replace existing antibiotics as well as the leading edge peptide design which our de novo designed template represents.</p>

PubMed Author Manuscript

Accessing Lipophilic Ligands in Dendrimer-Based Amphiphilic Supramolecular Assemblies for Protein-Induced Disassembly

Supramolecular nanoassemblies that respond to the presence of proteins are of great interest, as aberrations in protein concentrations represent the primary imbalances found in a diseased state. We present here a molecular design, syntheses, and study of facially amphiphilic dendrimers that respond to the presence of the protein, immunoglobulin G. It is of particular interest that the ligand functionality, utilized for causing the binding-induced disassembly, be lipophilic. Demonstration of binding with lipophilic ligands greatly expands the repertoire of binding-induced disassembly, since this covers a rather large class of ligand moieties designed for proteins and these provide specific insights into the mechanistic pathways that are available for the binding-induced disassembly process. Here, we describe the details of the binding induced disassembly, including the change in size of the assembly in response to proteins, concurrent release of noncovalently encapsulated guest molecules, and the specificity of the disassembly process.

accessing_lipophilic_ligands_in_dendrimer-based_amphiphilic_supramolecular_assemblies_for_protein-in

Introduction<!>Molecular design and synthesis<!>Self-assembling properties of dendrimers<!>Dendrimer\xe2\x80\x93protein interactions<!>Conclusion

<p>Stimuli-responsive supramolecular systems have gained significant attention in recent years, as these systems have implications in a variety of areas, especially in drug delivery.[1] Similarly, amphiphilic assemblies have been attractive because of the potential to encapsulate water-insoluble drug molecules within their interior, which can then be released at a specific location in response to a trigger.[2] In this regard, amphiphilic systems that respond to physical or chemical internal or external stimuli have been widely explored. Systems that respond to change in pH,[3] temperature,[4] redox environment,[5] and light[6] are popular in this area. Location-dependent variations in these factors could be considered to be secondary imbalances in biology. The primary imbalances in biology that result in a diseased state involve variations in protein concentrations. Therefore, it is interesting to design supramolecular assemblies that respond to proteins, an area that is relatively under-explored.[7]</p><p>Among responsive supramolecular assemblies, nanosized structures have been of great interest, since these have the propensity to accumulate in the inflamed tissues of certain diseases, notably cancer, due to the enhanced permeability and retention (EPR) effect.[8] This has spurred interest in molecular assemblies, based on surfactants,[9] lipids,[10] polymers,[2,11] and dendrimers.[12] While small molecule surfactant based amphiphilic assemblies exhibit the ability to sequester lipophilic molecules in aqueous media, these suffer from low stability and high critical aggregation concentrations (CACs). On the other hand, phospholipid based liposomes exhibit enhanced stability and low CACs.[10] However, their application is largely limited to the delivery of hydrophilic molecules or lipophilic molecules that can be rendered water soluble.[10,13] Considering the fact that most drug molecules are lipophilic, it is desirable that systems possessing lipophilic microenvironments, to accommodate guest molecules, are used. In this context, amphiphilic polymers have been widely studied for drug delivery applications due to their ability to form micelles and encapsulate lipophilic guest molecules. These efforts have resulted in some excellent contributions to the field.[14] However, dendrimers provide a distinct advantage in fundamentally understanding the structural factors that control amphiphilic supramolecular assemblies and stimuli-responsive disassemblies. This is mainly due to the excellent control over their size and, thus, the perfectly monodisperse nature of these macromolecules.[12,15]</p><p>With all these scaffolds, a limited number of reports exist on protein-sensitive assemblies.[7,16] These reports, however, are mainly based on the enzymatic activity of the protein that causes the disassembly rather than a specific ligand–protein interaction. Designing systems that respond to a protein-binding event will bring the large class of nonenzymatic proteins into the realm of stimuli-responsive supramolecular nanoassemblies and, thus, greatly expand the repertoire of these systems for applications, such as drug delivery and sensing.</p><!><p>Recently, we introduced a new class of facially amphiphilic biaryl dendrimers in which every repeating unit in the dendritic backbone contains both lipophilic and hydrophilic functionalities.[4a,12e] As a result of the orthogonal placement of these mutually incompatible units, these molecules exhibit a unique assembly switch, depending on their solvent environment. In other words, these dendrimers are able to form micelle-type assemblies in an aqueous milieu and inverted micelle-type assemblies in apolar solvents, such as toluene.[12e,17] In contrast to classical amphiphilic dendrimers,[12a,d] the micellar assemblies from our dendrimers are formed through aggregation of several dendrimer molecules. This feature presents a unique opportunity for stimuli-induced disassembly in these dendrimers through disaggregation.</p><p>Certain hydrophilic–lipophilic balance (HLB) is required for these facially amphiphilic dendrimers to self-assemble in the aqueous phase. Therefore, we envisaged the possibility of disturbing the HLB of a dendrimer, through interaction with a protein, to cause disassembly and afford a protein-sensitive amphiphilic nanoassembly. Our basic premise for the hypothesis is that a typical ligand is a relatively small moiety that can be incorporated on to a dendrimer side chain. This functionality makes a certain contribution towards the overall HLB of the dendron and thus the stability of the assembly. When a protein binds to this ligand moiety, the ligand is masked and a rather large protein is presented to the solvent surface. Since water-soluble globular proteins exhibit a hydrophilic surface, the small ligand functionality is essentially changed to a large hydrophilic moiety (Figure 1). We hypothesized that such an alteration should result in a drastic change of HLB and cause disassembly. Indeed, we have recently shown that disassembly of these amphiphilic aggregates can be achieved with a specific ligand–protein interaction, in which the ligand is hydrophilic and thus is displayed on the surface of the amphiphilic aggregate.[7c] This ligand display provides a convenient access for protein to bind to the ligand and thus cause the binding-induced disassembly (Figure 1).</p><p>A potential limitation of the above finding is that the presumed mechanism (Figure 1), allows for the binding-induced disassembly to be executed with hydrophilic ligands. However, most of the custom-designed ligands target the hydrophobic binding pockets of proteins; thus, most promising ligands are lipophilic in nature. When these lipophilic ligands are incorporated into our proteins, these functionalities will be buried within the interior of the assembly and thus might not be available for binding to complementary proteins (Figure 2, top).</p><p>We hypothesized that an alternate mechanistic possibility in these types of supramolecular assemblies might allow for binding-induced disassembly with lipophilic ligands. Here, we describe and test whether this alternate pathway is available for binding-induced disassembly. Note that noncovalent amphiphilic assemblies are in equilibrium with their corresponding monomers (illustrated by the equilibrium in Figure 2, left). If binding between the ligand and the protein is favored in the monomeric dendron, then binding can preferentially occur at the monomeric stage. In this case, the equilibrium would ultimately cause disassembly due to the Le Chatlier-type effect (Figure 2, bottom). If this pathway is available for binding-induced disassembly, then the versatility of this approach will greatly increase, as this approach would then not be limited to just hydrophilic ligands.</p><p>To test our hypothesis, we chose dinitrophenyl (DNP) moiety as the ligand functionality, because: 1) it is lipophilic and will be buried within the interior of the amphiphilic aggregate and thus the only reasonable pathway available for binding involves the equilibrium-driven disassembly; 2) DNP has been shown to bind to rat anti-DNP immunoglobulin G antibody (IgG) with subnanomolar binding affinity,[18] and 3) synthetic modification of the 2,4-DNP group is relatively straightforward and allows for the proof-of-concept to be achieved with relative ease. Structures of the targeted dendrons are shown as G1–DNP and G2–DNP. In these dendrons, the decyl chain acts as the lipophilic unit, and pentaethylene glycol (PEG) as the hydrophilic unit. PEG was chosen as a charge-neutral hydrophilic functionality to reduce nonspecific interactions between dendrimer and protein. The target dendrons are designed in such a way that these dendrimers are uniformly amphiphilic over the entire dendritic structure. To achieve this, the dendrons were constructed from a biaryl monomer, represented by structure 3 in Scheme 1. The biaryl monomer has several unique features: 1) it has the AB2 functional groups required for dendritic growth; 2) dendrimers constructed from this biaryl building block are fully amphiphilic because of the ability of the biaryl monomer to carry both lipophilic and hydrophilic functional groups. Thus, once the dendron is constructed every repeating layer in the dendritic backbone contains amphiphilic functionalities; 3) hydrophilic ligands can be placed in the dendrons by replacing the PEG unit with the ligand functionality, while the lipophilic ligand is placed by replacing the decyl moiety within a monomeric repeat unit.</p><p> </p><p>We were interested in having the syntheses of the dendrons modular, as this will allow for the incorporation of other lipophilic ligands on to the dendritic building block. We have previously utilized the 1,3-dipolar Huisgen cycloaddition reaction, the so-called click reaction, as the last step in our syntheses to install the ligand functionalities. Thus, the target building block unit for our dendron syntheses involves the presence of acetylene functionality at the lipophilic side of the dendron (Scheme 1, 3). The key step in the synthesis of 3 involves the formation of the biaryl bond. Thus, synthesis of biaryl compound 3 was achieved by using Stille coupling as the key step, from the aryl stannane 1 and bromoaryl ester 2 (Scheme 1). Reaction between the reported peripheral amphiphilic unit 4[4a] and biaryl building block 3[16b] in the presence of potassium carbonate afforded the G1 dendron, 5, in 88% yield. Similarly, the corresponding G2 dendron was synthesized from 3 and the brominated version of the G1 dendron 5.[4a] Once the first and second generation dendrons were assembled from the biaryl monomer 3, the 2,4-DNP ligand moiety was attached to these dendrons through the Huisgen reaction with 8 to obtain the targeted G1–DNP and G2–DNP dendrons. This reaction was chosen to install the DNP ligand to the dendrons, as it allows for easier future ligand variations. All dendrons were characterized by 1H and 13C NMR spectroscopy, and MALDI-ToF mass spectrometry, and details of the syntheses and characterizations are outlined in the Supporting Information.</p><!><p>We first studied the assembly properties of the dendrons G1–DNP and G2–DNP using Nile red as a fluorescent probe. Nile red is a lipophilic dye and is not soluble in water unless it is accommodated in a hydrophobic pocket of a micelle-like assembly. The emission spectra of Nile red, at different G1–DNP and G2–DNP dendron concentrations in water, indicate that these dendrons are capable of providing an apolar microenvironment that sequesters the lipophilic dye molecule (Figure S1 in the Supporting Information). The emission spectra at different dendron concentrations were used to calculate the critical aggregation concentrations (CACs) of the dendrons. Plotting the emission intensity of Nile red as a function of dendron concentration afforded an inflection point, which was taken to be the dendron's CAC (see the Supporting Information). Using this method, CACs for G1–DNP and G2–DNP dendrons were found to be 0.035 (18 μM) and 0.020 mgmL−1 (5 μM), respectively. Formation of the micelle-like assembly was further verified with dynamic light scattering (DLS) experiments. The dendron solutions (26 μM, above the CACs) were prepared in water and DLS results showed that 105 and 124 nm sized aggregates are formed for G1–DNP and G2–DNP dendrons, respectively (Figure 3a). This suggests that these dendrons are indeed aggregated to form micelle-like nanoassemblies in water, which are responsible for sequestering Nile red as the lipophilic guest.</p><!><p>Since the DNP ligand in the dendrons is known to bind anti-2,4-DNP IgG, we were interested in testing the effect of the presence of this protein on the self-assembled structures. Our hypothesis is that the binding interaction between the DNP ligand in the dendrons and the IgG protein will afford a dendron–protein complex, the HLB of which would be drastically different from that of the dendron itself. We anticipated that this change would result in disruption of the micelle-type assembly. In order to examine if these dendritic micellar aggregates are indeed responsive to IgG, we monitored the size of the assembly using DLS. Upon addition of anti-DNP IgG (7.5 μM) to the solution of G1–DNP (26 μM) a remarkable decrease in the size of the assembly was observed (Figure 3b). A similar change in the assembly size was also observed with G2–DNP. The sizes of the G1–DNP and G2–DNP dendrons decreased to approximately 10 nm; this corresponds to the size of the protein itself, and indicates that the dendritic assembly is disaggregated due to ligand–protein binding.</p><p>Although the disassembly of the micellar aggregates provides good evidence for the anti-DNP IgG sensitivity of the dendrons, it is necessary to determine whether this disassembly indeed takes place due to a specific ligand–protein interaction. Therefore, we examined the effect of the presence of noncomplementary proteins, pepsin (pI = 1.0), cytochrome c (CytC, pI=10.2), and α-chymotrypsin (ChT, pI = 8.8). These proteins were chosen for their diversity in pI values, since this is often the source of nonspecific interactions. Thus, solutions of G1–DNP and G2–DNP dendrons were exposed to these three proteins (Figure 4). We were gratified to find that the size of the dendritic assemblies were unchanged in the presence of these proteins. These results support our hypothesis that IgG binding indeed caused micellar disassembly.</p><p>Next, we investigated the possibility of binding-induced guest release from the micellar interiors. If the size change observed in DLS was indeed due to the disassembly of the micellar aggregate, then it is reasonable to anticipate that the disassembly event should cause a concomitant release of guest molecules noncovalently encapsulated within the dendritic interiors. For this purpose, Nile red encapsulated solutions of G1–DNP and G2–DNP dendrons were exposed to the anti-DNP IgG. Indeed, we observed Nile red release due to protein binding, as evident from the decrease in emission intensity of the solution (Figure 5). Interestingly, the guest release exhibited a time-dependent behavior, which is most likely due to the limited accessibility of the lipophilic ligand functionalities that are buried inside the cores of the micellar assemblies. Also, note that we observed only 50 and 45% of the dye molecules to be released within the total time frame, for G1–DNP and G2–DNP dendrimers, respectively. The lack of 100% release could be attributed to the fact that the dendron–protein complex still presents hydrophobic functionalities, and provides an opportunity for lipophilic guest molecules to be bound to the complex. Also, we were interested in finding if the guest release would be enhanced if the percentage of free dendrons were decreased by increasing the relative ratio of the protein to the dendron. The commercial form of the protein imposes an upper limit on its concentration. Fortunately, however, the lower CAC of G2–DNP dendron enables us to work with lower dendron concentration. Thus, when 13 μM G2–DNP dendron was incubated with the IgG (7.5 μM) for 12 h, we observed 52% dye release, as compared to 45% release with 26 μM of G2–DNP dendron (Figure 5b).</p><p>To determine whether dye release is specific only to IgG, we exposed G1–DNP and G2–DNP dendrons to pepsin, ChT, and CytC. We indeed found that exposure of G1–DNP and G2–DNP dendrons to these proteins did not cause any significant change in the fluorescence of Nile red; this indicates that there is no dye released from dendritic assembly (Figure 6). It should also be noted that CytC is a metallo-protein and we have previously shown that addition of positively charged CytC to negatively charged carboxylate polymers and dendrimers results in quenching of fluorescence of the noncovalently encapsulated fluorophores; this indicates the nonspecific binding of CytC to these assemblies.[19] Here, the lack of fluorescence change of Nile red in the presence of CytC provides additional support for the lack of nonspecific interactions between the dendritic assemblies and proteins.</p><p>To further examine whether the binding events and the concurrent dye release are specifically due to the DNP–IgG interactions, we also synthesized a G1-control[4a] dendron (Figure 7a). This dendron is structurally similar to G1–DNP, except that it lacks the DNP ligand functionality. When the IgG was added to the solution of Nile red encapsulated G1-control dendron (26 μM) we observed no guest release from this assembly (Figure 7b); this indicates that the release obtained with DNP-containing dendrons was indeed due to the specific ligand–protein interactions.</p><!><p>We have designed and synthesized facially amphiphilic biaryl dendrimers that contain lipophilic ligand functionalities that are complementary to anti-DNP immunoglobulin G (IgG). These dendrimers were shown to self-assemble into micelle-like aggregates in aqueous solution and are capable of encapsulating lipophilic guest molecules, such as Nile red. We have shown that these aggregates disassemble in response to the ligand–protein interaction. We attribute the disassembly event to the significant HLB change caused by the ligand–protein interaction. This disassembly process clearly suggests that the binding event between a protein and the ligand-containing dendron does not have to occur in the self-assembled aggregate stage. In this scenario, it is likely that the equilibrium between the monomeric state of the dendron and the micellar aggregate provides a viable pathway for the binding-induced disassembly process. Alternately, it is also possible that there is conformational flexibility within the building block units in the assembly that allows for transient exposure of the lipophilic ligand on the surface of the dendron and provides the opportunity for binding with the protein while remaining in the assembly. We do not have a way of discounting this possibility at this time. Nonetheless, it is clear that the binding-induced disassembly strategy is not limited to hydrophilic ligands. Demonstration of the protein-sensitive disassembly by using lipophilic ligands opens up the possibility of utilizing such molecular design for a variety of applications, particularly in drug delivery and sensing.</p>

PubMed Author Manuscript

Presence of Amorphous Carbon Nanoparticles in Food Caramels

We report the finding of the presence of carbon nanoparticles (CNPs) in different carbohydrate based food caramels, viz. bread, jaggery, sugar caramel, corn flakes and biscuits, where the preparation involves heating of the starting material. The CNPs were amorphous in nature; the particles were spherical having sizes in the range of 4-30 nm, depending upon the source of extraction. The results also indicated that particles formed at higher temperature were smaller than those formed at lower temperature. Excitation tuneable photoluminescence was observed for all the samples with quantum yield (QY) 1.2, 0.55 and 0.63%, for CNPs from bread, jaggery and sugar caramels respectively. The present discovery suggests potential usefulness of CNPs for various biological applications, as the sources of extraction are regular food items, some of which have been consumed by humans for centuries, and thus they can be considered as safe.

presence_of_amorphous_carbon_nanoparticles_in_food_caramels

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

<p>T he use as well as presence of nanoparticles (NPs) in food is a hotly debated area, owing to their short and long term effects on human health and the environment [1][2][3][4] . The promise of targeted and/or sustained release of drug, food colourants and flavours, while incorporated with NP, makes the pursuit of understanding of their functioning and fate a worthy exercise [5][6][7] . Although, substantial development in the engineering of consumable NPs [5][6][7] and their effects in vitro and in vivo have taken place 8 ; few biodegradable NPs have entered clinical trials and have been marketed [9][10] . While NP formulations for topical applications are accepted by majority of population 11 , the idea of the consumption of these particles, either for curing a disease or for having nutritional or flavouring benefit, creates an alarm for the public. The reason behind this seems to be their potential effect on human health following consumption, which has received little attention; and the lack of awareness, which has raised concerns regarding the safety of nanomaterials in biological and food applications [1][2][3][4] . A way around this problem, could originate out of direct derivation of nanomaterials from food products, especially from traditional food items. These materials could be considered safe for biological applications when there is no known toxicity and thus may possibly alleviate the misapprehension that all NPs are toxic.</p><p>History of nanotechnology is replete with examples of use of nanomaterials dating back to millennia [12][13][14] . The dye used in colouring hair to black, during the Greco-Roman period, is now known to have been consisted of PbS nanocrystals (NCs) 12 . Romans exhibited their mastery in technology in the Lycurgus cup by harnessing the optical properties of gold (Au) NPs 13 . The extraordinary mechanical strength and a sharp cutting edge in Damascus sabre have recently been attributed to the presence of carbon nanotubes (CNTs) and cementite nanowires 14 . In all the cases mentioned above, while the technology based on nanomaterials were known to different civilizations, the nanoscale nature of their functional constituents have only been revealed recently. The 'nano' dimensionality is not only confined to engineered materials or technology; nature also creates NPs or nanostructures which are present as functional components in an organism; either in the form of enzymes which catalyze most of the biological reactions or as ribosomes which act as the sites for protein synthesis. It was the invention of sophisticated microscopic and analytical techniques which has led to the discovery of these nanostructures. In this regard, the idea of searching for nanomaterials within regular food items cannot be inexplicit. This motivated us to search for NPs in food items, which can potentially be used for biological application, where the concern of the origin and toxicity of the nanomaterials can easily be waived off. Herein, we report the presence of carbon nanoparticles (CNPs) in regular carbohydrate based food caramels, such as in bread, jaggery, corn flakes and biscuits. The CNPs have been found to be present in those samples, where the preparation of food mainly involves heating of the starting ingredients in absence of water, leading to formation of caramels. Arguably; this discovery revealed that human consumption of nanomaterials in the form of food caramels has its history possibly from the period when human for the first time started eating bread.</p><p>Carbon dots (C-dots), which are CNPs below 10 nm are emerging as viable alternatives to semiconductor quantum dots (Qdots) owing to their important photoluminescent properties and lack of any known cytotoxicity [15][16][17] . The wavelength-tuneable emission properties have made them promising candidates as new 'nanolights' 17 . The optimism has led to increased recent interests in developing methods for their syntheses, involving both top-down and bottomup approaches 17 . Incidentally, there are also efforts to understand and tune their optical properties, based on the surface functional groups [18][19][20] . We have recently observed that caramelization of poly (ethylene glycol) under microwave irradiation constitutes formation of biocompatible C-dots 15 . This prompted us to analyse the components of different commercial and homemade caramel containing food items for the presence of CNPs. Amazingly, we found that the light to dark brown coloured caramels present in carbohydrate containing foods such as bread, jaggery, corn flakes and biscuit consist of amorphous CNPs, which were similar to those obtained from caramel produced upon heating of commercial sugar. The discovery of the prominent presence of CNPs in regular food items provides simpler and safer sources i.e. daily food items, where the extracted CNPs can be directly used for biological applications. Further, our observations of the hydrophilicity of the surface functional groups of the CNPs, containing carboxylic and alcoholic groups which provide easier alternatives for their conjugation with different therapeutics, could further make them preferred fluorescent candidates for biological applications [15][16][17] .</p><!><p>The CNPs isolated from the outer brown part of the bread bun and the caramels obtained from commercially purchased sugar (following caramelization) and jaggery were analysed by UV-vis and fluorescence spectroscopy. The UV-vis spectrum of each of the dispersions consisted of a peak (marked with asterisk) and a shoulder (marked with arrow) between 240 nm and 400 nm and is shown in Figure 1. In addition, there is the presence of a strong background till 540 nm. The peaks and the background extinction are known to occur for CNPs and they are consistent with the literature reports 17 . The exact assignment of the peaks is still not known and hence the difference among the individual samples could not be explained. CNPs were also extracted from commercially procured biscuits and corn flakes, the spectroscopic data for which are shown in Figure S1 (supplementary information).</p><p>Photographs of the original samples (bread, jaggery and sugar) which served as the preparatory ingredients are shown in Figures 2a, 2b and 2c. The photoluminescence spectra corresponding to the above dispersions (and other samples) are shown in Figures 2d-2i (and Figure S1). The pictures of the dispersions in the presence of white light and UV light are represented in Figures 2j-2o. Under normal white light the dispersions have the characteristic caramel colour, whereas, under UV light (l ex 5365 nm) it showed blue luminescence. All of the dispersions exhibited excitation dependent emission spectra as shown in Figures 2g-2i, which were similar to C-dots reported previously 17 . It was also observed that with increase in the wavelength of excitation from 325 to 375 nm the luminescence intensity increased, the maximum emission intensity being observed for the excitation wavelength of 375 nm, whereas, further increase of the excitation wavelength resulted in the decrease of emission intensity. Additionally, along with decrease in the fluorescence intensity with increasing excitation wavelength the emission maxima showed red-shift, displaying the property of excitation tuneable emission. The excitation dependent emission is an intrinsic property of CNPs, which has been widely reported by several research groups, including us 15,17,[21][22][23][24][25][26] . The quantum yields (QYs) of the CNPs obtained from different food sources were calculated using quinine sulphate as the standard 25 . At an excitation wavelength of 365 nm, the QYs of the CNPs are summarized in Table 1. The results indicated that these samples had QY typical of C-dots, which is on the order of 1%; with the highest being observed for samples from bread (1.2%) and that from jaggery had the lowest value (0.55%).</p><p>Transmission electron microscopy (TEM) images of the samples obtained from the dispersions of different caramel sources (bread, jaggery and sugar) are represented in Figures 3a-3c, which showed the presence of uniform spherical NPs. The particle distributions calculated from the images are shown in Figures 3d-3f. The average particle sizes as calculated from the TEM images for samples from bread, jaggery and sugar caramel, were determined to be 27.5 6 6.1, 20.3 6 7.5, 4.3 6 1.5 nm respectively. Similarly, samples from corn flakes and biscuits indicated the presence of NPs having sizes of 10.5 6 2.8 nm and 3.9 6 1.3 nm respectively (Figure S1, supplementary information). The results clearly indicated that NPs were present in the dispersions extracted from bread, jaggery, caramel of sugar and other materials. While the sample from bread had the highest particle size, the particles from sugar caramel produced at 200uC had the lowest size and the particle sizes of the sample from jaggery were in between the two. In addition, caramels from sugar, produced by heating at 180uC for 10 min, had particles of size 25.8 6 12.4 nm (Figure S2, supplementary information). Thermogravimetric analysis of sugar indicated decomposition starting at below 200uC with steady decrease in weight till 350uC (Figure S3, supplementary information). Loss of weight signifies the dehydration process of carbohydrate or formation of CO 2 . Thus, the NPs could possibly be produced at a temperature even lower than 200uC. Samples from bread, jaggery and caramel showed broad X-ray diffraction (XRD) peak at about 2h 5 18u (Figure S4, supplementary information), with no clear signature for crystalline nature of any of the samples. The above results indicated that NPs present in the caramels of bread, jaggery, corn flakes, biscuits and sugar possibly consisted of amorphous carbon.</p><p>Further, 13 C NMR (nuclear magnetic resonance) spectra of samples from bread, jaggery and caramel (Figures S5, S6 Further, in order to probe the extent of cytotoxicity of the extracted CNPs, we performed XTT based cell viability assay at varying concentrations of CNPs. The plot of percentage viability of cells to that of varying concentration of CNPs (0.05 mg/mL to 2.0 mg/mL) is shown in Figure S11. As is clear from the figure, no cytotoxicity was apparent even at the highest concentration of CNPs (2.0 mg/mL) used. In addition, one way ANOVA showed that the differences in the mean percentage viabilities of cells at different concentrations of CNPs extracted from jaggery (F5 0.652, P5 0.689) and bread (F 5 1.152, P 5 0.384) were not statistically significant.</p><!><p>The UV-visible and fluorescence spectra of the dispersion of CNPs extracted from different food sources displayed features similar to those of C-dots synthesized chemically and thereby suggesting the presence of CNPs in the samples. The fundamental mechanism of photoluminescence of CNPs is still a major question; however, it is thought that the presence of different surface trap sites could be one of the factors for the luminescence. 17,22 The origin of fluorescence from the obtained dispersion could only be attributed to the presence of CNPs because the analysis of the starting material for preparation of bread did not show any significant fluorescence (Figure S8, supplementary information). Sugar is known to be a nonfluorescent material, but the caramel prepared upon heating sugar showed the emergence of excitation tuneable luminescence, further confirming the formation of CNPs. Additionally, it was observed that the heating temperature for preparing the caramel had significant effect on the size of NPs formed. Caramels prepared at 180uC and 200uC had the sizes of 25.8 6 12.4 and 4.3 6 1.5nm respectively. This indicated that smaller particles were possibly formed at the higher temperature. In other words, the larger particle sizes of NPs obtained from bread and jaggery could be due to their low heating temperatures, whereas, the smaller particles sizes of NPs obtained from sugar caramel, corn flakes and biscuits could be due to higher heating temperatures. It may also be mentioned here that there could be other factors, such as the rate and duration of heating and chemical constituents of the samples, determining the sizes of the produced CNPs. The possibility of the formation of CNPs while preparing and analysing the sample in electron microscopy can be ruled out because when drop cast sample from sugar solution was observed in TEM no such particle formation was detected, even under the exposure of a 200 kV electron beam for several minutes. The images obtained at different time of irradiation, of the sample from sugar solution, in the electron beam of TEM are shown in Figure S9 (supplementary information). It is worth noting that similar extraction process was also used for determining the presence of CNPs, if any, in the interior white part of bread. TEM analysis revealed the presence of inhomogeneous particles (Figure S10a, supplementary information) which could be due to the suboptimal temperature in the inner zone. The fluorescence intensity of this dispersion was significantly low compared to that obtained from the brown part of the bread (Figure S10b, supplementary information). The size of the CNPs produced varied from sample to sample, indicating the possibility of heating temperature as the primary factor determining their sizes. However, it was interesting to observe that for all samples the particles produced were nearly uniform and spherical. To have an idea of the amount of CNPs which can be extracted from a food source we analysed the amount of particles obtained from 1 g each of jaggery and the brown layer of bread. It was observed that about 3 and 2 % w/w of CNPs, in the respective samples were present. The calculation is based on the amount of the starting ingredient taken for the isolation of CNPs and the sample recovered after purification. However, the amount recovered from these materials cannot solely be attributed to CNPs as polymeric layer will always remain surrounding these particles. Isolation of nude CNPs without the polymeric layer has not been possible in the present condition; even then it can give an approximate value about the fraction of particles extracted. Amorphous nature of NPs present in all the samples is demonstrated by the results of powder XRD data (Figure S4, supplementary information) as no peaks of crystalline origin was detected. The NMR studies revealed that the CNPs were coated with hydrophilic carbohydrate units. No peaks corresponding to the aromatic region was observed, which again supported the luminescence to be originating from the CNPs present in the dispersion.</p><p>In summary, our current work revealed the presence of CNPs in carbohydrate based regular food caramels from bread, jaggery, corn flakes and biscuit. The excitation wavelength dependent emission characteristic of the CNPs from food caramels were similar to those generated from sugar; however, the particle sizes varied indicating temperature -dependent formation of CNPs of different sizes. NMR spectroscopy revealed that the CNPs were coated with carbohydrate units. It is interesting to note that for centuries these caramels containing CNPs have been consumed by human beings with no known toxicity and thus it can be considered to have no or minimum risk on human health and may be used as a safe nanomaterial. Our finding of the presence of fluorescent CNPs in food caramels may also help their use in tracking and imaging conjugated biomolecules and drugs in vivo, without being imperilled.</p><!><p>Preparation and extraction of CNPs. Bread buns were purchased from the local market (Homa Bread, Guwahati, India) and analysed to check the presence of CNPs within it. The top brown layer of bread was carefully excised and 1 g of it was dissolved in 20 mL methanol by sonicating it at 35 kHz in a bath sonicator (Elmasonic TI-H-5 Elma, Germany) for 10 min. Following sonication, the volume of the methanol was reduced to 3 mL in a rotary evaporator before further purification. Jaggery (prepared from sugarcane juice) purchased from the market was heated following a traditional procedure which is as follows. Initially, jaggery (say 50 g) was mixed with water (about 10-15 mL) to make it sufficiently moist. The entire amount was then placed on a hot pan, which was being heated in the medium flame of a commercial gas stove. The mixture was constantly stirred using a kitchen spatula. In about 5 min, when the colour of the mixture turned dark brown, the entire amount was transferred to a pan containing a thin layer of oil and brought to room temperature. The oil layer helped in preventing aggregation of the mass and also in spreading the content over the pan. The jaggery caramel, which was ready for use then, was dissolved in methanol and allowed to stand for a few minutes to remove larger impurities. The sedimented particles from both the samples were removed by filtration. Centrifugation of the supernatant at 5000 rpm was performed further to remove impurities of smaller size. The yellow coloured supernatant thus obtained was further purified by column chromatography (using silica 60-120 mesh) with methanol:dichloromethane (2:3) mixture and finally dialysis (using 1 KDa membrane) was carried out to remove salts and other ions, if any. Similar procedures were followed for the extraction of CNPs from commercially purchased cornflakes and biscuits. Caramel was also prepared in laboratory by heating commercially available sugar. Sugar (Daurala Sugar Works, India) was taken in a glass vial and heated in an oil bath at 200uC for 10 min till the solid turned brown. The brown colored sticky mass was cooled down to room temperature and was then dissolved in methanol, followed by purification using column chromatography. The sample was then concentrated by evaporating the solvent in rotary-evaporator and then it was dissolved in water and finally dialysed before further analysis.</p><p>Characterization of CNPs. The extracted CNPs were characterized using TEM (JEOL 2100 UHR-TEM), UV-vis (Perkin Elmer Lambda 25) and fluorescence spectrophotometers (FluoroMax-4, Jobin Yvon). The TEM analysis was performed at an accelerating voltage of 80 kV, unless otherwise mentioned, and the sample was prepared by drop casting 5 mL of the respective sample on a 300 mesh carbon coated copper grids and subsequent air drying before analysis. 13 C NMR (100 MHz) of the dispersion was carried out in a Varian 400 MHz FT-NMR using D 2 O as the solvent. Thermogravimetric analysis (TGA) was performed in Q600 SDT Simultaneous DSC-TGA heat flow analyzer and powder XRD study was done using a Brucker D8 Advanced X-ray diffraction measurement system, with Cu Ka source (l 51.54 A ˚). The quantum yield (QY) was calculated using quinine sulphate in 0.10 M H 2 SO 4 solution as a standard, at an excitation wavelength of 365 nm, and the absorbance was kept below 0.15. The QY of the samples were determined according to equation 1.</p><p>Where, QY is the quantum yield, m is the slope of the plot of integrated fluorescence intensity vs absorbance and g is the refractive index of the solvent. For the aqueous solutions g S /g R 51. The subscript R refers to the reference fluorophore i.e. quinine sulphate solution and S for the sample. The values obtained are given in Table S1, supplementary information.</p><p>In vitro cytotoxicity assays. HeLa cells were obtained from National Center for Cell Sciences (NCCS), Pune, India and were cultured in Dulbecco's modified Eagle's medium supplemented with penicillin (50 units mL 21 ), streptomycin (50 mg mL 21 ), and 10% (v/v) fetal bovine serum. Cells were maintained in 5% CO 2 humidified incubator at 37uC. The cells were seeded in a 96 well culture plates at a density of 5000 cells/well and were allowed to grow overnight. The CNPs were then added into the wells in a concentration range of 50 ng/mL to 2 mg/mL and incubated in a humidified incubator for 24 h at 37uC and 5% CO 2 . XTT (Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) (Sigma-Aldrich, USA) based cell viability assay was carried out according to the manufacturer's protocol, to determine the percentage of viable cells. The assay is based on the metabolic activity of the cells to reduce the tetrazolium salt XTT to orange coloured compounds of formazan and the intensity of the dye is proportional to the number of metabolic active cells. The percentage cell viability of the untreated cells (control) was taken as 100%. All measurements were collected in triplicate and the values are expressed as mean 6 standard error (SE). Statistical analysis for ANOVA was performed using Sigma Plot.</p>

Scientific Reports - Nature

New paradigms in chemokine receptor signal transduction: moving beyond the two-site model

Chemokine receptor (CKR) signaling forms the basis of essential immune cellular functions, and dysregulated CKR signaling underpins numerous disease processes of the immune system and beyond. CKRs, which belong to the seven transmembrane domain receptor (7TMR) superfamily, initiate signaling upon binding of endogenous, secreted chemokine ligands. Chemokine-CKR interactions are traditionally described by a two-step/two-site mechanism, in which the CKR N-terminus recognizes the chemokine globular core (i.e. site 1 interaction), followed by activation when the unstructured chemokine N-terminus is inserted into the receptor TM bundle (i.e. site 2 interaction). Several recent studies challenge the structural independence of sites 1 and 2 by demonstrating physical and allosteric links between these supposedly separate sites. Others contest the functional independence of these sites, identifying nuanced roles for site 1 and other interactions in CKR activation. These developments emerge within a rapidly changing landscape in which CKR signaling is influenced by receptor PTMs, chemokine and CKR dimerization, and endogenous non-chemokine ligands. Simultaneous advances in the structural and functional characterization of 7TMR biased signaling have altered how we understand promiscuous chemokine-CKR interactions. In this review, we explore new paradigms in CKR signal transduction by considering studies that depict a more intricate architecture governing the consequences of chemokine-CKR interactions.

new_paradigms_in_chemokine_receptor_signal_transduction:_moving_beyond_the_two-site_model

1. Introduction<!>2.1. Origins of the two-site model and early studies of the site 1 interface<!>2.2. Site 1 interactions: chemokine allostery and conformational dynamics<!>2.3. Complex roles for receptor post-translational modifications<!>2.4. Complex interactions between receptor N-termini and the chemokine core<!>3.1. A more complicated site 2: the major and minor binding pockets<!>3.2. Role of subpocket specificity in receptor activation<!>3.3. Role of binding depth and chemokine N-terminal length in receptor activation<!>4.1. Defining unique, non-site 1, non-site 2 interactions at the receptor surface<!><!>4.1. Defining unique, non-site 1, non-site 2 interactions at the receptor surface<!>4.2. A \xe2\x80\x9cmulti-site model\xe2\x80\x9d accounts for diverse, interdependent chemokine-CKR interactions<!>4.3. Molecular switches at the extracellular surface: allosteric coupling to the TM region<!>Chemokine dimerization<!>Receptor dimerization<!>Stoichiometry of chemokine-receptor complex<!>5.2. Beyond G protein signaling: biased signaling at chemokine receptors<!>5.3. Beyond chemokines: binding and signaling by non-chemokine ligands<!>6. Conclusion

<p>Chemokine receptors (CKRs) are cell-surface seven transmembrane domain receptors (7TMRs) that mediate a diverse repertoire of functions, such as immune surveillance and embryonic development, by directly regulating cellular migration, adhesion, growth, and survival. They are also implicated in many pathological processes such as atherosclerosis, HIV infection, tumor metastasis, and autoimmune disorders [1]. Due to their prominent roles in so many disease processes, CKRs have been the target of considerable drug development efforts since the discovery of the chemokine-CKR system in the late 1980s [2, 3].</p><p>Chemokines and CKRs demonstrate widespread promiscuity, wherein chemokines may bind multiple receptors and vice versa. Of the nearly 50 chemokines and 20 CKRs identified in humans, most bind multiple counterparts, with a minority involved in monogamous interactions. Promiscuous interactions among chemokines and their receptors are increasingly recognized as a mechanism to generate diverse signaling or other functional outcomes using a discrete set of chemokine and CKR components [4, 5]. This characteristic promiscuity may be explained, in part, by their conserved tertiary structure, composed of an unstructured N-terminus, conserved mono- or di-cysteine motif (e.g. C, CC, CXC, CX3C, where X represents a non-cysteine residue), extended loop, three anti-parallel β-strands, and C-terminal α-helix (Fig. 1.A) [1]. One or two conserved disulfides constrain the chemokine fold by linking the cysteine motif with the β1–β2 turn (a.k.a. the 30s loop) and the β3-strand.</p><p>CKR binding and activation is described as proceeding via a two-step/two-site mechanism, a model which dates back to the mid-1990s. This model is alternatively framed by segregating chemokine-CKR interactions functionally (i.e. two-step) and spatially (i.e. two-site). In the functional formulation, site 1 provides affinity and specificity, followed by site 2 which elicits receptor activation. In the spatial formulation, site 1 refers to interactions between the CKR N-terminus (a.k.a., chemokine recognition site 1, CRS1) and the chemokine globular core, and site 2 refers to contacts between residues in the receptor transmembrane (TM) domain (a.k.a., CRS2) and the unstructured chemokine N-terminus [3]. Notably, interactions between chemokines and CKR extracellular loops (ECLs) are variously ascribed to site 1, site 2, or not included in these models at all [1, 6–9].</p><p>Isolation of the receptor N-terminal domain has enabled structure determination of several site 1 complexes but numerous difficulties hindered the characterization of full-length receptors. Since 2007, technical innovations have made possible the purification and crystallization of over 100 family A 7TMRs including CKRs. Until recently, only apo structures or those bound to small molecule antagonists were available [10–12]. In 2015, the first structures of chemokine-CKR complexes were solved, detailing chemokine interactions in the TM domain (i.e. site 2) [8, 9]. Combination of these site 1 and site 2 structures recently enabled construction of the most detailed chemokine-CKR model to date [13]; and together, these data highlight numerous contacts that fall outside of the conventional spatial and functional definitions of sites 1 and 2. This coupled with an increased awareness of biased agonism (i.e. preferential activation of G protein or β-arrestin pathways), non-chemokine ligands (e.g. ubiquitin, β-defensins), and the expanding roles of post-translational modifications (PTMs; e.g. sulfation, polysialylation) underscores how the two-site model may overlook the complexity and diversity of CKR signaling that we now appreciate two decades after it was proposed [4, 5, 14–17].</p><p>The goal of this review is to highlight instances in which the two-site model inadequately addresses more complex features of chemokine-CKR interactions. In doing so, we hope to broaden the reader's appreciation of the mechanistic details involved in CKR signal transduction. We emphasize that the two-site model has served as a useful framework to understand CKR activation, and in some cases may sufficiently describe binding and activation. Nevertheless, we believe it is advantageous to look beyond the functional and structural roles segregated into site 1 or site 2, to delineate new capacities for interactions that have not been well described by either site, and to include new features that have been discovered since the original conception of the two-site model.</p><!><p>The two-step/two-site model of chemokine-CKR interactions was realized almost 20 years ago through the work of three contemporaneous studies [18–20]. First, Monteclaro and colleagues used a chimera of the CCR2 N-terminal domain and the CCR1 TM region to show that the receptor N-terminus was sufficient to recognize CCL2 with high affinity and recapitulate the native interaction [18]. Notably, the complementary CCR1–CCR2 chimera exhibited a 30-fold decrease in G protein signaling, demonstrating that the CCR2 N-terminus is essential for chemokine recognition but not signaling. In a follow-up study they showed that high affinity CCL2 binding was completely dependent upon the presence of the CCR2 N-terminus and could be fully recapitulated using only a membrane tethered N-terminal peptide [19]. Crump and colleagues also hypothesized a two-site mechanism through studies of the chemokine rather than the receptor. They showed that mutation of the CXCL12 N-terminus attenuated signaling activity without significant loss of affinity (e.g. 3-13-fold increase in binding Kd) [20]. Taken together, these studies suggested that the site 1 and site 2 interactions were spatially and functionally independent, with site 1 conferring receptor specificity and affinity, and site 2 mediating receptor activation. Over time, other functional studies led to the consensus that this model was broadly applicable to the chemokine-CKR system [21]. Interestingly, the two site model of chemokine-CKR interactions was predated by an analogous model described for interactions between the inflammatory protein C5a and its receptor, suggesting broad applicability of this model among other GPCRs with protein ligands [22].</p><p>At the same time other studies began to probe the site 1 interface in greater detail. Alanine scanning of CXCL8 identified residues in an extended loop between the conserved N- terminal cysteine(s) and the 310-helix (a.k.a. the N-loop) [23]. Unlike CXCL8, CXCL1 is a high affinity CXCR2 ligand with weak affinity for CXCR1. Exchange of seven CXCL1 N-loop residues with those of CXCL8, a high affinity CXCR1 ligand, resulted in a molecule capable of recognizing both receptors [24, 25]. Using a similar chimera approach, Crump and colleagues showed that insertion of the CXCL12 N-loop into unrelated CXC-family chemokines (CXCL1 and CXCL10) rendered them capable of binding and activating CXCR4 [20]. Subsequent studies expanded the importance of this region for site 1 interactions to other CC and CXC chemokines, establishing the N-loop as a critical motif for CKR recognition [19, 20, 26, 27].</p><p>While these and related functional studies have helped define functional roles for the N-loop and other chemokine domains in signal transduction, NMR titration experiments have historically been used to define structural interactions contributing to site 1 recognition. One of the most common NMR-based approaches has been to titrate unlabeled, CKR N-terminal peptides into purified, [U-15N]-labeled chemokines to identify chemokine residues that participate in direct site 1 binding interactions. This and related approaches have been used to map the chemokine site 1 binding interactions for CCL11:CCR3 [28], CCL21:CCR7 [29], CCL24:CCR3 [30], CXCL8:CXCR1 [31, 32], CXCL10:CXCR3 [26], CXCL12:CXCR4 [13, 33–37], and CX3CL1:CX3CR1 [38]. Collectively, these studies demonstrate the essential role of the chemokine N-loop in directly binding CKR N-termini, and are validated by soluble chemokine- CKR structures (discussed in section 2.4).</p><!><p>While early studies of chemokine-CKR interactions suggested the functional and spatial independence of site 1 interactions, more recent studies suggest that site 1 interactions can alter functional outcomes. In a study of CXCL8 activation of CXCR2 and CXCR1, Rajarathnam and colleagues identified an important Gly-Pro (GP) sequence in the 30s-loop that had large conformational effects on CXCL8 when mutated, causing CXCL8 to activate CXCR1 and CXCR2 in unique ways [39]. While some GP mutants activated both receptors with similar potencies, two mutants (G31A and P32G) displayed a modest reduction in affinity at CXCR2 but completely lost the ability to elicit CXCR2-mediated calcium release. These same mutants lost all binding and calcium signaling at CXCR1. The study demonstrated that the change in signaling was due to intramolecular interactions between the GP motif of the 30s-loop and the conserved "ELR" motif of the CXCL8 N-terminus, which is known to be important for CXCR2 activation. The authors employed molecular dynamics (MD) simulations to show conformational switching for some CXCL8 mutants interconverted the 30s-loop between a type-I and type-II β-turn, thereby altering the conformation and orientation of the chemokine N-loop and N-terminus allosterically. They suggested that chemokines exist in conformational ensembles, and receptor binding and activation involves conformational selection on the part of the ligand and receptor. In this way, different CKR N-termini may selectively bind specific orientations of the chemokine ensemble, thereby eliciting unique functional outcomes at two separate receptors (see Section 5.1 on biased signaling).</p><p>In another study, the same group examined the role of the residue sandwiched between the conserved cysteines of CXCL8. Conversion of the CXC motif to a CC motif greatly reduced the binding affinity for both CXCR1 and CXCR2 and rendered it incapable of activating CXCR2 [40]. The mutation did not affect the chemokine fold, dimerization, or glycosaminoglycan (GAG) binding, suggesting that the attenuated binding and signaling properties were a consequence of altered intramolecular dynamics. Supported again by MD simulations, the authors suggested that allosteric site 1 interactions may in effect 'steer' the orientation of the chemokine N-terminus within the receptor TM bundle. Similar studies of vMIP-II and CX3CL1 cysteine motifs suggest that "conformational switching" may be a more general phenomenon among the chemokine family [41]. These studies demonstrate that subtle structural changes in one chemokine domain can significantly alter receptor activation by eliciting conformational changes in another domain. In effect, these studies challenge the structural and functional independence of site 1 and site 2 interactions.</p><!><p>Farzan and colleagues expanded the scope of interactions underlying site 1 recognition by showing that sulfation of tyrosines in the CCR5 N-terminus enhanced affinity for CCL3 and CCL4 [42]. This and later studies broadened the repertoire of CKR post-translational modifications (PTMs) to include glycosylation, demonstrating that in addition to enhancing chemokine affinity, PTMs can regulate functional outcomes of site 1 interactions [17, 21].</p><p>CKRs undergo enzymatic, O-sulfate modification by tyrosyl protein sulfotransferase (TPST) during processing in the trans-Golgi network. The presence of these sulfotyrosine (sTyr or sY) PTMs at receptor N-termini enhances binding of chemokine ligands and affects receptor activation in many chemokine-CKR systems [16, 42–49]. The structural contributions of sTyr modifications to site 1 interactions were defined by the NMR solution structure of a covalently-linked CXCL12 dimer bound to the first 38 amino acids of CXCR4 that was enzymatically sulfated at three tyrosine positions. The CXCL12 locked dimer was engineered out of necessity, as CXCR4 peptide binding altered the CXCL12 monomer-dimer equilibrium, thereby hindering NMR experiments. The observation that tyrosine sulfation enhanced CXCR4 peptide binding, which in turn promotes CXCL12 dimerization, defined an allosteric model in which binding of sTyr peptides at the conserved sTyr pocket causes CXCL12 self-association. For instance, Ziarek and Getschman, et al., found that a sulfated heptapeptide corresponding to the Tyr21 region of CXCR4 specifically and preferentially binds to the CXCL12 dimer while promoting dimerization of WT-CXCL12. These studies represent the first evidence that binding at a pocket, disconnected from the CXC dimer interface, could allosterically regulate chemokine self-association [50]. Specifically, dynamic NMR studies of CXCL12 revealed that to accommodate dimer formation, the α-helix of CXCL12 rearranges to an almost 90° angle, perpendicular to the β-strands [51]. This large motion is triggered by two residues adjacent to the sulfotyrosine binding pocket that serve as a link or "microswitch" back to the C-terminal α-helix of CXCL12 [52]. Importantly, self-association of CXCL12 has significant functional effects, as the locked, dimeric version of CXCL12 elicits a unique signaling profile compared to WT-CXCL12 [36].</p><p>Glycosylation of CKR extracellular domains occurs during processing in the endoplasmic reticulum (i.e. N-linkage of asparagine residues) or Golgi (i.e. O-linkage of serine/threonine residues). Recent studies described a novel functional role for chemokine receptor glycosylation in which polysialic acid (polySia) addition to receptor glycans allows CKRs to discriminate between chemokine binding partners [17]. CCR7 is polysialyated by the enzymes ST8Sia II and ST8Sia IV on the surface of patrolling dendritic cells. This rare PTM is utilized by CCL21, which has an unusual extended C-terminal tail not shared by the other CCR7 ligand, CCL19. When CCR7 is polysialylated, CCL21 binds CCR7 with high affinity due to an interaction between the polySia of CCR7 and the C-terminus of CCL21. This interaction is thought to release CCL21's tail from an autoinhibitory interaction with its chemokine core, freeing its N-terminus to bind and activate CCR7. In the examples described for both tyrosine sulfation and polysialylation, interactions between these receptor PTMs and chemokine site 1 domains dictate unique functional outcomes. Despite some shared features of PTMs in chemokine-CKR signal transduction (e.g. sTyr enhancement of chemokine affinity/potency [16]), many features appear to be context-specific (e.g. polySia alteration of CCL21 activity [17]) or even contradictory (e.g. sTyr promotes and inhibits chemokine dimerization for different chemokine-CKR pairs [16]), preventing the prediction of PTMs on biological activity in the absence of experimental data.</p><!><p>An early model for the interaction of CXCL8 with the CXCR1 N-terminus set the structural precedent for site 1 formation, corroborating the direct involvement of the N-loop and expanding the interface to include the chemokine cleft, formed by the N-loop and β2/β3 turn [32]. Nevertheless, this NMR structure required a biased NMR structure refinement procedure due to weak binding of their modified CXCR1 peptide, suggesting the need for more site 1 complexes to validate the site 1 interface [32]. To date, six site 1 complexes (four NMR [13, 32, 35, 53] and two crystallographic [8, 9]) have been determined in which the receptor peptide adopts three different orientations. Indeed, in all structures except the CCL11:CCR3 complex, the receptor lies nearly perpendicular to the β-sheet axis primarily contacting the N-loop, chemokine cleft and β-strands, validating the site 1 interface defined by the CXCL8:CXCR1 structure. Despite this common interface, the site 1 complexes demonstrate considerable architectural diversity. For instance, the orientation of the N- and C-termini of the CXCR4 peptide is inverted when bound to CXCL12 compared to other site 1 complex structures. While a recent review has suggested that this orientation is incompatible with chemokine N-terminal insertion into the TM domain [54], flexible docking of the monomeric CXCL12:CXCR41–38 structure into CXCR4 demonstrates that this distinct directionality may facilitate rotation of CXCL12 relative the CXCR4 TM bundle so that it is positioned to form extensive site 2 and intermediate-site interactions [13]. Specifically, our model predicts that CXCR4 assumes a bent conformation adjacent to the conserved N-terminal Pro-Cys (PC) motif relative to other chemokine-CKR complexes (Fig. 1B, discussed in Section 4.1) [13]. Despite their differences, all six receptors form apolar and electrostatic contacts, often through a highly conserved tyrosine with the chemokine cleft.</p><p>Dimeric CXCL12 was recently identified as a biased agonist that induces G protein signaling but is incapable of promoting β-arrestin recruitment or cellular migration [13, 36, 55]. Regardless of quaternary structure CXCR4 makes specific contacts with the N-loop and chemokine cleft, contributing 50% of the total site 1 binding energy [50], but residues of the CXCR4 N-terminal domain adopt two distinct conformations when bound to CXCL12 monomer and dimer (Fig. 1C–E). For example, CXCR4 residues 7–9 contribute an intermolecular β-strand to monomeric CXCL12 and residues 4–6 tuck into a hydrophobic pocket abutting the C-terminal helix (Fig. 1C, D) [13]. These contacts are supported by mutagenic and NMR experiments with the full-length receptor [13, 56, 57]. Self-association with a second CXCL12 molecule competes for the monomer-specific β1 and helix contacts and displaces those residues of the receptor, which instead form less stable contacts with the second CXCL12 protomer (Fig. 1E) [35]. Inspection of the CXCL12:CXCR4 hybrid model suggests dimeric CXCL12's distinct signaling profile may result from unique contacts with the ECL and TM regions (discussed in Section 4).</p><p>It is reasonable to assert that the degree to which the receptor N-terminus wraps around the globular core modulates the chemokine's orientation and interactions with the receptor ECL and TM regions. In the context of biased agonism, the receptor N-terminus may mask or expose epitopes to the receptor ECLs. Taken together, the site 1 interactions may generate functional complexity via unique interactions rather than simply tethering the chemokine and contributing binding energy. Classic definitions of site 1 fail to recognize the diversity of binding modes and unique domains that can interface with receptor N-termini, suggesting a greater spatial and functional complexity than the traditional model would suggest.</p><!><p>The recent crystal and NMR structures of chemokine-CKR complexes provide clues that far from following a two-site convention, interactions are diverse and highly specific for each individual chemokine-CKR pair at the extracellular surface [8, 9, 13]. Similarly, a surfeit of 7TMR crystal structures over the past decade is defining how the conserved TM architecture recognizes diverse ligand types and triggers unique signaling outcomes [58, 59]. In particular, the early 7TMR crystal structures divided the orthosteric-binding site (i.e. the 'main' ligand binding pocket) into two subpockets [58, 60–62]: the major subpocket consists of the cavity defined by TMs 4, 5, and 6, and the minor subpocket by TMs 1 and 2. TMs 3 and 7 occupy the interface between the two subpockets and stabilize ligand-CKR interactions in either subpocket [3, 58, 63, 64].</p><p>A 2013 analysis of over 40 7TMR structures revealed the majority of co-crystallized family A 7TMR ligands contacted the major subpocket (especially TMs 3, 5, and 6) with few ligands forming even one contact in the minor subpocket [65]. It should be noted this analysis was enriched for antagonist contact points since inactive 7TMR structures are more abundant. Likewise, peptide-binding receptors (including CKRs) represented a small minority of the crystallized receptors in this study. Nevertheless, reviews of CKR binding determinants show a more equitable distribution of contact points among major and minor subpockets, with many CKR agonists and antagonists alike preferentially binding the minor subpocket alone [3, 7–10, 12, 64]. This trend is borne out among the five CKR-ligand co-complexes, with three of the five co-crystallized ligands primarily occupying the minor subpocket (IT1t:CXCR4, vMIP-II:CXCR4, and CX3CL1:US28), one ligand primarily occupying the major subpocket (CVX15:CXCR4), and one ligand straddling the two subpockets (maraviroc:CCR5) [8–10, 12].</p><p>CKRs possess a number of unique features that may explain why ligands more readily sample the minor binding pocket relative to other family A 7TMR subtypes. Firstly, the extracellular portion of TM1 in all CKR structures is inwardly oriented toward the center of the TM bundle, with CXCR4-IT1t displaced 9 Å relative to a prototypical family A member, β2-adrenergic receptor (β2AR) [10]. TM1 is positioned closer to the adjacent TM7 and creates a more contiguous helix-helix interface [8–10, 12]. Secondly, compared to the β2AR, TM1 is 1–2 turns longer extracellularly when the receptor (CXCR4 or US28) is bound to a chemokine ligand (vMIP-II or CX3CL1) and TM7 is 1–2 turns longer regardless of the associated ligand (discussed in Section 4.1). The overall effect of the elongated TM1 and TM7 helices, and the inward orientation of TM1, is to create a larger minor pocket. Another feature that may enrich minor subpocket contacts is that chemokines almost universally bind receptor N-termini [7]. By forming extensive interactions with CKR N-termini, which themselves are linked to TM7 via a disulfide bond, chemokines are positioned directly above the minor subpocket (Fig. 2A, discussed in Section 5.1). Still, small molecule ligands also demonstrate enriched utilization of the minor binding pocket despite making no contacts with CKR N-termini, suggesting ectodomain interactions are not solely responsible [3].</p><!><p>The diversity of CKR ligand binding sites emphasizes the additional level of regulation built into receptor activation compared to major subpocket-biased 7TMRs. The major and minor subpockets contain unique sets of residues that comprise molecular switches [4, 64, 66]. Molecular switches are conserved receptor 'hotspots' that undergo conformational rearrangements following agonist binding, helping to drive global conformational rearrangements required for 7TMR activation [66]. To complicate matters, CKR agonists and antagonists frequently share a subset of receptor contacts, a general feature of 7TMR ligands [67]. For instance, Glu7.39 and Trp2.60 (Ballesteros-Weinstein nomenclature [68]) frequently serve as contact points for small molecule CKR ligands [3, 64, 69]. Notably, our recent hybrid model of CXCL12 bound to CXCR4 (based on the IT1t:CXCR4 X-ray and LM:CXCR41–38 NMR structures) suggests that unique positional and rotameric states of multiple CXCR4 residues, relative to antagonist-bound structures, contribute to receptor activation [13]. Consequently, site 2 binding might itself be broken down into a series of "choices" dictated by the ligand: 1) selection of the CKR binding-pocket (i.e. major subpocket, minor subpocket or a combination of both), and 2) stabilization of subpocket residues in active (or inactive conformations), both of which will have the effect of engaging (or preventing engagement of) a particular subset of molecular switch residues required to elicit the ligand-associated functional response (Fig. 2B).</p><p>The diverse binding modes in the major and minor subpockets place each ligand in the proximity of a unique subset of molecular switches. Considering the major binding pocket, the "conserved core" interaction between Pro5.50, Ile3.40, and Phe6.44, directly below the ligand binding sites of the β2AR and μ-opioid receptor (MOR), propagates conformational changes to the intracellular receptor surface for activation [70–72]. Similarly, below the minor pocket is the TxPxW motif (i.e. Thr2.56-x-Pro2.58-x-Trp2.60) conserved among most chemokine receptors, although its specific role in receptor activation is not well understood [64, 73]. Pro2.58 is important for receptor activation in multiple receptors, including CCR5 [64, 74]. Trp2.60 has been consistently identified as a principal binding contact for CKR small molecule antagonists, and when mutated, disrupts their inhibitory effects [3, 75]. Finally, our CXCL12:CXCR4 hybrid model suggests that interactions between the TxPxW motif and residues in TMs 3 and 7 may initiate a concerted rearrangement of a group of hydrophobic residues in the TM region, resulting in receptor activation [13]. Given the unique distribution of both ligand contacts and residues involved in conserved motifs among different TM domains, it is becoming clear that ligand-specific receptor outcomes are a consequence of the stabilization of specific rotameric states in the receptor-binding pocket followed by engagement of unique subsets of molecular switches.</p><p>A chemokine's subpocket "preference" may also depend upon its N-terminal cysteine motif. Qin and colleagues aligned multiple chemokine structures belonging to the CC and CXC subgroups, and noted that CXC chemokines display a characteristic bend immediately preceding the CXC motif, causing their N-terminus to run parallel to the N-loop [8]. In models of CXCL12 bound to CXCR4, they predicted that the bend directs the N-terminus toward the major pocket, whereas vMIP-II (a viral CC chemokine) directs its N-terminus towards the minor subpocket. Since most chemokines are thought to form interactions with receptor N-termini, CC chemokines might be predicted to preferentially utilize the minor pocket, whereas CXC chemokines would be able to take advantage of the major binding pocket by redirecting their N-termini via the CXC bend. Despite possessing a distinct "bulge" at its CX3C motif, The N-terminus of CX3CL1 inserts into US28's minor subpocket [9, 38]. Nevertheless, the drastic deviations in chemokine orientations from those seen in recent structures and models suggests that subpocket preference may be more complicated than can be predicted by the CC/CXC/CX3C motif [8, 13]. More structures of chemokine-CKR complexes will be needed to see if the cysteine-motif elicits subpocket binding preferences.</p><!><p>In addition to the ligand's "choices" to 1) specify a binding pocket, and 2) stabilize subpocket residues, a ligand may also "choose" to bind at a particular depth within that pocket. While four of five CKR-ligand complexes bind high in the orthosteric-binding pocket relative to other 7TMRs, maraviroc binds CCR5 at a depth resembling that of many aminergic ligands [8–10, 12]. A review of mutagenic and functional studies suggests diversity in the depths at which different chemokines contact their respective receptors, with some N-termini potentially achieving depths comparable to those of deep-binding aminergic ligands [7]. Additional complex structures will be needed to validate that chemokines may contact receptors at different depths within the TM domain, however current data suggest that depth variation presents yet another level of complexity within site 2 manipulated by chemokines to achieve specific signaling outcomes.</p><p>Chemokine N-terminal length does not necessarily correlate with the chemokine's binding depth, or its functional properties. Early chemokine structure-function studies showed that truncation of the chemokine N-terminus transforms chemokine agonists into antagonists [20, 76, 77]. Recent studies of the CCL5 N-terminus demonstrate that extension of the chemokine N-terminus produces variant-specific functional outcomes, such as receptor internalization, degradation, recycling, or biased signaling [54, 77]. Similar approaches have since been applied to other chemokines [77]. A recent study by Hanes and colleagues utilized phage display and modeling to suggest how N-terminal length influences receptor function [78]. The authors screened two phage display libraries of CXCL12 for CXCR4 antagonists: a "N-addition" library with a single amino acid addition to CXCL12 and the first four residues of the lengthened chemokine randomized, and a "N-truncation library," where the first four residues were deleted and residues 5–8 randomized. Two results were conclusively found: 1) the N-addition library produced more antagonists, whereas the N-truncation library produced none, and 2) of the N-addition antagonists found, many bound with greater affinity than WT-CXCL12. Interestingly, the screen selected for a subset of variants possessing neutral polar and aliphatic residues, independent of amino acid sequence. The authors propose that despite the "scrambled sequence," similar intermolecular contacts form due to the conformational dynamics of the chemokine N-terminus and receptor pocket. These results are consistent with recent NMR studies of the MOR peptide agonist dynorphin, which was highly dynamic even in a receptor-bound state [78, 79]. In sum, these studies suggest that it may be difficult to make generalizations with respect to chemokine N-terminal length as it relates to CKR activation, as examples of elongated and shortened chemokine variants demonstrate diverse outcomes. Moreover, the dynamic nature of the chemokine N-terminus suggests that elongated peptides may adopt a more folded structure in the orthosteric pocket, as opposed to "diving" more deeply into the TM bundle [78]. Indeed, comparison of the vMIP-II:CXCR4 structure and our CXCL12:CXCR4 model suggests that despite vMIP-II's two additional N-terminal residues, both chemokines reach the same depth by virtue of vMIP-II forming a short N-terminal helix [13].</p><!><p>The first chemokine-CKR crystal structure showed that in contrast to recognizing spatially distinct receptor domains, the chemokine formed interactions spanning from the receptor N-terminus (i.e. site 1) to the receptor TM domain (i.e. site 2) [8]. Noting a region that lacked precedence as either site 1 or site 2, Qin and colleagues named an interaction between the chemokine's CC motif and the receptor N-terminal base chemokine recognition site 1.5 (CRS1.5) (Fig. 2.A) [8]. Similar interactions were observed in the CX3CL1:US28 structure, confirming previous predictions that the N-terminal stalk region (in the context of its disulfide interaction with TM7) serves a direct and essential role in chemokine recognition [9, 80]. More recently we identified analogous sites (i.e. CRS1.5-like) in our hybrid CXCL12:CXCR4 model and experimentally validated their role in binding and activation [13]. The existence of multiple structurally-validated intermediate interfaces calls into question the assumed spatial and functional separation between sites 1 and 2, suggesting that other interactions may be overlooked by the two-site model. This section will highlight these and other intermediate chemokine-CKR interactions that do not fall into traditional spatial designations of sites 1 or 2, and will speculate on the functional implications of these interactions.</p><!><p>Diverse chemokine orientations: The most striking difference between the vMIP-II:CXCR4 and CX3CL1:US28 structures is the substantial deviation in chemokine orientation relative to the orthosteric pocket of the two receptors [8, 9]. Specifically, vMIP-II and CX3CL1 are rotated ~35° about the C-terminal ends of their C-terminal α-helices (Fig. 3A, B). The CXCL12:CXCR4 hybrid model diverges even more drastically, with CXCL12 rotated ~80° relative to vMIP-II [13]. The CXCL12:CXCR4 model was produced through a combination of rigid body docking and computational relaxation such that the backbone, side chain and rigid body positions were optimized. Extensive hydrophobic, polar and charged interactions spanning nearly half of CXCL12's total surface area supports the plausibility of a substantial deviation in orientation relative to vMIP-II. Importantly, variation in chemokine orientation allows these ligands to form unique but overlapping subsets of interactions with receptor ECLs and TM domains.</p><p>ECL2 links a 'disulfide network': The orientations of CX3CL1 and vMIP-II relative to their receptor binding pockets demonstrates that CX3CL1, but not vMIP-II, is ideally positioned to interact with ECL2 (Fig. 3A, B). CX3CL1 forms multiple interactions between its 30s-loop and ECL2 of US28, which, intriguingly, completes a disulfide network spanning from TM3 to TMs 1 and 7. In addition to a family A-conserved disulfide bond between ECL2 and TM3, most CKRs possess a disulfide that connects the N-terminal stalk with TM7 to form an additional ECL, termed "ECL4" (reviewed in 52) [80]. Owing to these two CKR disulfides, rigid body motions of receptor TM domains elicited by a chemokine at one site (e.g. N-loop interactions with TM7) could be efficiently communicated to a distant receptor domain (e.g. TM3) via a third interface (i.e. ECL2-30s-loop interactions), as suggested by Rajagopalan and colleagues [6]. In short, this structure may provide a glimpse of how multiple-site coupling at extracellular domains might influence chemokine receptor conformation via lateral (through-chemokine) allostery.</p><p>Chemokine loop engagement: The two chemokine-CKR co-crystal structures exhibit an extended TM interface (relative to other non-CKR family A 7TMRs), formed by 1–2 additional α-helical loops at the extracellular portions of TMs 1 and 7, and the disulfide bond linking TM7 to the receptor N-terminus (Fig. 2.A) [8, 9]. This extended TM1–TM7 interface may also be important for chemokine binding and orientation, analogous to the 30s-loop-ECL2 interactions of CX3CL1 and US28. For instance, Leu13 in the vMIP-II N-loop forms contacts, albeit weakly, with the extended TM1–TM7 interface (i.e. residues Cys2747.25 and Glu2777.28), as well as the closely positioned Gly273ECL3. These interactions in turn may contribute to the rotation of vMIP-II relative to CX3CL1, causing the vMIP-II 30s-loop to be spatially sequestered, preventing the formation of multiple interactions with CXCR4 (i.e. vMIP-II is 30s-loop deficient). In effect, these chemokine-CKR co-crystal structures suggest that chemokines utilize different loops to stabilize intermediate (i.e. non site 1/site 2) interactions with CKRs, thereby guiding chemokine orientation and likely influencing the signaling behavior of unique chemokine-CKR complexes.</p><p>Diverse uses of ECL2: Some descriptions of the two site model categorize chemokine interactions with ECLs as site 1 interactions due to their contribution toward specificity and affinity, as well as their interactions with the chemokine body [3, 9]. Nevertheless, ECLs also interact with chemokine N-termini and strongly influence CKR activation, as was recently shown by Chevigné and colleagues at CXCR4 [6, 7, 81, 82]. Chemokine-CKR structures also support the resistance of ECL interactions to site 1 or site 2 classification: CXCR4 preferentially uses ECL2 to stabilize the vMIP-II N-terminus, whereas US28 preferentially uses ECL2 to stabilize the CX3CL1 30s loop, making few N-terminal contacts (Fig. 3A, B) [8, 9]. Evidently, each CKR utilizes ECL2 for different purposes, likely contributing to the unique binding modes of the respective chemokines.</p><!><p>These structural examples illustrate that in some instances, spatial delineation of site 1 and site 2 may be artificial. Moreover, each chemokine-CKR complex utilizes distinct combinations of structural domains to specify unique functional outcomes. Having now seen the first comprehensive structural evidence of multiple intermediate, non-site 1/2 interactions, we will consider how receptor ECLs take on functional characteristics of sites 1 and 2 alike.</p><!><p>While we are only now beginning to appreciate the extent and diversity of chemokine-CKR interactions following high-resolution structural data, pharmacological evidence predating the two-site model supported a more complex "multi-site model" of CKR activation [83, 84]. Studies of CXCR1 and CXCR2 in the 1990s established a number of important principles concerning CKR recognition and activation as they relate to the receptor extracellular surface, including: 1) different chemokines utilize unique combinations of extracellular domains for the binding and activation of a single CKR [85, 86], 2) a single chemokine may utilize unique combinations of extracellular domains when binding different CKRs [85], and more generally 3) CKR binding and activation is a consequence of multiple, interdependent variables, particularly the identity of the chemokine and the simultaneous interactions it makes with all adjacent extracellular domains (i.e. N-terminus, ECLs 1–3) (Fig. 2A, B) [83–86]. Similar pharmacological and structural studies expanded these principles to other chemokine-CKR pairs, including CCR1 [81, 87, 88], CCR2 [18, 89, 90], CCR3 [91, 92], CCR5 [18, 88, 93–96], CXCR1 [97–99], CXCR2 [97, 98], CXCR3 [100], CXCR4 [82, 101], CX3CR1 [102].</p><p>CXCR3 provides an illustrative example of the interdependence of chemokine "multi-site" binding, chemokine preference for unique CKR conformations, and associated functional outcomes. Xanthou and colleagues disputed the universality of the two-site model for chemokine ligands, proposing instead a "multi-site model in which several distinct extracellular domains are required for efficient ligand binding and receptor activation" [103]. The authors created "gain-of-function" chimeras by individually swapping the N-terminus, ECL1, ECL2, and ECL3 of CXCR3 into CXCR1 (which does not share ligands with CXCR3), and vice versa to create "loss-of-function" chimeras. They found that while absence of ECL2 did not abolish chemokine binding, it completely attenuated CXCR3-mediated signaling, supporting a role beyond chemokine recognition. In addition, they showed that each chemokine required a unique subset of interactions to activate CXCR3: CXCL9 required ECL2 and ECL3; CXCL10 required all EC domains; and CXCL11 required the N-terminus, ECL1, and ECL2.</p><p>From these experiments it is clear that in addition to possessing spatial characteristics of sites 1 and 2, ECL2 may in some instances possess functional characteristics of sites 1 and 2. Moreover, this study suggests a "multi-site" mechanism by which different chemokines could, in principle, elicit functionally distinct downstream outcomes. Two lines of evidence support such a mechanism. A recent study demonstrated that the three CXCR3 ligands elicit unique patterns of Gαi activation, β-arrestin recruitment, and internalization following CXCR3 stimulation [5]. Secondly, CXCL10 preferentially binds inactive conformations of CXCR3, whereas CXCL11 binds both inactive and active conformations [104]. Intriguingly, the distinct functional outcomes elicited by CXCL10 and CXCL11 downstream of CXCR3 can drive opposing cellular and physiologic consequences. For instance, CXCL11 signaling promotes Th1 cell polarization into an immunotolerant T cell subset, resulting in an anti-inflammatory phenotype in a mouse model of multiple sclerosis, whereas CXCL10 promotes pro-inflammatory effects [105].</p><p>In all, these data show that chemokines can preferentially bind unique subsets of EC domains and/or available receptor conformations to elicit specific functional outcomes. By illustrating the interdependence of multiple extracellular domains on CKR signal transduction, these studies undermine the functional separation of sites 1 and 2 into recognition and activation, and imply that cooperative interactions influence chemokine recognition at extracellular CKR domains and subsequent functional outcomes. Moreover, unique interactions between each chemokine-CKR pair suggest therapeutic approaches that might target non-overlapping chemokine binding sites to selectively disrupt one chemokine-CKR interaction while maintaining another. In fact, several such selective allosteric modulators have been described for CCR1 and CCR5, demonstrating the feasibility of this approach for the development of highly targeted CKR therapies [3, 106–108].</p><!><p>As suggested in the previous section, the role of ECLs is more nuanced than static stabilization of chemokine ligands. Dynamic interactions between ECL and TM residues may act as molecular switches regulating receptor activation, especially in the case of ECL2. While ECL2 displays high variability in sequence, length, and structure even among related receptor subtypes, an impressive number of family A receptors seem to utilize ECL2 in this capacity, including rhodopsin [109–112], the serotonin 5-HT4 receptor [113], the V(1a) vasopressin receptor (V1aR) [114], the cannabinoid receptor 1 (CB1) [115–117], the β2AR [118], the angiotensin II type 1a receptor (ATII1aR) [119, 120], the D2 dopamine receptor (D2R) [121], the C5a complement receptor (C5aR) [122], and protease activated receptor 1 (PAR-1) [123], among others [124, 125]. Considering CXCR4, we recently suggested that manipulation of ECL2 by CXCL12 draws ECL2 toward the orthosteric pocket, thereby moving TMs 2 and 3 closer to one another [13]. These TM movements may then help initiate receptor activation, in an analogous mechanism to that predicted for CCR5 [126].</p><p>In addition to ECL2, ECLs 3 and 4 have been suggested to act as a tandem molecular switch required for CXCR4 activation [80, 101]. Using mutagenesis and a yeast-based Gαi protein activation screen, Rana and colleagues showed that interaction between TM7 and the CXCR4 N-terminus is essential for receptor activation, and that replacement of the disulfide-bonded cysteines with an electrostatic pair (Arg-Glu) conserves CXCR4 signaling. The authors suggest a model in which the N-terminal-TM7 disulfide undergoes a conformational change during receptor activation that is transmitted to ECL3 and TM6. Comparison of the active state CX3CL1:US28 and inactive state vMIP-II:CXCR4 structures supports this model (Fig. 3.B) [8, 9]. Compared to CXCR4, US28 demonstrates an inward (i.e. toward the TM domain) motion of ECL4, seemingly driving an inward motion of ECL3 and the extracellular portion of TM6. In a well characterized mechanism, inward motion of the top of TM6 causes it to rotate about a conserved proline "kink," resulting in substantial outward movement at the intracellular face to accommodate G protein binding [70].</p><p>Similar ECL4 motions may contribute to CXCR4 activation, as suggested by our recent CXCL12:CXCR4 model. Hydrogen bonding between the backbone carbonyl of CXCR4 Phe29N-term, which is adjacent to the N-term-TM7 disulfide, and CXCL12 Ser6, "pulls" the extracellular domain of TM7 toward the orthosteric pocket, which in turn may facilitate rearrangement of residues in CXCR4's TM domain during activation [13]. These examples suggest that chemokine binding to extracellular domains "primes" the receptor for activation by stabilizing an intermediate conformation, followed by chemokine N-terminal insertion into the receptor TM core and intracellular coupling of signaling effectors (i.e. G protein or β-arrestin). More structural studies will be needed to validate the role of ECL4 in the activation of CXCR4, US28, and expand its role to other CKRs. Nevertheless, it is becoming clear that chemokine-ECL interactions serve complex functional roles in CKR recognition and signaling.</p><!><p>Initially thought to be a crystallization artifact, chemokine dimerization has been reexamined over the past years in numerous structural and biochemical studies. It appears now that the vast majority of chemokines are able to form dimeric species, with the monomer-dimer equilibrium being regulated by factors such as pH, anions and interactions with glycosaminoglycans [127, 128].</p><p>Depending on the family, chemokines adopt two main oligomeric states with unique structural arrangements and interaction interfaces. CC chemokines form flexible and extended dimers mainly through residues surrounding the cysteine motif [129, 130], whereas CXC chemokines self-associate in more compact dimers via interactions involving the first β-strand [128, 131, 132]. In both types of interactions the N-termini of the two monomers are pointing in opposite directions. Chemokines of the C family, XCL1 and XCL2, have recently been shown to exist in a monomer-dimer equilibrium, unusually requiring complete protein unfolding. XCL dimers adopt a novel dimer conformation that also creates a six-stranded β-sheet [133, 134]. CX3CL1, the only member of the CX3C family, dimerizes in a similar manner to that of CC chemokines [135]. Additionally, some chemokines have been observed to form tetramers (e.g. CCL2, CCL5, CCL27, CXCL4, and CX3CL1) or higher-order oligomers [130, 135–139]. Heterodimers of two different CC or CXC chemokines as well as cross-family CC/CXC heterodimers have also been reported [140–142]. Furthermore, HMGB1 (High mobility group protein B1) protein was reported to form complexes with CXCL12 promoting different conformational rearrangements of CXCR4 from that of CXCL12 alone [143]. These findings further challenge the two-step binding model for chemokine-CKR interactions and complicate the question of which stoichiometries are capable of generating functional responses.</p><p>Immobilization of chemokines on glycosaminoglycans (GAGs) is an important step for chemokine function as it creates a gradient to direct cell migration and regulates the local chemokine concentration and availability for their receptors. Likewise, GAG binding can favor dimer formation as demonstrated for CCR2-binding chemokines [141], CXCL8 [144, 145], CXCL12 [146–149], XCL1 and XCL2 [133, 134]. Oligomerization has also been shown to increase GAG affinity by creating a more extensive surface for interactions [128].</p><p>The biological relevance of chemokine dimerization is still a matter of debate and its impact on receptor binding, stoichiometry and biased signaling remains to be unraveled [128, 132, 150, 151]. As an illustration it has been demonstrated that monomeric and dimeric CXCL12 induce different intracellular signaling responses and opposite effects on cell migration, but other recent studies suggested that this receptor interacts with CXCL12 in a 1:1 stoichiometry [36, 57].</p><!><p>Throughout the past two decades, it has been assumed that CKRs exist as monomers, which behave as fully competent signaling units. This assumption, in part, forms the basis of the classical two-site binding model. However, a number of studies demonstrated that CKRs can form homodimers and/or heterodimers (Fig. 4A) [152]. CKR dimerization has been investigated by various biochemical approaches such as co-immunoprecipitation (co-IP) [153–156], protein fragment complementation (PFC) [157, 158], Förster/Bioluminescence Resonance Energy Transfer (FRET/BRET) [159–161] and GPCR Heteromer Identification Technique (GPCR HIT) [162, 163]. The first structural evidence of CKR dimerization however was provided by the first inactive-state crystal structures of CXCR4 in which the receptor was present as a dimer with the interface between the subunits located at the top of TM5 and TM6 and stabilized by hydrogen bonds [10]. CKRs from all four subfamilies (C, CC, CXC, CX3C) have now been described to form homo- or heterodimers in vitro [154, 164–166] and some of them, including CXCR4 and CCR5, were shown to interact with other families of GPCRs such as the α1A/B-adrenergic receptors [167], opioid receptors [168] or non-GPCR membrane proteins that modulate the activity of the receptor or act as coreceptor for certain non-conventional ligands (Fig. 4A) [169]. Receptor dimerization has been shown to modify ligand binding properties [155, 170] and receptor signaling [153, 167, 171, 172] as well as intracellular trafficking [158]. However, so far there is no in vivo data reporting the existence of CKR dimers and therefore their biological relevance remains controversial [152, 173].</p><!><p>Another poorly understood and highly debated facet of chemokine-CKR interactions is their stoichiometry in functional signaling complexes. As both chemokines and receptors can homo- and heterodimerize, novel hypotheses around the stoichiometry of their interactions have emerged, leading to more complex models than the initially proposed two-step/two-site model. Among them, the 1:2 stoichiometry model where one chemokine binds two receptors simultaneously (Fig. 4B), the 2:1 stoichiometry model in which a chemokine dimer binds one receptor (Fig. 4C), and finally, the 2:2 stoichiometry model in which both the chemokine and the receptor interact as dimers (Fig. 4D) [8, 21, 57, 174]. Complementation studies carried out with CXCR4 mutants partially deficient in site 1 (i.e. CRS1) or site 2 (i.e. CRS2) were inconsistent with a 1:2 stoichiometry model and supported CXCR4 monomers as fully competent signaling units [57]. These results were later supported by the crystal structure resolution of the viral chemokine vMIP-II in complex with CXCR4 and CX3CL1 in complex with US28, both revealing a 1:1 stoichiometry interaction and an extensive contact surface between the two partners [8, 9]. However, more recent investigation of the preferential binding of monomeric CXCL12 to either monomeric (1:1) or dimeric (1:2) CXCR4 by molecular dynamics simulations proposed that in the 1:2 stoichiometry model, the N-terminus of the chemokine could make more tight contacts with the CRS2 of the second monomer to more efficiently favor signaling than in the 1:1 stoichiometry [174]. Finally, studies of CXCR4:ACKR3 heterodimers suggest that upon CXCL11 binding to CXCR7, conformational changes propagate through the dimer interface activating CXCR4 without the need of its own ligand (1:2*stoichiometry) (Fig. 4E) [172]. Gathering structural and mechanistic information on receptor dimerization, chemokine/receptor stoichiometry and relating it to functional observations remains challenging and necessitates state-of-the-art techniques to strengthen or to invalidate the currently accepted but oversimplified two-step/two-site model [9, 21, 57].</p><!><p>Once proposed to serve redundant signaling and functional roles, promiscuous chemokine-CKR interactions are widely believed to confer signaling and functional complexity to the CKR axis [1, 4–6]. Individual chemokines regulate multiple essential functions via independent CKR interactions. In turn, the consequences of each unique interaction depends upon concurrent spatial and temporal expression of both partners [175]. To complicate matters, CKRs were until recently accepted to signal exclusively through canonical G protein pathways and to couple exclusively to the Gαi/o G protein subtype. However, mounting evidence shows that some CKRs may also signal through other G protein subtypes (Gαs, Gαq/11 or Gα12/13) and activate G protein-independent signaling cascades (e.g. via β-arrestin) in ligand- and cell-specific contexts [176–178]. Analogous findings have been described for countless non-CKR 7TMRs for over a decade, signifying the new paradigm known as biased signaling or functional selectivity [179, 180]. Biased signaling appears to be ubiquitous among CKRs, and it provides a framework for understanding how a finite cast of ligands generates myriad functionally diverse outcomes. Biased signaling has been subdivided into three categories: ligand bias, receptor bias and cellular or tissue bias [4, 5, 181, 182].</p><p>Chemokine ligand bias occurs when different chemokines bind the same receptor to elicit distinct cellular responses. Ligand bias has been well documented for both CC and CXC chemokines, including CCL19 and CCL21 at CCR7 [177, 183, 184], CCL27 and CCL28 at CCR10 [5], three chemokine ligands at CXCR3 [5, 184], CXCL7 and CXCL8 at CXCR2 [185], as well as for chemokine ligands at CCR1 [5, 186], CCR2 [187] and CCR4 [188, 189]. Intriguingly, biased responses may be elicited by chemokines bearing unique PTMs, including truncation, citrullination or dimerization as reported for CCL14 [190] and CXCL12 [36]. The characteristic promiscuity of the chemokine-CKR network and poor sequence identity among chemokines, may partly explain the pervasiveness of ligand bias among chemokine ligands [5]. Indeed, variation of the structural interactions discussed in the article (summarized in Fig. 2B) such as chemokine orientation, ECL contacts, and major/minor subpocket selection likely stabilizes distinct active forms of the receptor, eliciting preferential coupling to different intracellular effectors [191]. While we are far from defining the precise structural mechanisms underpinning biased signaling, studies of other family A 7TMRs suggest a role for helical movements of and direct physical interactions with TMs 5 and 6 for G protein and TM7 for β-arrestin coupling, respectively [4, 70, 191–194].</p><p>In a peculiar twist, a chemokine agonist at one receptor can antagonize another receptor. Chemokines activating CXCR3 can also bind CCR3, blocking CCL11-induced cell migration and G protein signaling [195]. Similarly, CXCL11 and CCL7, well-characterized agonists at CXCR3 and CCR1/CCR2, respectively, were found to be antagonists at CCR5 [196–198]. In another example of dual activity chemokines, vMIP-II binds human and viral CKRs across all four families, acting as an antagonist or an agonist at different receptors [199–201]. Dual activity is often observed in cross-family interactions and could relate to differences in chemokine N-terminal orientation among CC and CXC chemokines (discussed in Section 3.2) [8]. Nevertheless, antagonism within the same family has also been reported, suggesting that other determinants may also play a role [198, 202].</p><p>Receptor bias occurs when a particular receptor preferentially or exclusively couples to a particular effector even in the context of multiple different ligands. Receptor bias has been well- characterized at atypical and viral CKRs [203]. For instance, CXCL12 binding to CXCR4 elicits both G protein and β-arrestin signaling [176, 204], whereas binding to ACKR3 (a.k.a. CXCR7) elicits G protein-independent β-arrestin signaling exclusively [205].</p><p>Cellular or tissue bias occurs when the same chemokine-CKR triggers distinct signaling pathways or cellular responses in different cellular contexts. For instance, CCL19 binding to CCR7 induces chemotaxis only in certain cell types [206, 207]. Such cellular bias is unsurprising considering the large variety of cells expressing CKRs, each of which carry unique expression profiles of signaling effectors (e.g. G protein subtypes and β-arrestin isoforms), receptor modifying enzymes (e.g. GRKs, TPSTs), as well as of other CKRs or receptor modulating partners involved in dimeric receptor interactions.</p><!><p>The chemokine receptors CCR2, CCR3, CCR5, CXCR2, CXCR4 and ACKR3 bind endogenous or virus-encoded ligands other than chemokines. These unconventional ligands vary widely in size, ranging from large proteins (e.g. > 100 kDa) to peptides, and often have no sequence or structural similarities with chemokines [15, 169, 208, 209]. Despite their structural dissimilarities, non-chemokine CKR ligands can trigger signaling pathways similar to those induced by endogenous chemokines, although in some cases they initiate unconventional signaling responses [15, 169, 208–212]. For some, binding and signaling relies on the CKR alone [15], while for others, the CKR operates in tandem with another membrane protein that usually serves as primary receptor [210, 213].</p><p>One of the best-known examples of non-chemokine CKR ligands is the HIV envelope protein gp120 (120 kDa), which uses CCR5 and CXCR4 as co-receptors for cell type-specific recognition and entry into host cells. To initiate viral membrane fusion with host cell membranes, gp120 first binds to CD4, a primary single TM segment receptor, initiating conformational changes (Fig. 4F). These changes then expose gp120's third variable loop (i.e. V3 loop), which in turn interacts with CCR5 or CXCR4 [213–215]. The second interaction was suggested to occur in a two-site binding mechanism similar to that initially proposed for chemokines, with common interacting determinants [211]. Importantly, not only is the interaction of gp120 with CXCR4 or CCR5 required for cell-specific HIV entry but it also leads to the activation of signaling pathways such as JNK and MAPKs, facilitating the early steps of viral replication [216, 217]. Tat, the HIV-trans-activating protein (14 kDa) released extracellularly by infected cells, triggers G protein-mediated signaling and chemotaxis through CCR2 and CCR3 [218, 219] and acts as an antagonist of CXCR4 [220]. Similarly, the HIV-1 matrix protein p17 binds CXCR1 and CXCR2, inducing chemokine-like activity on monocytes through Rho/ROCK activation [221, 222].</p><p>More recently, the pseudo-chemokine MIF (macrophage migration inhibitory factor), a pleiotropic and proinflammatory chemotactic cytokine of 12.3 kDa highly expressed by tumor cells, has been identified as a ligand for CXCR2 [209], CXCR4 [169] and ACKR3 [210], inducing ERK1/2 and ZAP-70 signaling and chemotaxis. As with gp120, the binding of MIF to CKRs requires a primary receptor, CD74, a single segment membrane-spanning protein also known as HLA class II histocompatibility antigen gamma chain (Fig 4G). Although MIF possesses some chemokine-like features, including a pseudo-ELR motif (D45-X-R129) and an N-loop-like region (amino acids 48–57), it lacks the canonical cysteine motif and is therefore classified among the chemokine-like function (CLF) chemokines.</p><p>In addition to CXCL12, CXCR4 also binds extracellular ubiquitin (eUb, 8.6 kDa). The eUb-CXCR4 interaction was proposed to follow a two-site binding mode, leading to G protein signaling similar to that induced by CXCL12 [15]. Other endogenous non-chemokine ligands such as human β3-defensin (HDB-3) (5.1 kDa) [223] and EPI-X4 (1.8 kDa) a 16-amino acid peptide derived from human albumin [224] also interact with CXCR4 but fail to induce intracellular signaling. Finally, human cytosolic proteins such as histidyl- and asparginyl-tRNA synthetases, released in some inflammatory pathologies, were shown to induce leukocyte migration through CCR3 and CCR5 [225]. Similar results were reported for parasitic asparginyl-tRNA synthetases which act as agonists for CXCR1 and CXCR2 [226].</p><p>The identification of non-cognate ligands for CKRs, some exclusive to a single receptor, others interacting with several receptors across several subfamilies, further emphasizes the complexity of the chemokine receptor network, which seems now more promiscuous and predisposed to bias than initially thought. These new ligands will certainly help to uncover other important physiological and pathological functions for this family of receptors, explain past observations and provide new therapeutic opportunities to modulate chemokine/CKR activity.</p><!><p>The two-site model has served as a valuable conceptual framework in which to understand chemokine-CKR signal transduction over the past twenty years. Nevertheless, a growing body of evidence supports a more complex model regulating how chemokines interact with their receptors to mediate an increasingly diverse complement of outcomes. The first structures depicting the complete extracellular and TM chemokine-CKR interfaces identified new interactions resisting site 1 and site 2 categorization. In parallel, advances in our understanding of 7TMR structure, dynamics, and activation are helping to define mechanisms by which chemokine binding is translated into receptor activation. Finally, paradigm shifts from within and outside of the chemokine realm are altering how we understand the complexity of the chemokine system. A more complete, "multi-site model" [103] of how chemokine-CKR interactions elicit functional outcomes will require active-state CKR structures and complementary studies probing the dynamics and functional specificity of unique ligand-receptor pairings.</p>

PubMed Author Manuscript

Fumarate-based metal-organic frameworks as a new platform for highly selective removal of fluoride from brick tea

Adsorption and removal of fluoride from brick tea is very important but challenging. In this work, two fumarate-based metal-organic frameworks (MOFs) were synthesized for the selective removal of fluoride from brick tea infusion. MOFs were examined for adsorption time, effect of dose, and uptake capacity at different initial concentrations and temperatures. Remarkably, over 80% fluoride removal was achieved by MOF-801 within 5 min at room temperature, while no significant adsorption occurred for the catechins and caffeine in the brick tea infusion. Further, with the use of the Langmuir equation, the maximum fluoride uptake capacity for the nontoxic calcium fumarate (CaFu) MOF was calculated to be as high as 166.11 mg g −1 at 373 K. As observed from FTIR, EDX and XPS results, hydroxyl group in MOFs were substituted by fluoride. This work demonstrates that the novel fumarate-based MOFs are promising materials for the selective removal of fluoride from brick tea infusion.

fumarate-based_metal-organic_frameworks_as_a_new_platform_for_highly_selective_removal_of_fluoride_f

<!>Results and Discussion<!>Adsorption behavior of fumarate-based MOFs for fluoride removal from brick tea infusion.<!>Conclusion<!>Synthesis of the MOF-801.<!>Effect of dose.

<p>Tea is a popular and healthy beverage due to that drinking tea has numerous health benefits, such as lower cardiovascular risk, reduce body fat, as well as decrease the risk of tumors 1,2 . However, Tea plants can accumulate and store a large amount of fluoride in mature leaves by adsorbing it from the soil and air without toxicity symptoms 3 . An abundance of fluoride can be released during tea infusion and the bioavailability of the released fluoride is nearly 100% to consumers, owing to the soluble fluoride ions from tea are easily adsorbed through the gastrointestinal track 4 . As we all known that fluoride is an essential element to mammals and a moderate amount of fluoride helps bone development whereas excessive intake of fluoride can lead to various diseases such as dental and skeletal fluorosis 5,6 . The fluoride content in tea is thought to be safe and could contribute to human health when the concentration of fluoride at low levels of 100-300 mg kg −1 , but it contained in some special tea (i.e., brick tea) is usually extremely high with levels up to 600 mg kg −1 7 . Brick tea based fluorosis is mainly found in the northwestern of China, such as Qinghai, Tibet, Inner Mongolia, and Sinkiang, where most of minorities are habitual consumer of brick tea with high level of fluoride 8 . To date, brick tea type fluorosis is still considered as a severe health problem in some parts of China, due to it is impossible to change these minorities brick tea habitual consumption. Hence, it is necessary to develop suitable methods to strictly control and remove the fluoride from tea. It is well known that adsorption method is one of the most applicable methods due to its low cost and simple operation 9,10 . Recently, Zhao et al. reported on a plant polyphenol-Ce hybrid adsorbent for the effectively adsorption of fluoride during the period of tea plants growing 7 . However, for these plant polyphenol-Ce-based adsorbents, it was difficult to separate the adsorbents from the soil and their fluoride adsorption capacity is still low, making them difficult to widespread industrial use. Therefore, a simple strategy to develop high capacity and selective fluoride adsorbents is highly desired.</p><p>As a new class of crystalline inorganic-organic porous hybrid materials, metal-organic frameworks (MOFs) have emerged as one kind of promising material to develop novel adsorbents 11,12 . Due to the exceptional internal surface area, tailored structure, tunable pore architectures combined with diverse framework functionalities, MOFs experienced fast development and have displayed a vast range of promising applications such as catalysis 13 , gas storage and separation 14 , drug delivery 15 , as well as adsorption and removal of hazardous materials 16 . For the adsorption of contaminant-related applications, MOFs have been widely exploited for the selective adsorption and removal of toxic dyes 17 , pharmaceuticals 18 , nitrogen compounds 19 , sulfur compounds 20 , and heavy metal ions 21 , because the pore size and shape of MOFs can be easily controlled to facilitate the uptake of targeted guest molecules. Moreover, in the recent years, a few pioneering studies demonstrating the promise of MOFs in the removal of fluoride from water have also been reported. For example, Liu et al. first reported on the fabrication of MIL-96(Al) for the selective defluoridation of drinking water 22 . Later, Lin and co-workers reported on an amine-functionalized zirconium MOF (named UiO-66-NH 2 ) used as enhanced adsorbents for fluoride removal 23 . Further, De groups reported a promising aluminium fumarate MOF (AlFu) adsorbent for the removal of fluoride from groundwater 24 . These MOFs-based porous adsorbents exhibit fast and excellent adsorption abilities for the fluoride removal from water system. However, to date, employment of MOFs as adsorption materials for the selective removal of fluoride from tea system has not been reported.</p><p>Herein, we report two fumarate-based MOFs (i.e., MOF-801 and CaFu) for the highly selective adsorption of fluoride from brick tea infusion. As a subfamily of porous MOFs materials, a series of zirconium(IV)-based MOFs (Zr-MOFs) have been developed since 2008 due to their inherent thermally and chemical stability 25 . MOF-801 is the smallest member of Zr-MOFs with a formula of Zr 6 O 4 (OH) 4 (fumarate) 6 and fcu topology 26 . MOF-801 possesses hydrophilic adsorption sites since plenty of hydroxyl groups are involved in the nodes of Zr(IV). Given the presence of zirconium-bound hydroxyl groups in the nodes of framework which are expected to facilitate the adsorption of fluoride via the anion exchange behavior. Impressively, the prepared MOF-801 exhibits excellent adsorption performance and stability toward fluoride removal from brick tea infusion. Furthermore, a homologous nontoxic calcium fumarate (CaFu) MOF was also synthesized and employed to efficient remove fluoride from tea infusion. These fumarate-based MOFs not only show high efficiency and selectivity towards the fluoride removal, but also experimentally simple and easy to handle for the uptake of fluoride by using tea bag model. To the best of our knowledge, this is the first example of MOF-based adsorbents for the selective adsorption of fluoride from brick tea infusion.</p><!><p>Synthesis and characterization of the fumarate-based MOFs. MOF-801 is a typical microporous Zr-based MOF consisting of Zr 6 nodes bridged by fumarate linker to give the three dimensional (3D) structure (Fig. 1a) 27 . Fumaric acid was chosen as the organic linker in this work because it is an important biologically occurring molecule and could be used as a typical food additive 28 . MOF-801 has two crystallographically independent tetrahedral cage sizes of 5.6 Å and 4.8 Å and one octahedral cage size of 7.4 Å 26 . The phase purity of the as-synthesized product was checked by PXRD. For comparison, the pattern of the simulated from the crystallographic data of MOF-801 is also shown. As shown in Fig. 2a, all of the experimental PXRD pattern diffraction peaks are well matched with the MOF-801 standard literature values 26 . No miscellaneous PXRD peaks were observed, indicating that the as-synthesized MOF is undoubtedly MOF-801. The XPS was employed to identity the chemical compositions, especially the valence state of the elements of the MOF. The XPS full spectrum, as presented in Fig. 2c, confirms that the existence of C, O, and Zr in the MOF-801 sample. The high-resolution of Zr 3d spectrum is shown in Fig. 2d, the binding energy peaks located at around 182.89 and 185. 21 eV can be ascribed to 3d 5/2 and 3d 3/2 of Zr(IV), which is similar with the reported Zr-based MOFs 29 . Therefore, the results suggest that these peaks only belong to Zr(IV) in the MOF-801 framework. The N 2 adsorption-desorption isotherm was also implemented to measure the surface area of MOF-801 and the result is displayed in Fig. 2b. The N 2 adsorption-desorption isotherm of MOF-801 exhibits typical type-I behavior (Fig. 2b), which is related to microporous material. The Brunauer-Emmett-Teller (BET) surface area is 755 m 2 g −1 , and the calculated pore volume is 0.44 cm 3 g −1 .</p><p>The morphology of the sample was then characterized by SEM and TEM. These SEM images reveal that the obtained MOF-801 nanoparticles (NPs) are of spherical shape with a uniform size and good dispersity (Fig. 3a and b). The morphology of these NPs were further identified by TEM (Fig. 3c and d). One can see that the MOF-801 nanospheres exhibit a narrow size distribution, which are in good agreement with the SEM observation. Preliminary statistics based on the product shown in Fig. 3 indicates that the average size of the MOF-801 nanospheres is 150 nm.</p><!><p>Studies regarding the removal of fluoride from water system have shown that porous MOFs are good adsorbents for the adsorption of fluoride [22][23][24]30 . However, to date, investigations used MOFs to adsorb fluoride from tea infusion are still scarce up. Therefore, in this study, MOF-801 adsorbent was checked for the adsorption of fluoride from brick tea infusion. The pH of the solution plays a key factor in the fluoride removal, which influences the surface charge of the adsorbents. In order to evaluate the influence of pH on the adsorption of fluoride, 40 mg of MOF-801 was suspended in 25 mL of brick tea infusion with the initial fluoride concentration of 8 mg L −1 at various pH (from 2 to 8). As can be seen from Fig. 4a that the adsorption is higher at a lower pH and drops drastically after pH 5. This can be attributed to the fact that at pH 5.5, the MOF NPs become neutral in charge and at a higher pH, the adsorbent NPs become negatively charged as shown in Fig. 4b. In acidic pH conditions, the MOF-801 is positively charged facilitating the adsorption of fluoride. At pH 6, the fluoride adsorption was reduced, which due to the competitive adsorption of OH − in the brick tea infusion. Since we are concerned about drinking tea and the pH value of the actual prepared brick tea infusion is 5.4, we did not adjust the pH during the following experiment. To deepen the understanding of the equilibration time for maximum uptake of fluoride and to have a better understanding of adsorption kinetics, the adsorption of fluoride on MOF-801 from brick tea infusion was investigated as a function of contact time. As shown in Fig. 4c, 40 mg of MOF-801 is used as adsorbents to capture 25 mL brick tea infusion with the initial fluoride concentration of 8 mg L −1 . Accordingly, the fluoride adsorption capacity increased rapidly during the first 2 min and gradually attained the adsorption equilibrium only in 5 min. In can be found that MOF-801 can remove nearly 80% of the fluoride ions present in the respective brick tea infusion within 5 min. Tea has been well studied for its health benefits on human because tea leaves contain large amounts of catechins, including (−)-epicatechin gallate (ECG), (+)-catechin (C), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC), (+)-gallocatechin gallate (GCG), and (−)-epigallocatechin (EGC), which are typical powerful antioxidants 2 . As can be seen from Fig. 4d, no significant loss of catechins and only minor loss of caffeine (Caf) can be observed within 30 min, indicating that the MOF-801 adsorbents possess excellent selective adsorption and removal of fluoride from brick tea infusion. The residual zirconium concentration of the brick tea infusion was determined with ICP. Significantly, there was no residual zirconium ion can be detected in the brick tea infusion. The results indicate that MOF-801 will be a promising candidate to adsorption of fluoride from brick tea infusion.</p><p>Further, the adsorption kinetic data was modeled by the pseudo-second-order kinetic equation (Equation S1) 31 . As can be seen from Fig. S1, the parameter values of this kinetic model can be calculated with the plot of t/q t versus t, and the obtained correlation coefficient is 0.9999, revealing that the selective removal of fluoride from brick tea infusion onto the MOF-801 frameworks follows this kinetic model very well. The kinetic rate constant (k 2 ) and the equilibrium adsorption capacity (q e ) values of MOF-801 under this brick tea infusion condition were determined to be 0.69 g mg −1 min −1 and 3.91 mg g −1 , respectively.</p><p>The adsorption capacity is important for the application of adsorbents. The amount of fluoride adsorbed per gram of MOF-801 was investigated by exposing the MOF to the brick tea infusion under a wide range of fluoride concentrations. In order to make sufficient time for removal to occur, tea infusion were tested after 60 min of exposure. It was found that the adsorption capacities remarkably increased as the fluoride initial concentration increased in the brick tea infusion (Fig. 5a), suggesting the favorable selective adsorption of fluoride by MOF-801 at high concentrations. Further, to quantitatively predict the adsorption capacity of MOF-801, we employed the Langmuir model to fit the adsorption isotherm data and high correlation coefficients can be obtained from Fig. S2. In the case of Langmuir model, the adsorption process of adsorbent is occurred as a mono-layer over the adsorbent surface 23 . When the adsorption sites of MOF-801 are occupied by the fluoride in the tea infusion, then these occupied sites cannot be used for the adsorption anymore. Thus, the maximum adsorption capacity of MOF-801 for fluoride can be calculated using the Langmuir equation (Equation S2) 23 . Figure S2 shows the plots of C e /q e versus C e over MOF-801 for fluoride removal at different temperatures, and the maximum adsorption capacity values (q m ) can be determined from the slops. According to Equation S2, the q m of MOF-801 for fluoride in the brick tea infusion is 32.13 mg g −1 at 298 K (Table 1). This value is superior to that of the many conventional fluoride adsorbents [32][33][34][35] , and even comparable to some of the novel MOF based adsorbents which were used in the simple water system 22,23,36 . Additionally, as displayed in Fig. 5a, the fluoride adsorption capacity over MOF-801 increases with increasing the temperature, and the q m becomes 38.60 and 45.72 mg g −1 at 308 and 318 K, respectively (Table 1). This trend shows the positive effect of MOF-801 on the adsorption isotherm at higher temperatures, suggesting that the removal of fluoride of MOF-801 in tea infusion could be endothermic reaction [37][38][39] . To gain a better understanding of the thermodynamic feasibility and the adsorption process, three basic thermodynamic parameters like Gibbs free energy change (ΔG) (Equation S3), entropy change (ΔS) (Equation S4), and enthalpy change (ΔH) (Equation S4) were calculated from the removal of fluoride on MOF-801 by using standard methods 22 . As shown in Table 1, the obtained ΔG values of MOF-801 are -1.99, -2.25, and -2.49 kJ mol −1 at 298, 308, and 318 K, respectively. The negative values of ΔG at all temperatures reveal that the adsorption process of fluoride to MOF-801 can be spontaneous adsorption within this temperature range. Significantly, ΔG value becomes more negative when the temperature is increased, suggesting that the adsorption process is more favorable at high temperatures.</p><p>The Van't Hoff plot, constructed according to Equation S4, gave straight line which is shown in Fig. 5b, and the values of ΔS and ΔH can be determined from the intercept and slop of the plot. It can be seen from Table 1 that ΔH and ΔS were determined to be 4.54 kJ mol −1 and 21.97 J mol −1 K −1 . As we know that the value of ΔH range 2.1-20.9 kJ mol −1 is regarded as physical adsorption while range 20.9-418 kJ mol −1 can be corresponded to chemical adsorption 40 . Thus, the ΔH value in this work implies that the fluoride adsorption process occurred by MOF-801 due to the physical adsorption. The positive ΔH value (Table 1) indicated that the adsorption of fluoride over MOF-801 was an endothermic process, which was in accord with the increasing adsorption capacity associated with increasing adsorption temperature (Fig. 5a). The endothermic process may be due to a stronger interaction between pre-adsorbed water and the MOF than the interaction between fluoride and the MOF. At the same time, the obtained positive value of ΔS (21.97 J mol −1 K −1 ) further confirmed that the increase of randomness at the solid adsorbent/tea infusion solution interface during the fluoride adsorption reaction over MOF-801. This phenomenon can be attributed to the released water molecules at the interface is greater than the adsorbed fluoride ions by the MOF-801 adsorbents 41 . Therefore, the driving force of fluoride adsorption (negative ΔG) on MOF-801 is due to an entropy effect (positive ΔS) rather than an enthalpy change (positive ΔH).</p><p>The reusability is one of the important issues for the practical application of adsorbents. A crucial problem in the use of adsorbents is that they suffer from the low efficiency of separation. The tea bag model described here is designed to overcome these challenges. In a simple and easy to use design, a tea bag containing MOF-801 NPs were prepared and dipped in fluoride contaminated brick tea infusion as shown in Fig. S3b. After adsorption, the tea bag was first removed from the brick tea infusion, washed with diluted NaOH (0.01 M) and water, and then dried at 70 °C. After this treatment, the tea bag containing MOF-801 NPs were used again for other consecutive runs under the same adsorption conditions for 1 h. As shown in Fig. S3a, no significant loss in the adsorption efficiency of fluoride from brick tea infusion can be observed in the subsequent five consecutive cycles, indicating that the MOF-801 adsorbents possess excellent long-term adsorption stability and could be reused for multiple rounds.</p><p>Since the adsorbent dose is an important factor for the control of fluoride removal efficiency, the parameter of MOFs dose were tested and the results are presented in Fig. 6. In an easy and simple method for practical application, the effects of MOFs dose were carried out by exposing MOFs and brick tea leaves to deionized water directly with boiling at 373 K for 30 min. It was obvious that the final adsorbed percentage of fluoride increased with the amount of MOF-801 (Fig. 6a). As the amount of MOF-801 increased from 0.4 to 2.0 g L −1 , the efficiency of fluoride uptake gradually increased from 18% to 70%. The higher fluoride adsorption efficiency at the higher MOF-801 dose was due to the more active sites of MOFs available present in the tea infusion. Significantly, the losses of the catechins and caffeine were all lower than 5% (Fig. 6c), suggesting that the MOF-801 adsorbents could highly selective adsorption of fluoride from brick tea infusion. When the amount of MOF-801 increased to 4.0 g L −1 , the efficiency of fluoride removal went up to 92%. However, the losses of the catechins and caffeine were increased to around 20% in this conditions. This comparison implies that the MOFs dose is a key factor for selective adsorption of fluoride from brick tea system. Although a higher dose of MOFs is beneficial to removal of fluoride, an over MOFs dose must be avoided due to the increase of catechins and caffeine loss at higher dose. Therefore, it is obviously that the best MOF-801 dose range for the selective removal of fluoride from brick tea infusion is below 2.0 g L −1 .</p><p>One may criticize the fact that the Zr-based MOFs adsorbents were not bio-compatible for practical applications, nevertheless, homologous nontoxic calcium fumarate (CaFu) MOF was also synthesized and tested to obtain a good performance in the field of fluoride removal from tea infusion. The structure of the as-synthesized CaFu was characterized by PXRD (Fig. S4) and SEM (Fig. S5). It is clearly seen that the CaFu material consists of irregular shape particles with the size around 4.5 μm. Similarly, a high CaFu dose exhibited a high fluoride adsorption from the tea infusion and the percentage of fluoride removal increased to 37% with 2.0 g L −1 of CaFu (Fig. 6b). Although this value is lower than that of above obtained MOF-801, it is still superior to that of Tea-Al biosorbent which we reported recently 42 . In our previous work, we have reported the synthesis of aluminum oxide decorated tea waste based biosorbent (e.g., Tea-Al), which is promising for the fluoride removal from the brick rea infusion 42 . However, a critical drawback of Tea-Al is non-selective fluoride removal from brick tea infusion. The fumarate-based MOFs adsorbents described here are designed to overcome this challenge. As shown in Fig. 6d, no significant losses of the catechins and caffeine were observed with the dose of CaFu below 2.0 g L −1 .</p><p>Furthermore, the initial fluoride concentration-dependent removal capacity was also obtained to investigate the adsorption isotherm of fluoride on CaFu adsorbent. 30 mg of CaFu and 0.5 g of brick tea were mixed with 25 mL of 8-512 mg L −1 fluoride solution. The adsorption isotherms of CaFu adsorbent were obtained after boiling in tea infusion for 30 min at 373 K. Figure 7 shows that the adsorption capacity of CaFu also increased as the initial concentration of fluoride increased in the tea infusion. As displayed in the inset of Fig. 7, the isotherm data fit the Langmuir model well, and the correlation coefficient is 0.9886. Remarkably, the maximum adsorption capacity of CaFu for fluoride in the brick tea infusion is 166.11 mg g −1 at 373 K. To date, there have been only two pioneering studies on the fluoride removal from tea infusions (e.g., Tea-Al 42 and Fe 3 O 4 /Al 2 O 3 -PUF 43 ). The maximum adsorption capacity value of CaFu is the highest value ever reported for fluoride removal from the brick tea infusion system 42,43 . The present work may provide potential of synthesis of such nontoxic MOFs-based adsorbents for application in fluoride removal from brick tea.</p><p>To shed light on the mechanism of fluoride adsorption on MOFs, FT-IR (Fig. 8a), EDX (Fig. S6), and XPS spectra (Fig. 8b-d) were used to characterized CaFu before and after adsorption of fluoride. Prior to adsorption, the IR spectrum of CaFu contains two strong bands around 1594 and 1405 cm −1 corresponding to the -O-C-Ogroup, suggesting that the Fu species is coordinated to the Ca atoms. The sharp band of CaFu around 3465 cm −1 and a small band around 2745 cm −1 are assigned to the stretching of Ca nodes terminal -OH group and the hydrogen-bonding between the -OH in the Ca nodes and aqua, respectively 44 . After adsorption of fluoride, the stretching of -OH group at 3465 cm −1 is remarkably diminished and the -OH stretch of the hydrogen-bonding based at 2745 cm −1 disappeared completely (Fig. 8a). Furthermore, the FT-IR spectra were also used to characterize MOF-801 before and after fluoride adsorption (shown in Fig. S7). The FT-IR spectra results are similar with what was observed on the CaFu before and after adsorption of fluoride systems. Based on these observations, we propose a simple mechanism displayed in Fig. 1b for fluoride removal over MOFs: first, fluoride ions can be adsorbed onto the porous fumarate-based MOFs via interactions between the fluoride ions and the activity metal center in the framework. There are abundant of hydroxyl groups around the nodes of MOFs. Then the fluoride replaces hydroxyl group on the metal-node in the structure of MOFs through the anion exchange behavior. (Fig. 8c), which is similar with that of Ca 2+ ions 45 . For the sample of CaFu after fluoride adsorption, apart from those binding energy peaks belonging to pure CaFu frameworks, singles of F 1 s appeared at 684.8 eV can be detected (Fig. 8b,d). In particular, it is worth to mention that Ca 2p 3/2 and Ca 2p 1/2 of the sample of CaFu after fluoride adsorption were shifted to 348.3 and 351.9 eV (Fig. 8c), demonstrating that the bonding environment of Ca nodes was changed after adsorption of fluoride. These results are good consistent with the IR observation, and further confirming that the adsorption reaction depended on the fluoride and the Ca-node coordinatively unsaturated centers of CaFu.</p><!><p>In summary, two fumarate-based MOFs have been synthesized and used in the highly selective removal of fluoride from brick tea infusion. The adsorption capacity of MOF-801 for fluoride from the tea infusion was 32.13 mg g −1 at 298 K. Besides, the adsorption capacity of CaFu was 166.11 mg g −1 at 373 K. Furthermore, the two fumarate-based MOFs showed a highly selective fluoride adsorption from the tea infusion and no significant losses of the catechins and caffeine were observed with the dose of MOFs below 2.0 g L −1 . FTIR and XPS results point to the key importance of numbers of node-based coordinatively unsaturated adsorption sites for the effective fluoride adsorption to occur. Present study suggests that these fumarate-based MOFs have great potentially useful for the fluoride adsorption from brick tea leaves. The structure characterization of the samples were collected by the powder X-ray diffraction (PXRD) patterns with Cu target from 5 to 50°. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images were performed by a Hitachi S-4800 and JEOL JEM 2100 at 200 kV, respectively. X-ray photoelectron spectroscopy (XPS) were performed on the Catalysis and Surface Science Endstation of National Synchrotron Radiation Laboratory (NSRL). Nitrogen adsorption-desorption isotherms were obtanied on a micromeritics TriStar II 3020 adsorption analyzer at 77 K. Fourier transform infrared spectrometer (FTIR) were recoreded on a Nicolette is 50 FTIR spectrometer. The fluoride concentration was measured by a fluoride ion selective electrode (9609 BNWP). Catechins and caffeine concentrations were determined by the high performance liquid chromatography (HPLC, Waters 2695) with a 2489 ultraviolet (UV)-visible detector.</p><!><p>The MOF-801 was prepared according to a recently published with some modifications 25 . 1.6 g of ZrOCl 2 ⋅8H 2 O and 0.58 g of fumaric acid were dissolved in 27 mL DMF-formic acid (v/v = 20:7) mixed solution. After dissolved thoroughly, the clear solution was put into an autoclave for crystallization at 130 °C for 6 h. After reaction, the obtained products were harvested by centrifugation, washed with DMF and ethanol, and then dried overnight at 100 °C under vacuum.</p><p>Synthesis of the CaFu. 0.6964 g of fumaric aicd and 0.9491 g of Ca(CH 3 COO) 2 were dissolved in distilled water (30 mL). After dissolved thoroughly, the clear solution was put into an autoclave for crystallization at 65 °C for 16 h. After cooling to room temperature, the products were obtained by centrifugation, washed with ethanol, and then dried overnight at 100 °C under vacuum.</p><p>Fluoride adsorption kinetic screening. Firstly, the initial fluoride stock brick tea infusion was prepared by dispersing 0.5 g of brick tea into 25 mL of deionized water (1:50 g mL −1 ) with boiling for 30 min at 373 K. After that, the mixture solution was filtered, and the initial brick tea infusion was obtained. The fluoride concentration in the brick tea infusion was measured to be 8 mg L −1 by a fluoride ion selective electrode. The 10000 mg L −1 of fluoride solution was prepared by dissolving NaF in deionized water.</p><p>Kinetic experiments were performed by exposing 40 mg of MOF-801 to 25 mL of brick tea infusion with the initial fluoride concentration of 8 mg L −1 in a 50 mL polypropylene centrifuge tube. The mixture solutions were placed in a vapour-bathing constant temperature oscillator at 298 K under a speed of 250 rpm. Then the solutions were filtered after a certain of adsorption time, both initial and the remaining fluoride ion, catechins and caffeine concentrations were determined with fluoride ion selective electrode and HPLC, respectively.</p><p>Fluoride adsorption isotherm and thermodynamic. The maximum adsorption capacity of MOF-801 was performed by exposing 40 mg of MOF-801 to 25 mL of the brick tea infusion in a 50 mL polypropylene centrifuge tube with fluoride concentrations of 8, 16, 32, 64, 128, 256 mg L −1 . The solutions were placed in a vapour-bathing constant temperature oscillator at 298 K under a speed of 250 rpm for 60 min. Then the adsorbents were separated by filtration, and the remaining fluoride concentrations were measured with fluoride ion selective electrode. To further get thermodynamic parameters (ΔG, ΔS, ΔH) of MOF-801, the adsorption was also performed at 308 K and 318 K.</p><p>Experimental procedure for reusability tests. For the reusability of the MOF-801 for the fluoride removal from brick tea infusion, easy to use tea bag containing 40 mg of MOF-801 NPs were prepared. The tea bag containing MOF-801 was dipped in brick tea infusion in a 50 mL polypropylene centrifuge tube with fluoride concentrations of 8 mg L −1 . At the end of the adsorption, the tea bag was removed from the brick tea infusion and the adsorbent was washed with NaOH solution (0.01 M, 5 mL × 3). After sonication for 30 min, the tea bag containing adsorbents was collected, washed with distilled water three times, and then re-dipped in the brick tea infusion (25 mL, 8 mg L −1 ) for the next cycle. To test the adsorption potential of the regenerated MOF-801 adsorbent, five cycles of regeneration studies were carried out.</p><!><p>The effects of MOFs dose were carried out by exposing 10-100 mg (0.4-4 g L −1 ) of MOFs (MOF-801 or CaFu) and 0.5 g of brick tea to 25 mL of deionized water. The mixture solutions were then boiled at 373 K for 30 min. Then the solutions were filtered, and the remaining fluoride ion, catechins and caffeine concentrations were determined with fluoride ion selective electrode and HPLC, respectively.</p><p>CaFu maximum uptake per gram. The CaFu adsorption isotherm experiments were determined by exposing 30 mg of CaFu and 0.5 g of brick tea to 25 mL of deionized water in a polypropylene centrifuge tube with initial fluoride concentrations of 8-512 mg L −1 . These solutions were then boiled at 373 K for 30 min. Then the solutions were filtered, and the remaining fluoride ion concentrations were determined with fluoride ion selective electrode.</p>

Scientific Reports - Nature

Room-temperature chemical synthesis of C 2

Diatomic carbon (C 2 ) exists in carbon vapour, comets, the stellar atmosphere, and interstellar matter, but although it was discovered in 1857, 1 it has proved frustratingly difficult to characterize (Figure 1), since C 2 gas occurs/exists only at extremely high temperatures (above 3500°C). 2 Since 1930, several experimental methods to generate C 2 have been developed by using extremely high energy processes, such as electric carbon arc and multiple photon excitation, 3,4 and the C 2 species obtained were reported to exhibit singlet dicarbene (double bond) and/or triplet biradical (triple bond) behavior. 5-7 In contrast, recent theoretical simulations suggest that C 2 in the ground state should have a singlet biradical (quadruple bond) character. 8,9 Here, we present a straightforward room-temperature/pressure synthesis of C 2 in a flask. We show that C 2 generated under these conditions behaves exclusively as a singlet biradical, as predicted by theory. We also show that spontaneous, solvent-free reaction of in situ-generated C 2 under an argon atmosphere results in the formation of graphene, carbon nanotubes (CNTs) and fullerene (C 60 ) at room temperature. This is not only the first chemical synthesis of nanocarbons at ordinary temperature and pressure, but also provides experimental evidence that C 2 may serve as a key intermediate of various sp 2 -carbon allotropes.

room-temperature_chemical_synthesis_of_c_2

<p>Diatomic carbon (C 2 ) is historically an elusive chemical species. Considerable efforts have been made to generate/capture C 2 experimentally and to measure its physicochemical properties. The first successful example of artificial generation of C 2 , which was confirmed spectroscopically, involved the use of an electric carbon arc under high vacuum conditions. 10 Subsequent chemical trapping studies pioneered by Skell indicated that C 2 behaves as a mixture of singlet dicarbene (double bond) and triplet biradical (triple bond) states in a ratio of 7:3 to 8:2 (Supplementary Fig. S1A). 3,6 Multiple photon dissociation of two-carbon small molecules (acetylene, ethylene, tetrabromoethylene, etc.) by infrared or UV irradiation in the gas phase was also developed to generate C 2 , but this photo-generated C 2 also exhibited several electronic states. 4 Recently, other approaches for the isolation of C 2 have been reported, using potent electron-donating ligands to stabilize C 2 by means of dative interactions (L:→C 2 ←:L), but such stabilized complexes no longer retain the original character of C 2 (Supplementary Fig. S1B). [11][12][13][14] Instead, theoretical/computational simulation has been applied recently, and the results indicated that C 2 has a quadruple bond with a singlet biradical character in the ground state (Fig. 1). These various theoretical and experimental findings have sparked extensive debate on the molecular bond order and electronic state of C 2 in the scientific literature, probably because of the lack of a method for the synthesis of ground-state C 2 .</p><p>For the present work, we focused on hypervalent iodane chemistry, aiming to utilize the phenyl-λ 3 -iodanyl moiety as a hyper-leaving group (ca. 10 6 times greater leaving ability than triflate (-OSO 2 CF 3 ), a so-called super-leaving group). 15 We designed [β-(trimethylsilyl)ethynyl](phenyl)-λ 3 -iodane 1a, 16 in the expectation that it would generate C 2 upon desilylation of 1a with fluoride ion to form anionic ethynyl-λ 3 -iodane 11, followed by facile reductive elimination of iodobenzene (Fig. 2A). Gratifyingly, exposure of 1a to 1.2 equivalents of tetra-n-butylammonium fluoride (Bu 4 NF) in dichloromethane resulted in smooth decomposition at -30 °C with the formation of acetylene and iodobenzene, indicating the generation of C 2 ! However, all attempts to capture C 2 with a range of ketones and olefins, such as acetone (3), 1,3,5,7-cyclooctatetraene (4), styrene (7), and 1,3,5-cycloheptatriene, failed, though they smoothly reacted with arc-generated C 2 on an argon matrix at -196 °C (Supplementary Fig. S1). 7,17 These findings immediately suggested that the putative C 2 synthesized here at -30 °C has a significantly different character from C 2 generated under high-energy conditions (Supplementary Fig. S2). Taking account of the fact that quantum-chemical calculations suggest a relatively stable singlet biradical C 2 with quadruple bonding in the ground state, we next examined an excellent hydrogen donor. 9,10-Dihydroanthracene (12) has very weak C-H bonds (bond dissociation energy of 12: 76.3 kcal mol -1 vs CH 2 Cl 2 : 97.3 kcal mol -1 ) 18,19 that might effectively trap the putative singlet biradical C 2 . When 12 was added to the reaction mixture, anthracene (13) was obtained accompanied with the quantitative formation of acetylene (Fig. 2A), which clearly suggests that the generation of C 2 and subsequent hydrogen abstraction from 12 gave acetylene. The formation of acetylene was confirmed by Raman spectroscopy after AgNO 3 trapping, and the amount of acetylene was estimated by the quantitative analysis of Ag 2 C 2 thus generated. These results strongly support the relatively stable (singlet) biradical nature of our C 2 , in accordance with the theoretical calculations. Thus, we turned our attention to the galvinoxyl free (stable) radical 14 in order to trap C 2 directly. To our delight, O-ethynyl ether 15 was obtained in 14% yield, accompanied with the formation of acetylene (84%) (Fig. 2B). The structure of 15 was fully characterized by 1 H/ 13 C NMR spectra: an upfield-shifted acetylenic proton was seen at 1.78 ppm in the 1 H NMR, as well as considerably separated 13 C NMR chemical shifts of two acetylenic carbons (C α : 90.4 ppm, C β : 30.0 ppm), clearly indicating the presence of an ethynyl ether unit. 20 In solution, di-galvinoxyl alkyne 16 was undetectable or barely detectable even when excess amounts of 14 were used, though 15 was obtained as almost the sole product in all cases. On the other hand, when we performed the trapping reaction in the presence of 2 equivalents of 14 under solvent-free conditions, 16 was clearly observed by atmospheric pressure chemical ionization (APCI) mass (MS) spectrometry, although in very small quantity (Supplementary Fig. S3). 21 These findings are consistent with the valence bond model of a singlet biradical species, according to which the energy barrier of the second hydrogen abstraction is lower by approximately 10 kcal/mol compared with the first hydrogen abstraction, which has to overcome the bonding energy of the singlet biradical. 22 It should be noted that the O-phenylated product was not formed at all, excluding alternative single electron transfer (SET) pathways, such as those via ethynyl(phenyl)-λ 2 -iodanyl radical (Supplementary Fig. S4). 23 In order to obtain more direct information about the generation of C 2 "gas", we designed a connected-flask, solvent-free experiment (Fig. 2C): a solvent-free chemical synthesis of C 2 using 1a with 3 equivalents of CsF was carried out in one of a pair of connected flasks (Flask A), and 3 equivalents of 14 was placed in the other flask (Flask B). The reaction mixture in Flask A was vigorously stirred at room temperature for 72 hours under argon. As the reaction proceeds in Flask A, generated C 2 gas should pass from Flask A to Flask B. Indeed, the color of 14 in Flask B gradually changed from deep purple to deep brown as the reaction progressed. After 72 hours, the formation of 15 and 16 was confirmed by APCI-MS analysis of the residue in Flask B.</p><p>We then performed a 13 C-labeling experiment using 1b- 13 C β , which was synthesized from H 3 13 C-I in 8 steps. 24 Treatment of 1b- 13 C β (99% 13 C) with Bu 4 NF in the presence of 14 in CH 2 Cl 2 gave a mixture of 15-13 C α and 15-13 C β , suggesting that C 2 is generated before the O-ethynyl bond-forming reaction with 14 (Fig. 2D). The observed O-13 C/ 12 C selectivity (71:29) may be related to very fast radical pairing between C 2 and 14 prior to ejection of iodobenzene from the solvent cage. 25 We also carried out 13 C-labeling experiments using 1b- 13 C β in solvents of different viscosities. The observed O-13 C/ 12 C selectivity decreased as the viscosity decreased, and the regioselectivity was almost lost (52:48) under solvent-free conditions. Similarly, the O-13 C/ 12 C selectivity was 51:49 in the connected-flask experiment. All these findings rule out stepwise addition/elimination mechanisms (Supplementary Fig. S5). Given that C 2 generated at room temperature or below behaves exclusively as a A Reaction of 1a with Bu 4 NF in the presence of 9,10-dihydroanthracene (12) B Reaction of 1a with Bu 4 NF in the presence of galvinoxyl free radical ( 14) D 13 C-Labeling experiment using 1b- 13 singlet biradical, as theoretically predicted for the ground state, we examined whether this ground-state C 2 would serve as a molecular element for the formation of various carbon allotropes. Today, sp 2 -carbon allotropes such as graphene, carbon nanotubes (CNT) and fullerenes, in which sp 2 -carbon takes the form of a planar sheet, tube, ellipsoid, or hollow sphere, are at the heart of nanotechnology. 26 But, in contrast with the rapid growth of their practical applications, the mechanisms of their formation remain unclear. Various models and theories for the growth of sp 2 -carbon allotropes have been proposed, most of which include the addition/insertion of C 2 into a growing carbon cluster as a key step. 27,28 However, this idea lacks experimental verification. To investigate this issue, we examined the solvent-free reaction of the present singlet biradical C 2 in order to avoid hydrogen quenching. Notably, simple grinding of CsF and 1.5 equivalents of 1a in a mortar & pestle at ambient temperature for 10 min under an argon atmosphere resulted in the formation of a dark-brown solid containing various sp 2 -carbon allotropes, as determined by resonance Raman spectroscopy (Supplementary Fig. S6), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) (Fig. 3A) and electrospray ionization (ESI) MS (Supplementary Fig. S7). Careful examination of the Raman spectra and high-resolution transmission electron micrograph (HRTEM) images indicated that high-quality graphite with few defects and an interlayer distance of 0.33 nm (Fig. 4A-C) and amorphous carbon had been mostly synthesized (Supplementary Fig S6A), together with very small amounts (<0.0001%) of C 60 and CNTs (ca. 0.5-7 nm in diameter, Fig. 4D and Supplementary Fig. S6B, S7, S8, and S9A), and we did not detect larger fullerenes, such as C 70 , C 76 , C 78 , and C 84 . This specificity may reflect the ambient temperature/pressure condition, as the electric carbon arc method generally affords a fearsome mixture of sp 2 -carbon allotropes. By using 1b- 13 C β , we further confirmed that C 60 is synthesized from C 2 .</p><p>Grinding of 1b- 13 C β with CsF under the same reaction conditions as above afforded C 60 - 13 C 30 , which was detected by means of MALDI-TOF and ESI MS, while non-labeled C 60 was not detected at all (Fig. 3B, Fig. S9B). The formation of this unique fullerene is solid evidence for the role of C 2 , as its occurrence probability in nature is extremely small (0.01) 30 . 1 Swan W. On the prismatic spectra of the flames of compounds of carbon and</p>

ChemRxiv

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

nitric_oxide_photo-donor_hybrids_of_ciprofloxacin_and_norfloxacin:_a_shift_in_activity_from_antimicr

Introduction<!><!>Introduction<!><!>Introduction<!>Design and Synthesis of Novel Ciprofloxacin and Norfloxacin Hybrids<!>Synthetic Strategy for the Synthesis of Compounds 1–3a,b<!>Design and Synthesis of Novel Ciprofloxacin and Norfloxacin Hybrids<!>Synthetic Strategy for the Synthesis of Final Compounds 6a–6d and 7a–7d<!>Spectroscopic and Photochemical Characterization of Novel Ciprofloxacin and Norfloxacin Derivatives<!><!>In Vitro Antitumor Activity Evaluation of Novel Fluoroquinolone Derivatives<!><!>In Vitro Antitumor Activity Evaluation of Novel Fluoroquinolone Derivatives<!><!>In Vitro Antitumor Activity Evaluation of Novel Fluoroquinolone Derivatives<!><!>In Vitro Antitumor Activity Evaluation of Novel Fluoroquinolone Derivatives<!><!>In Vitro Antitumor Activity Evaluation of Novel Fluoroquinolone Derivatives<!><!>In Vitro Antimicrobial Activity on Pseudomonas aeruginosa<!><!>In Vitro Antimicrobial Activity on Pseudomonas aeruginosa<!>Computational Studies<!><!>Computational Studies<!>Conclusions<!>General Remarks<!>General Procedure for the Synthesis of Carboxylic Acids 1a and 1b<!>1-Cyclopropyl-6-fluoro-7-(4-(4-nitro-3-(trifluoromethyl)phenyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (1a)<!>1-Ethyl-6-fluoro-7-(4-(4-nitro-3-(trifluoromethyl)phenyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (1b)<!>General Procedure for the Synthesis of Ciprofloxacin and Norfloxacin Methyl Esters 2a and 2b<!>Methyl 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (2a)<!>Methyl 1-Ethyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (2b)<!>General Procedure for the Synthesis of Methyl Esters 3a and 3b<!>Methyl 1-Cyclopropyl-6-fluoro-7-(4-(4-nitro-3-(trifluoromethyl)phenyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (3a)<!>Methyl 1-Ethyl-6-fluoro-7-(4-(4-nitro-3-(trifluoromethyl)phenyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (3b)<!>2-((4-Nitro-3-(trifluoromethyl)phenyl)amino)ethanol (4a)<!>3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propan-1-ol (4b)<!>General Procedure for the Synthesis of Methanesulfonates 5a and 5b<!>2-((4-Nitro-3-(trifluoromethyl)phenyl)amino)ethyl Methanesulfonate (5a)<!>3-((4-Nitro-3-(trifluoromethyl)phenyl)amino)propyl Methanesulfonate (5b)<!>General Procedure for the Synthesis of Methyl Esters 6a–6d<!>Methyl 1-Cyclopropyl-6-fluoro-7-(4-(2-((4-nitro-3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (6a)<!>Methyl 1-Cyclopropyl-6-fluoro-7-(4-(3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (6b)<!>Methyl 1-Ethyl-6-fluoro-7-(4-(2-((4-nitro-3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (6c)<!>Methyl 1-Ethyl-6-fluoro-7-(4-(3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylate (6d)<!>General Procedure for the Synthesis of Carboxylic Acids 7a–7d<!>1-Cyclopropyl-6-fluoro-7-(4-(2-((4-nitro-3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (7a)<!>1-Cyclopropyl-6-fluoro-7-(4-(3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (7b)<!>1-Ethyl-6-fluoro-7-(4-(2-((4-nitro-3-(trifluoromethyl)phenyl)amino)ethyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (7c)<!>1-Ethyl-6-fluoro-7-(4-(3-((4-nitro-3-(trifluoromethyl)phenyl)amino)propyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (7d)<!>Griess Test<!>Cell Lines and In Vitro Culture Conditions<!>Cell Viability Assay<!>Minimum Inhibitory Concentration Determination<!>Bacterial Binding Assay<!>Antimicrobial Photo-Inactivation Test<!>Molecular Docking<!>Molecular Optimization<!>Molecular Dynamics Simulations<!>MM/PBSA Calculation of the Energies of Binding during the MD Simulation<!><!>Author Contributions<!>

<p>Since their early discovery as byproducts of chloroquine synthesis,1 quinolones have represented one of the most important classes of antibiotics for urinary and respiratory infection treatment.2−4 Quinolones exert their bactericidal activity by interfering with DNA gyrase in Gram-negative bacteria and topoisomerase IV in Gram-positive bacteria.5 Both enzymes belong to the topoisomerase family, which plays an essential role in the regulation of the DNA topological state, in DNA replication, and in the condensation and segregation of chromosomes.6,7 In the presence of quinolone, the enzyme forms a DNA/enzyme/drug ternary complex that perturbs DNA replication, leading to bacterial death or eukaryotic cell apoptosis.</p><p>Structural modifications of the first marketed compound of this class of molecules, nalidixic acid, generated compounds with greater potency, a broader spectrum of activity, improved pharmacokinetics, and lower frequency of acquired resistance. In particular, Norfloxacin (Nor) and Ciprofloxacin (Cip), belonging to the second-generation fluoroquinolones (Figure 1), displayed increased potency and affinity for Gram-negative bacteria due to the introduction of a fluorine atom at position 6. Subsequent structure modifications led to third- and fourth-generation fluoroquinolones, with improved efficacy against Gram-positive organisms.2,8,9 Also, structure–activity relationship (SAR) studies highlighted the importance of the N-1 substituent on the quinolone core together with the presence of either a carboxylic acid function in position 3 and a ketone function in position 4. Furthermore, to expand their spectrum of action and improve pharmacokinetics, a saturated heterocyclic ring containing an amine function was introduced at the 7-position from the second-generation fluoroquinolones.10</p><!><p>Chemical structures of Ciprofloxacin, Norfloxacin, and Vosaroxin.</p><!><p>Of note, recent evidence indicates that higher doses of these drugs exert anticancer effects,11,12 and as expected, this effect is related to their ability to interfere with DNA topoisomerase II, the gyrase human counterpart, which is a well-known target of several anticancer drugs, such as epipodophyllotoxins (etoposide), anthracyclines (doxorubicin and daunorubicin), amsacrine, and mitoxantrone. In this view, we envisioned the design and synthesis of a novel class of 4-quinolone-based topoisomerase II inhibitors endowing the enhancement of their cytotoxic activity.</p><p>SAR studies on anticancer quinolones (Figure 2A,B)4,13−15 allowed identifying the types of structure modifications boosting anticancer activity, such as the reduction of their zwitterionic character by modifying the carboxylic group at the 3-position or by adding a proper substituent on the aliphatic heterocyclic amine at the 7-position. Out of these studies, Vosaroxin16 has reached phase III clinical trial investigation (Figure 1).17 Interestingly, Vosaroxin, such as other molecules of the same class, appears to be devoid of the typical side effects of topoisomerase II inhibitors, namely, significant cardiotoxicity and cross-resistance with other topoisomerase II inhibitors, while preserving the cytotoxic effect in multidrug-resistant (MDR) or inactivated p53 cell lines.13</p><!><p>Antimicrobial (A) and antitumoral (B) structure–activity relationships of 4-quinolones.</p><!><p>The combination of different therapeutic functionalities with synergic or additive features within the same molecular scaffold represents a fascinating opportunity to improve the overall treatment efficacy. Furthermore, the possibility of administering a single drug bearing multiple biological "effectors" could allow higher control on pharmacokinetic and side effects while boosting the treatment outcome.18−22</p><p>In this context, the use of molecules able to release nitric oxide (NO) under the exclusive control of light, e.g., NO photo-donors, has been recently described for antibacterial and anticancer treatment.23,24 NO is physiologically produced by the NO synthase enzyme family from l-arginine and O225 and performs multiple physiological roles ranging from the control of the vascular tone to neurotransmission.26,27 Moreover, NO possesses antimicrobial properties28 and has been shown to be a key player in cancer biology, where its role seems to be regulated by several factors, such as the tumor cell subtype, NO cell sensitivity, exposure time, and cellular concentration.29 In fact, unlike pico- and nanomolar NO concentrations are known to boost cancer progression and invasiveness, micromolar NO concentrations promote cytotoxicity30 and interfere with P-glycoprotein activity,31 thus representing a promising option to tackle MDR phenomena. Due to the intrinsic difficulties posed by the direct administration of gaseous NO, specific NO donors,32 alone or included in nanomaterials, have been actively investigated.33,34 In particular, molecules able to release NO upon the application of an external stimulus, such as light, have attracted increasing attention due to the possibility of precisely controlling the NO production and release only at the site of interest.35−42</p><p>Based on the above, we herein report the design, synthesis, characterization, and molecular modeling studies of 12 Cip and Nor derivatives, endowed with a 4-nitro-3-trifluoromethyl-aniline moiety for the light-triggered release of NO. The new derivatives' biological activity has also been evaluated, both in prokaryotic and tumor cells, along with the effect of NO release on cell viability and compared with Cip and Nor.</p><!><p>The new Cip and Nor derivatives are characterized by a carboxylic or a methyl ester group at the 3-position and a NO photo-donor directly linked to the N-4 atom of the piperazine ring (compounds 1a, 1b, 3a, and 3b) or connected through an alkyl spacer made of two or three methylene units (compounds 6a–6d and 7a–7d) (Table 1).</p><p>The strategy developed for synthesizing final compounds 1a, 1b, 3a, and 3b is depicted in Scheme 1. Carboxylic acids 1a and 1b were prepared following a one-step procedure involving an aromatic nucleophilic substitution between 4-fluoro-1-nitro-2-(trifluoromethyl)benzene and Cip or Nor in DMSO at 120 °C for 1 h. Methyl esters 3a and 3b were synthesized in two steps, including a Fisher esterification between Cip or Nor with refluxing methanol, and p-toluenesulfonic acid (22 h) to afford esters 2a and 2b that were subsequently reacted with 4-fluoro-1-nitro-2-(trifluoromethyl)benzene in refluxing acetonitrile overnight to provide final compounds 3a and 3b.</p><!><p>Reagents and conditions: (i) 4-fluoro-1-nitro-2-(trifluoromethyl)benzene, DMSO, 120 °C, 1 h; (ii) CH3OH, p-TsOH, 70 °C, 22 h; (iii) 4-fluoro-1-nitro-2-(trifluoromethyl)benzene, CH3CN, 80 °C, overnight.</p><!><p>Scheme 2 reports the synthetic pathway to achieve final compounds 6a–6d and 7a–7d. Starting from 4-fluoro-1-nitro-2-(trifluoromethyl)benzene, intermediates 4a and 4b were prepared through an aromatic nucleophilic substitution with 2-aminoethanol or 3-aminopropan-1-ol in acetonitrile at 60 °C overnight. Mesylation of the alcoholic function of compounds 4a and 4b provided the intermediates 5a and 5b that were reacted with derivatives 2a and 2b in refluxing acetonitrile overnight. The obtained methyl esters 6a–6d were hydrolyzed with a refluxing NaOH aqueous solution (2 M) for 24 h to afford the corresponding carboxylic acids 7a–7d.</p><!><p>Reagents and conditions: (i) 1-aminoethanol or 1-aminopropan-3-ol, CH3CN, 60 °C, overnight; (ii) CH3SO2Cl, TEA, dry CH2Cl2, 0 °C, then room temperature, 1 h; (iii) 2a and 2b, CH3CN, reflux, overnight; (iv) aqueous NaOH 2 M, reflux, 24 h.</p><!><p>To investigate the spectroscopic behavior of the synthesized compounds, carboxylic acids 1a, 1b, and 7a–7d were selected for recording absorption and fluorescence spectra, showing the characteristic peaks at 400 and 450 nm, respectively (Figure S25). The amount of NO released from the selected compounds upon light irradiation was quantified using the Griess assay, in which NO2, generated upon the reaction of released NO with oxygen, reacts with the Griess reagent, generating a purple azo dye that can be spectroscopically monitored following its absorption peak at ∼540 nm.43 Therefore, aqueous solutions of compounds 1a, 1b, and 7a–7d (80 μM) were treated with the Griess reagent and irradiated with a white lamp for different time intervals (15 min, 1 h, and 2 h). All performed analyses showed the development of a light purple coloration, confirming a weak NO release, especially after 1 h irradiation time. Although the absorption peak of the analyte solution may weakly skew absorbance measurements at 540 nm, the obtained data indicate a trend in NO production; in particular, the amount of NO released seems to be in the order 7b > 1b > 7d > 1a = 7a > 7c. Furthermore, the nitrite concentration was quantified by measuring the absorbance at 540 nm with respect to a standard curve of NaNO2 in H2O. Data reported in Figure 3 confirmed a significant nitrite production by 7b, 1b, and 7d following 1 h irradiation, particularly evident for 7b ([NO2–] = 6.2 μM), while the extent of NO2 generated by other compounds was almost negligible. Except for compound 1b, these results suggest that longer spacers between the piperazine ring and the NO-donor moiety might favor the NO production yield.</p><!><p>NO detection by a Griess test. Effects of 80 μM solution of 1a, 1b, and 7a–7d on NO production after 1 h of white light irradiation (mean ± SD of three independent experiments). Nitrite concentration was determined by comparing the test samples' absorbance values to a standard curve generated by serial dilution of 50 μM NaNO2.</p><!><p>The effect of the novel hybrid derivatives on cellular viability of a panel of tumor cell lines of different tissue origins (DU145 and PC3: prostate; MCF-7, MCF7/ADR, and MDA-MB231: breast; HCT116: colon) was investigated through the MTT assay following 3 days treatment with the compounds. Histograms reported in Figures 4 and 5 represent the IC50 values extrapolated from the corresponding concentration–response curves. In each graph, IC50 values of new derivatives were compared to the reference compounds, e.g., Cip or Nor. All compounds showed effects at micromolar concentrations (Tables S1 and S2).</p><!><p>IC50 values obtained following 72 h treatment with Ciprofloxacin (C) and its derivatives in the MTT assay (mean ± ES 4/5 independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001 vs Cip).</p><p>IC50 values obtained following 72 h treatment with Norfloxacin (N) and its derivatives in the MTT assay (mean ± ES 4/5 independent experiments; °p < 0.05, °°p < 0.01, °°°p < 0.001 vs Nor).</p><!><p>Figure 4 shows that all Cip derivatives were significantly more potent than the parent compound in DU145 and PC3 prostate cancer cells in HCT116 colorectal cancer cells (the more potent being compound 7b, with an IC50 = 1.83 μM) and in MDA-MB231 breast cancer cells (IC50 values lower than 2.50 μM for compounds 7a and 7b) while only compounds 7a and 7b were more potent than Cip in MCF7 cells (Table S1). In general, carboxylic acids 7a and 7b, where the alkyl linker connects the fluoroquinolone scaffold to the NO photo-donor moiety, showed lower IC50 values when compared to their corresponding methyl esters 6a and 6b. Specifically, this trend was more pronounced for compound 7a, which was 2- to 7-fold more potent than 6a in the tested cell lines. In contrast, the difference in potency was lower when comparing 6b and 7b. These data indicate that a shorter alkyl bridge improves the cytotoxic effect (7avs7b). On the other hand, results obtained for methyl esters 6a and 6b suggest that a longer alkyl chain provides better cytotoxic properties. Finally, compounds lacking the alkyl bridge (1a and 3a) displayed an ambiguous trend on the tested cell lines. Indeed, DU145 and HCT116 cell lines were more sensitive to the methyl ester 3a, whereas the PC3 and MDA-MB231 cell lines displayed a slight susceptibility toward the carboxylic acid 1a.</p><p>A similar trend was observed for the SAR of the novel Nor hybrids, except for the better activity of carboxylic acid 1b in all the tested cell lines when compared to its methyl ester analog 3b. In particular, results obtained in DU145 and PC3 cells showed that all Nor derivatives were significantly more potent than the reference compound, with best results obtained for 7d in the DU145 cell line (IC50 = 1.56 μM) and 7c in the PC3 cell line (IC50 = 2.33 μM). In MCF7 and MDA-MB231 cells, compounds 1b and 7d showed a significantly lower IC50 than Nor, while 7c was the most potent among all the novel hybrids in both breast cancer cell lines (IC50 = 2.27 and 1.52 μM for MCF7 and MDA-MB231, respectively). Interestingly, in HCT116 cells, only 7c and 7d resulted to be more potent than Nor (Figure 5 and Table S2).</p><p>Last, the effects of all the newly synthesized compounds on cell viability were not limited to cancer cell lines; indeed, as shown on Table 2, which report the IC50 values obtained by the MTT assay following 72 h treatment with the compounds, also the non-tumorigenic HBL100 and the WH1 fibroblast cell lines responded to Cip, Nor, and to their derivatives at similar extent compared to cancer cell lines (Table 2).</p><!><p>Mean ± ES 4/5 independent experiments.</p><p>p < 0.05 vs Nor.</p><!><p>To evaluate if NO released upon light irradiation could affect cell viability, the MTT assay was performed on DU145 and PC3 cells following treatment with two compounds that showed interesting potency against the two prostate cancer cell lines and different degrees of NO release. Specifically, the Cip and the Nor derivative that showed to develop the highest and the lowest NO levels by the Griess assay, namely, 7b and 7c, respectively, were selected for this experiment. Treatment was followed by 1 h irradiation with white or blue light and 48 h incubation in a drug-free medium. As shown in Figure 6, no differences in IC50 values were observed in cells treated with 7b and 7c and irradiated (both with white or blue light) with respect to cells treated and kept in the dark. However, compound 7c seems to be more active on DU145 cells when irradiated under blue light with respect to white light.</p><!><p>IC50 values obtained following 24 h treatment with compound 7b or 7c with 1 h irradiation and 48 h incubation in the drug-free medium and MTT assay (mean ± ES 3/4 independent experiments. *p < 0.05 vs white light).</p><!><p>Overall, these results might indicate that the amount of NO released could be inadequate to induce cell death, thus confirming that the NO release does not play an additive role in the new fluoroquinolone derivatives' toxic effect.</p><p>A significant obstacle to the successful chemotherapeutic treatment of tumors is their inadequate response to anticancer drugs due to intrinsic or acquired resistance phenomena. Tumor cells can be insensitive to drug treatment at the therapy onset (intrinsic resistance), or they can initially respond to anticancer agents, becoming refractory on subsequent treatment cycles (acquired resistance). For instance, the development of MDR following an initial drug response severely limits the success of doxorubicin (DOX) treatment of breast cancers.44,45 DOX is an anthracycline antibiotic targeting topoisomerase II and represents a mainstay in clinical management of early-stage and metastatic breast cancer. MDR to DOX involves few biochemical alterations, such as reduced drug accumulation, increased detoxification, increased DNA repair, topoisomerase II alterations, or in cell cycle regulation. In this scenario, identifying MDR-modulating agents or drugs able to escape MDR phenomena is of utmost importance in anticancer research.</p><p>As expected, the DOX-resistant MCF7/ADR cell line, obtained by selecting MCF7 cells exposed to increasing DOX concentrations, is significantly less sensitive to anthracycline treatment with respect to the wild-type cell line, with a resistance index (R.I.) of 22 (Figure 7). Interestingly, MCF7/ADR cells showed similar sensitivity to several Cip and Nor derivatives (Table 3) as observed for the MCF7 cell line, with the best IC50 values obtained for carboxylic acids 1a and 1b (8.72 and 5.63 μM, respectively).</p><!><p>IC50 values obtained following 72 h treatment with DOX and the MTT assay (mean ± ES 4/5 independent experiments; ***p < 0.001 vs MCF7).</p><p>Mean ± ES 4/5 independent experiments.</p><p>p < 0.05 vs reference compound.</p><!><p>In summary, the in vitro experiments showed that derivatives 7a and 7b are the most effective Cip analogs against all considered cell lines and with respect to the reference compound, indicating that regardless of the linker length, the carboxylic acid moiety outperforms the ester function (Figure 8). In line with these results, Nor derivatives 7c and 7d displayed a similar trend, and compound 7c was the best performing of the series (Figure 8). Cytotoxicity studies performed on MCF7/ADR resistant cell lines confirmed that the carboxylic moiety is essential for the anticancer activity and that only derivatives 1a and 1b, where the p-nitro-trifluoromethyl aniline is directly linked to the fluoroquinolone scaffold, can provide IC50 values in the low micromolar range (Figure 8).</p><!><p>Summary of the most potent compounds synthesized in this work with their IC50 values on the tested cancer cell lines.</p><!><p>Considering that fluoroquinolones, such as Cip and Nor, are among the few antibiotics used to control the growth of P. aeruginosa, an opportunistic pathogen representing one of the most relevant agents of nosocomial infections, we preliminarily determined whether the novel derivatives could also be effective against the model strain PAO1. Our results indicate that, while Cip and Nor had minimal inhibitory concentration (MIC) values of 1.82 ± 1.19 and 4.16 ± 1.80 μg/mL, respectively, none of our newly designed derivatives showed antimicrobial activity comparable or better than reference compounds (MIC ∼25 μg/mL). These results might indicate that the fluoroquinolone scaffold's structural changes compromise the antimicrobial activity, probably due to an impaired interaction with the microbial cell wall and/or with the bacterial target, e.g., the DNA gyrase. To rule out the first hypothesis, we investigated the ability of our novel compounds to bind the PAO1 cell wall. Binding experiments showed that upon incubation of P. aeruginosa PAO1 cells with our fluoroquinolones, a decrease in absorbance was observed in the supernatants (Figures S26 and S27), indicating good binding rates with the outer layer of the cell wall. However, it should be underlined that a good interaction between the molecules and the bacterial cell wall is not predictive of an effective uptake.</p><p>Since most compounds showed an absorption peak in the 390–395 nm region, we next investigated their activity upon irradiation with blue light. For a preliminary evaluation, we selected compound 7c, showing the highest absorption peak in the violet-blue range and some degree of activity against DU145 cancer cells. Upon irradiation with a light-emitting diode at 405 ± 10 nm and a fluence not toxic to cells (20 J/cm2), compound 7c did not show any antimicrobial activity against P. aeruginosa PAO1 (Figure 9a), indicating that despite its interaction with the cell wall, this is not sufficient to induce killing by photo-oxidative stress.</p><!><p>Photodynamic treatment of P. aeruginosa PAO1 (a) and S. aureus ATCC 6538P (b). Bacteria were incubated in the dark with or without 7c (10 μM) for 10 min and then irradiated under blue light (20 J/cm2). Dark controls were not irradiated. Viable cells are expressed as CFU/mL. Values represent the mean of at least three independent experiments, *p < 0.05.</p><!><p>It is well established that one of the major challenges in treating Gram-negative bacteria, with respect to the Gram-positive ones, is the difficulty of antimicrobial agents to strongly bind and cross their cell wall, which significantly differs in composition as compared to the latter. In order to shed light on the specific behavior of our fluoroquinolones, we then evaluated the effectiveness of compound 7c in killing Gram-positive Staphylococcus aureus. Interestingly, the photoactivation of 7c caused a significant (p < 0.05) 2 log unit decrease in S. aureus with respect to the dark incubated control (Figure 9b). This observation might support the hypothesis that compound 7c is able to efficiently bind the cell wall and probably cross the cytoplasmic membrane of S. aureus, ultimately inducing a photo-oxidative stress at the cell-wall level and/or at the cytoplasmic level, thus causing cellular death upon light irradiation. This result indirectly further demonstrates that despite the fact that our fluoroquinolones bind the cell wall of P. aeruginosa, this is not sufficient to induce bacterial killing by photoactivation. The selective activity of derivative 7c toward Gram-positive bacteria indicates that our derivatives act as neutral antimicrobial photosensitizers (aPSs). In fact, it has been reported that neutral photosensitizers are usually active toward Gram-positive microorganisms and not in the Gram-negative ones, being able to overcome the murein barrier of the first, but not the outer membrane of the cell wall of the latter.46 Based on the observed selectivity of 7c, it could be hypothesized that despite the fact that molecular modeling studies (see the following paragraph) account for the effective binding of fluoroquinolones with both topoisomerase II and gyrase, their biological activity against P. aeruginosa is hampered by an ineffective uptake. In the future, this could be overcome by incorporating these molecules within suitably designed carriers.</p><!><p>Molecular modeling studies were carried out to investigate the interactions with the reference cellular and bacterial targets. The calculated free energies of binding (ΔG) to the catalytic site of the human topoisomerase IIα (Topo IIα), bacterial topoisomerase IIA (Topo IIA), and bacterial gyrase (Gyr) for the novel compounds are reported in Table 4. First, the crystal structure of the Topo IIα isoform (PDB ID: 5GWK) was used for docking studies.47</p><p>All compounds displayed better in silico affinity toward Topo IIα than Cip and Nor, while DOX showed greater affinity, as demonstrated by experimental data. The Cip derivatives have lower free energies of binding than the Nor derivatives, except for compounds 6d and 7d. The 1a carboxyl group coordinates Mg2+ via a salt bond of 1.73 Å (Figure 10A). Furthermore, the complex is stabilized, within the catalytic site, by the H-bond with the Gly760 and Asp541 backbone, while the nitro group establishes electrostatic interactions with Arg487 (Figure 10B).</p><!><p>3D superposition of the best-docked pose for 1a bound to the Topo IIα (A) and binding site interactions (B). RMSD superposing on the receptor (C) and starting structure (D).</p><!><p>The protein aids the anchoring of the ligand within the pocket by a cation-π link formed between Arg487 and the nitro group portion of the ligand. The neighboring DNA bases also contribute to the stabilization of the complex.</p><p>Once the general interaction model was established, equilibrium molecular dynamics (MD) simulations (100 ns) were performed to analyze the Topo IIα/DNA/1a ternary model system's evolution and stability. The RMSDs of the tertiary structures (Topo IIα/DNA/1a) compared to the first ones at time 0 were analyzed and plotted during the 100 ns MD simulation (Figure 10C). The overall RMSD for the protein system appeared to have reached the equilibrium after 10 ns (Figure 10D), and the stabilization of the protein–ligand complex was reached after 7 ns, keeping the complex's extensive hydrogen-bonding network constant. The energies of binding, calculated by the Molecular Mechanics Poisson–Boltzmann Surface Area (MM/PBSA) methodology (see the Experimental Section), including the time average, along MD simulation trajectories were employed to assess the strength of the interactions between the ligand and the binding pocket in the dynamic environment. Compound 1a shows a stable fluctuation that settles after the first 10 ns and records an average value of −17.4 kcal/mol in the remaining 90 ns (Figure S28).</p><p>The docked laying of 1b is very similar to that of 1a despite a loss of 0.8 kcal/mol, probably due to the ethyl group's worse stacking. It is interesting to note that compounds 7a, 7b, and 7d have lower calculated free energies of binding than other compounds against human Topo IIα. Indeed, these compounds have shown excellent results in the tested cancer cell lines.</p><p>All the novel compounds were anchored in the active sites of Topo IIA (PDB ID: 2XCT) of S. aureus and DNA gyrase B (PBD ID: 6MS1) of P. aeruginosa. Anchored poses with the lowest bond energy, hydrogen bonds, noncovalent interactions, such as the π–π interactions, and the details of the π-cationic interactions were recorded and validated.</p><p>All the compounds analyzed showed better in silico affinity on bacterial topoisomerase than Cip and Nor, except compound 3a, while all compounds showed lower in silico activity against DNA gyrase. Despite the good in silico results, the compounds have a worse antimicrobial activity in vitro than the reference drugs (Cip and Nor). It seems that the novel compounds bind to the outer membrane of the Gram-negative cell wall, but the crossing of the cytoplasmic membrane to reach the cytoplasmic environment and then the molecular target could represent the issue to overcome. Thus, further investigations are needed to shed light on the observed impairment of novel derivatives' antimicrobial activity.</p><!><p>In this work, we successfully managed to design and synthesize twelve novel Cip and Nor derivatives endowed with a NO photo-donor moiety. The light-triggered release of NO has been demonstrated by spectroscopic and photochemical studies, showing the release of this gasotransmitter in the micromolar range, especially for compound 7b. Docking studies confirmed that these novel chemical entities effectively bind to both bacterial and human topoisomerases, with better calculated free binding energies with respect to the parent compounds. P. aeruginosa PAO1 was not sensitive to the novel derivatives, and photoactivation experiments support the hypothesis that this could be ascribable to an inefficient uptake.</p><p>As far as anticancer activity is concerned, all novel fluoroquinolone derivatives displayed strong anticancer potency on a panel of different cancer cell lines, which was especially remarkable for compounds 7a–7d. On the other hand, the light-triggered release of NO from compounds 7b and 7c did not grant an additional cytotoxic effect on PC3 and DU145 prostate cancer cell lines, although a better response to the compounds was observed following blue light irradiation with respect to white light irradiation. Further studies focused on the precise mechanism of action of these compounds and on the role of NO in these cell lines are in progress.</p><p>Importantly, our data showed that some of the tested compounds, including compounds 1a, 1b, and 7a–7d, exhibit cytotoxic effects on MDA-MB231 cells, which are representative of triple-negative breast cancer (TNBC), one of the most aggressive and refractory forms of breast cancer. TBNC does not respond to endocrine therapy or other currently available targeted agents;48−50 thus, alternative therapeutic options that can selectively address this tumor subset are urgently needed. In this view, the promising results obtained with our novel derivatives on MDA-MB231 cells represent an encouraging starting point for developing and optimizing more effective treatment. Furthermore, compounds 1a and 1b also displayed a strong cytotoxic effect on DOX-resistant MCF7/ADR breast cancer cells, making these hybrids promising candidates for MDR breast cancer treatment. As expected, most of our fluoroquinolone derivatives displayed a certain extent of toxicity on healthy cells; this issue could be overcome in the future by encapsulating those molecules within suitably designed delivery systems.</p><!><p>Reagent-grade chemicals were purchased from Sigma-Aldrich or Fluorochem and were used without further purification. All reactions were monitored by thin-layer chromatography (TLC) performed on silica gel Merck 60 F254 plates; the spots were visualized by UV light (λ = 254 and 366 nm) and an iodine chamber. Melting points were determined on a Büchi B-450 apparatus in capillary glass tubes and are uncorrected. Flash chromatography purification was performed on a Merck silica gel 60, 0.040–0.063 mm (230–400 mesh) stationary phase using glass columns with a diameter between 1 and 4 cm. Nuclear magnetic resonance spectra (1H NMR and 13C NMR recorded at 500 and 125 MHz, respectively) were obtained on Varian INOVA spectrometers using CDCl3, acetone-d6, CD3OD, and DMSO-d6 with a 0.03% of TMS as an internal standard. Coupling constants (J) are reported in hertz. Signal multiplicities are characterized as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad), and app (apparent). Purities of all compounds were ≥95% as determined by microanalysis (C, H, and N) that was performed on a Carlo Erba instrument model E1110; all the results agreed within ±0.4% of the theoretical values.</p><!><p>To a suspension of Cip or Nor (0.100 g, 0.3 mmol) in 5 mL of DMSO was added 4-fluoro-1-nitro-2-(trifluoromethyl)benzene (0.063 g, 0.3 mmol) under stirring. The color of the suspension immediately turned yellow. The reaction was carried out in a sealed Pyrex vial at 120 °C for 60 min. After cooling to room temperature, the product was precipitated with deionized water and decanted. The solid yellow residue was repeatedly washed in sequence with deionized water, isopropanol, and diethyl ether. The obtained solid was dried under N2 and did not require any further purification. According to this procedure, the following products have been obtained.</p><!><p>Yellow solid (89%): mp 296–298 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.67 (s, 1H), 8.13 (d, J = 9.5 Hz, 1H), 7.94 (d, J = 13.5 Hz, 1H), 7.59 (d, J = 7.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 9.5, 2.5 Hz, 1H), 3.86–3.82 (m, 1H), 3.78 (t, J = 5.0 Hz, 4H), 3.54 (t, J = 4.8 Hz, 4H), 1.32 (q, J = 7.0 Hz, 2H), 1.22–1.19 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 176.35, 165.92, 152.89, 148.10, 144.59 (d, JCF = 9.9 Hz), 139.19, 135.59, 128.85, 124.33, 121.36, 118.59, 115.29, 111.67 (d, JCF = 5.1 Hz), 111.08 (d, JCF = 22.3 Hz), 106.76, 106.18, 48.53, 45.96, 35.87, 7.60. Anal. Calcd. for C24H20F4N4O5: C, 55.39; H, 3.87; N, 10.77. Found: C, 55.42; H, 3.89; N, 10.74.</p><!><p>Yellow solid (74%): mp 310–312 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.97 (s, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.97 (d, J = 13.0 Hz, 1H), 7.36 (s, 1H), 7.31 (d, J = 9.0 Hz, 1H), 7.22 (d, J = 7.0 Hz, 1H), 4.61 (q, J = 7.0 Hz, 2H), 3.77 (br s, 4H), 3.53 (br s, 4H), 1.43 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 176.25, 166.06, 153.06, 148.58, 144.86, 137.19, 135.77, 128.80, 124.35, 119.81, 119.32, 117.47, 115.32, 111.68 (d, JCF = 6.25 Hz), 111.42 (d, JCF = 28.7 Hz), 105.78, 48.64, 46.06, 41.00, 14.29. Anal. Calcd. for C23H20F4N4O5: C, 54.33; H, 3.96; N, 11.02. Found: C, 54.50; H, 3.94; N, 10.99.</p><!><p>To a warmed suspension of the starting fluoroquinolone (1.00 g, 3.00 mmol) in 100 mL of CH3OH was dropped p-toluenesulfonic acid (3.00 g, 15.7 mmol) dissolved in 5 mL of CH3OH via a syringe. The resulting solution was refluxed for 22 h. The reaction mixture was then cooled to room temperature, and the reaction solvent was removed under vacuum. To the resulting yellow oil was added a saturated solution of Na2CO3 (50 mL), and the aqueous phase was extracted with CH2Cl2 (3 × 50 mL). The organic phase was dried over anhydrous Na2SO4, filtered, and concentrated at reduced pressure. The crude was purified by flash chromatography using a CH2Cl2/CH3OH gradient eluting system. According to this procedure, the following products have been obtained.</p><!><p>White solid (91%): mp 230–233 °C; 1H NMR (500 MHz, CDCl3): δ 8.54 (s, 1H), 8.02 (d, J = 13.3 Hz, 1H), 7.29–7.27 (m, 1H), 3.91 (s, 3H), 3.46–3.42 (m, 1H), 3.29–3.27 (m, 4H), 3.15–3.13 (m, 4H), 2.40 (s, 2H), 1.33 (t, J = 6.5 Hz, 2H), 1.14 (q, J = 6.5 Hz, 2H); 13C NMR (125 MHz, CDCl3): δ 173.24, 166.61, 153.61 (d, JCF = 247.5 Hz), 148.46, 145.15 (d, JCF = 10.0 Hz), 138.18, 123.14 (d, JCF = 7.5 Hz), 113.46 (d, JCF = 22.5 Hz), 110.21, 104.87, 52.20, 51.19, 46.06, 34.65, 8.28. Anal. Calcd for C18H20FN3O3: C, 62.60; H, 5.84; N, 12.17. Found: C, 62.78; H, 5.86; N, 12.19.</p><!><p>White solid (92%): mp 189–190 °C; 1H NMR (500 MHz, CDCl3): δ 8.42 (s, 1H), 8.06 (d, J = 13.0 Hz, 1H), 6.73 (d, J = 7.0 Hz, 1H), 4.20 (q, J = 7.5 Hz, 2H), 3.92 (s, 3H), 3.22–3.21 (m, 4H), 3.10–3.08 (m, 4H), 1.92 (s, 2H), 1.54 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, CDCl3): δ 173.15, 166.72, 153.39 (d, JCF = 238.0 Hz), 148.30, 145.36 (d, JCF = 10.5 Hz), 136.22, 123.87 (d, JCF = 6.6 Hz), 113.80 (d, JCF = 22.9 Hz), 110.26, 103.85, 52.17, 51.36, 49.10, 46.09, 14.50. Anal. Calcd for C17H20FN3O3: C, 61.25; H, 6.05; N, 12.61. Found: C, 61.32; H, 6.06; N, 12.58.</p><!><p>To a solution of the appropriate fluoroquinolone methyl ester (2a or 2b) in anhydrous CH3CN (10 mL) at 40 °C was added 4-fluoro-1-nitro-2-(trifluoromethyl)benzene under stirring. The color of the solution immediately turned yellow. The temperature was raised up to 80 °C, and the reaction mixture was left under stirring overnight. The solvent was evaporated, and the crude was diluted with CH2Cl2 (50 mL) and washed with deionized water (3 × 25 mL). The organic phase was dried over Na2SO4, filtered, and reduced under vacuum. The residue was purified by flash chromatography using a CH2Cl2/CH3OH gradient eluting system. According to this procedure, the following products have been obtained.</p><!><p>The title compound was obtained using 0.265 g (0.77 mmol) of 2a and 0.172 g (0.82 mmol) of 4-fluoro-1-nitro-2-(trifluoromethyl)benzene. Yellow solid (61%): mp 265–267 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.12 (d, J = 9.5 Hz, 1H), 7.78 (d, J = 13.5 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 9.5, 2.5 Hz, 1H), 3.77–3.74 (m, 4H), 3.73 (s, 3H), 3.68–3.63 (m, 1H), 3.44–3.42 (m, 4H), 1.27–1.23 (m, 2H), 1.13–1.10 (m, 2H); 13C NMR (125 MHz, CDCl3): δ 173.12, 166.52, 153.44 (d, JCF = 247.5 Hz), 153.15, 148.65, 143.88 (d, JCF = 12.5 Hz), 138.15 (d, JCF = 10.0 Hz), 128.70, 126.46 (d, JCF = 33.8 Hz), 123.9 (d, JCF = 7.5 Hz), 123.47, 121.29, 115.41, 113.91 (d, JCF = 22.5 Hz), 112.72 (d, JCF = 6.3 Hz), 110.56, 105.03, 52.30, 49.54, 47.29, 34.67, 8.36. Anal. Calcd. for C25H22F4N4O5: C, 56.18; H, 4.15; N, 10.48. Found: C, 56.31; H, 4.13; N, 10.51.</p><!><p>The title compound was obtained using 0.500 g (1.5 mmol) of 2b and 0.314 g (1.5 mmol) of 4-fluoro-1-nitro-2-(trifluoromethyl)benzene. Yellow solid (52%): mp 236–238 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.65 (s, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 13.0 Hz, 1H), 7.37 (d, J = 2.5 Hz, 1H), 7.31 (dd, J = 9.5, 3.0 Hz, 1H), 7.08 (d, J = 7.0 Hz, 1H), 4.42 (q, J = 7.0 Hz, 2H), 3.76–3.74 (m, 7H), 3.43–3.41 (m, 4H), 1.38 (t, J = 7.5 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 171.46, 165.10, 153.28, 152.93, 151.31, 148.85, 143.63 (d, JCF = 10.0 Hz), 136.16, 135.54, 128.83, 124.59, 123.54, 122.74 (d, JCF = 6.3 Hz), 115.34, 111.92 (d, JCF = 22.5 Hz), 111.72 (d, JCF = 6.3 Hz), 109.15, 105.88, 51.16, 48.86, 48.05, 46.17, 14.23. Anal. Calcd. for C24H22F4N4O5: C, 55.17; H, 4.24; N, 10.72. Found: C, 55.28; H, 4.25; N, 10.75.</p><!><p>To a solution of 2-aminoethanol (1.00 g, 16.4 mmol) in anhydrous CH3CN (10 mL) was added 4-fluoro-1-nitro-2-(trifluoromethyl)benzene (2.32 g, 11.1 mmol). The reaction was left under stirring at 60 °C overnight. Then, the reaction solvent was removed under reduced pressure and the residue was dissolved in EtOAc and washed with a saturated solution of NaHCO3 (3 × 25 mL). The organic phase was dried over Na2SO4, filtered, and concentrated under vacuum. The crude was purified by flash chromatography eluting with a 20% of Cy in EtOAc to give the desired product. Yellow solid (72%): 1H NMR (500 MHz, CD3OD): δ 8.01 (d, J = 9.0 Hz, 1H), 7.04 (d, J = 2.5 Hz, 1H), 6.81 (dd, J = 9.0, 2.5 Hz, 1H), 3.74 (t, J = 5.5 Hz, 2H), 3.35 (t, J = 5.5 Hz, 2H); 13C NMR (125 MHz, CD3OD): δ 154.65, 136.41, 130.34, 127.27 (t, JCF = 18.4 Hz), 125.08, 122.92, 113.05, 112.26, 61.15, 46.26. Anal. Calcd. for C9H9F3N2O3: C, 43.21; H, 3.63; N, 11.20. Found: C, 43.12; H, 3.64; N, 11.22.</p><!><p>To a solution of 3-aminopropan-1-ol (0.143 g, 1.90 mmol) in anhydrous CH3CN (5 mL) was added 4-fluoro-1-nitro-2-(trifluoromethyl)benzene (0.200 g, 0.95 mmol). The reaction was left under stirring at 60 °C for 12 h in a closed glass Pyrex vial. After the reaction was complete, the reaction solvent was removed under reduced pressure and the residue was repeatedly triturated with n-hexane till the formation of a yellow solid. The solid was decanted, solubilized in EtOAc, and washed with a saturated solution of NaHCO3 (3 × 25 mL). The organic phase was dried over Na2SO4, filtered, and reduced under pressure, affording the pure desired product that did not require any further purification. Yellow solid (quantitative): 1H NMR (500 MHz, acetone-d6): δ 8.03 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 3.0 Hz, 1H), 6.88 (dd, J = 6.5, 3.0 Hz, 1H), 6.67 (br s, 1H), 3.82–3.68 (m, 3H), 3.40 (q, J = 6.5 Hz, 2H), 1.90–1.85 (m, 2H); 13C NMR (125 MHz, acetone-d6): δ 154.19, 136.02, 130.26, 126.74 (t, JCF = 38.3 Hz), 123.68 (d, JCF = 270.8 Hz), 112.00 (d, JCF = 145.5 Hz), 60.01, 40.97, 29.45. Anal. Calcd. for C10H11F3N2O3: C, 45.46; H, 4.20; N, 10.60. Found: 45.33; H, 4.21; N, 10.63.</p><!><p>To a solution of the appropriate alcohol (4a or 4b) in anhydrous CH2Cl2 was added TEA at 0 °C. The reaction mixture was left under stirring for 30 min, and then methanesulfonyl chloride was added dropwise using a dropping funnel. The reaction was left under stirring at room temperature for 60 min. After this time, the solvent was removed under reduced pressure, the resulting residue was dissolved in CH2Cl2 and washed in sequence with a saturated solution of NH4Cl (25 mL) and a saturated solution of NaHCO3. The organic phase was dried over Na2SO4, filtered, and evaporated. The crude products were directly used in the next step with no further purification or characterization. According to this procedure, the following products have been synthesized.</p><!><p>The title compound was obtained using 0.100 g (0.4 mmol) of 4a in 5 mL of anhydrous CH2Cl2, 166 μL (1.2 mmol) of TEA, and 62 μL (0.8 mmol) of methanesulfonyl chloride. Yellow oil (quantitative).</p><!><p>The title compound was obtained using 0.300 g (1.13 mmol) of 4b in 10 mL of anhydrous CH2Cl2, 0.345 g (3.40 mmol) of TEA, and 0.328 g (2.26 mmol) of methanesulfonyl chloride. Yellow oil (quantitative).</p><!><p>In a two-neck round-bottom flask, the appropriate fluoroquinolone methyl ester (2a or 2b) was added to the appropriate methanesulfonate solution (5a or 5b) in anhydrous CH3CN under a N2 atmosphere. The reaction mixture was refluxed under stirring overnight. Then, the solvent was removed under reduced pressure, and a saturated solution of NaHCO3 was added to the residue. The aqueous phase was extracted with CH2Cl2 (3 × 25 mL), and then the organic phases were dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by flash chromatography using a CH2Cl2/CH3OH gradient eluting system. According to this procedure, the following products have been obtained.</p><!><p>The title compound was synthesized using 0.276 g (0.8 mmol) of 2a in 15 mL of anhydrous CH3CN and 0.129 g (0.39 mmol) of 5a. Yellow solid (69%): mp 231–233 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.08 (d, J = 9.5 Hz, 1H), 7.75 (d, J = 13.0 Hz, 1H), 7.47 (t, J = 5.0 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.16 (s, 1H), 6.88 (dd, J = 9.0, 2.5 Hz, 1H), 3.73 (s, 3H), 3.66–3.62 (m, 1H), 3.37 (app. q, J = 6.0 Hz, 2H), 3.28–3.24 (m, 4H), 2.70–2.65 (m, 4H), 2.63 (t, J = 6.0 Hz, 2H), 1.26–1.24 (m, 2H), 1.11–1.08 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 171.52, 164.95, 153.57, 152.38 (d, JCF = 193.0 Hz), 148.22, 143.85 (d, JCF = 9.6 Hz), 138.05, 133.55, 129.70, 124.82 (d, JCF = 32.9 Hz), 123.63, 121.79 (d, JCF = 6.1 Hz), 121.46, 111.50 (d, JCF = 22.6 Hz), 108.98, 106.18, 55.97, 52.42, 51.23, 49.52, 34.71, 7.51. Anal. Calcd. for C27H27F4N5O5: C, 56.15; H, 4.71; N, 12.13. Found: C, 55.94; H, 4.70; N, 12.17.</p><!><p>The title compound was synthesized using 0.350 g (1.01 mmol) of 2a in 15 mL of anhydrous CH3CN and 0.195 g (0.57 mmol) of 5b. Yellow solid (50%): mp 202–204 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.44 (s, 1H), 8.08 (d, J = 9.5 Hz, 1H), 7.75 (d, J = 13.5 Hz, 1H), 7.59 (t, J = 5.5 Hz, 1H), 7.43 (d, J = 7.5 Hz, 1H), 7.07 (s, 1H), 6.85 (dd, J = 9.5, 2.5 Hz, 1H), 3.73 (s, 3H), 3.67–3.62 (m, 1H), 3.29–3.23 (m, 6H), 2.61–2.56 (m, 4H), 2.46 (t, J = 7.0 Hz, 2H), 1.77 (m, 2H), 1.27–1.23 (m, 2H), 1.11–1.08 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 171.53, 164.96, 152.42 (d, JCF = 203.8 Hz), 148.22, 143.88 (d, JCF = 10.0 Hz), 138.06, 133.38, 129.77, 125.02, 123.62, 121.77 (d, JCF = 6.3 Hz), 121.45, 111.50 (d, JCF = 22.5 Hz), 108.99, 106.16, 54.87, 52.48, 51.24, 49.57, 40.42, 34.70, 25.40, 7.51. Anal. Calcd. for C28H29F4N5O5: C, 56.85; H, 4.94; N, 11.84. Found: C, 57.01; H, 4.95; N, 11.81.</p><!><p>The title compound was synthesized using 0.400 g (1.2 mmol) of 2b in 15 mL of anhydrous CH3CN and 0.197 g (0.6 mmol) of 5a. Yellow solid (61%): mp 245–247 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.64 (s, 1H), 8.08 (d, J = 9.5 Hz, 1H), 7.79 (d, J = 13.5 Hz, 1H), 7.47 (t, J = 5.0 Hz, 1H), 7.15 (s, 1H), 7.03 (d, J = 7.0 Hz, 1H), 6.88 (dd, J = 9.5, 2.5 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H), 3.37 (q, J = 6.0 Hz, 2H), 3.27–3.25 (m, 4H), 2.67–2.65 (m, 4H), 2.62 (t, J = 6.0 Hz, 2H), 1.37 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 171.46, 165.11, 153.40, 152.29 (d, JCF = 213.5 Hz), 148.78, 144.16 (d, JCF = 10.3 Hz), 136.16, 133.53, 129.71, 124.81 (d, JCF = 31.9 Hz), 123.62, 122.54 (d, JCF = 6.3 Hz), 121.45, 111.81 (d, JCF = 22.4 Hz), 109.10, 105.69, 55.96, 52.45, 51.14, 49.54, 48.02, 14.19. Anal. Calcd. for C26H27F4N5O5: C, 55.22; H, 4.81; N, 12.38. Found: C, 55.40; H, 4.80; N, 12.40.</p><!><p>The title compound was synthesized using 0.465 g (1.4 mmol) of 2b in 15 mL of anhydrous CH3CN and 0.240 g (0.7 mmol) of 5b. Yellow solid (50%): mp 216–218 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.64 (s, 1H), 8.08 (d, J = 9.5 Hz, 1H), 7.78 (d, J = 13.5 Hz, 1H), 7.59 (t, J = 5.5 Hz, 1H), 7.07 (s, 1H), 7.03 (d, J = 7.0 Hz), 6.84 (dd, J = 9.0, 2.0 Hz, 1H), 4.40 (q, J = 7.0 Hz, 2H), 3.73 (s, 3H), 3.28–3.20 (m, 6H), 2.60–2.54 (br m, 4H), 2.45 (t, J = 7.0 Hz, 2H), 1.77 (m, 2H), 1.37 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 171.47, 165.11, 153.40, 152.33 (d, JCF = 223.4 Hz), 148.78, 144.18 (d, JCF = 10.3 Hz), 136.16, 133.37, 129.81, 125.00, 123.61, 122.52 (d, JCF = 6.3 Hz), 121.44, 111.79 (d, JCF = 22.5 Hz), 109.10, 105.66, 54.88, 52.50, 51.14, 49.60, 48.01, 40.43, 25.39, 14.19. Anal. Calcd. for C27H29F4N5O5: C, 55.96; H, 5.04; N, 12.08. Found: C, 56.08; H, 5.05; N, 12.06.</p><!><p>In a round-bottom flask, the appropriate methyl ester (6a–6d) and a solution of 2 M NaOH were refluxed for 24 h under vigorous stirring. After this time, the reaction mixture was cooled, and a solution of 2 M HCl was added up to the isoelectric point. The obtained precipitate was filtered under vacuum and washed in sequence with deionized water, isopropanol, and diethyl ether. The solid was then purified by flash chromatography using a CH2Cl2/CH3OH gradient eluting system. According to this procedure, the following products have been obtained.</p><!><p>The title compound was obtained starting from 0.159 g (0.28 mmol) of 6a and 25 mL of 2 M NaOH. Yellow solid (75%): mp 229–231 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.59 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.83 (d, J = 13.5 Hz, 1H), 7.52–7.47 (m, 2H), 7.16 (s, 1H), 6.89 (d, J = 8.5 Hz, 1H), 3.70 (br s, 1H), 3.29 (m, 6H), 2.70–2.65 (br s, 4H), 2.63 (t, J = 5.5 Hz, 2H), 1.28–1.27 (br m, 2H), 1.09 (br s, 2H); 13C NMR (125 MHz, DMSO-d6): δ 175.45, 166.42, 153.18, 147.48, 146.44, 138.66, 133.53, 129.73, 124.96, 124.71, 123.65, 119.32, 111.11 (d, JCF = 21.3 Hz), 105.87, 55.97, 53.84, 52.43, 49.53, 40.11, 7.53. Anal. Calcd. for C26H25F4N5O5: C, 55.42; H, 4.47; N, 12.43. Found: C, 55.61; H, 4.48; N, 12.40.</p><!><p>The title compound was obtained starting from 0.112 g (0.19 mmol) of 6b and 25 mL of 2 M NaOH. Yellow solid (74%): mp 235–237 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.66 (s, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 13.0 Hz, 1H), 7.63 (s, 1H), 7.57 (d, J = 7.0 Hz, 1H), 7.08 (s, 1H), 6.86 (dd, J = 9.0, 2.0 Hz, 1H), 3.86–3.80 (br m, 1H), 3.41–3.33 (br s, 4H), 3.29–3.25 (m, 2H), 2.61 (br s, 4H), 2.48–2.42 (br m overlapped with DMSO, 2H), 1.79 (br s, 2H), 1.33–1.30 (m, 2H), 1.20–1.17 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ 176.32, 165.88, 153.96, 152.59 (d, JCF = 152.5 Hz), 147.99, 139.15, 133.40, 129.79, 124.8 (d, JCF = 32.5 Hz), 123.61, 121.44, 118.57, 110.94 (d, JCF = 23.8 Hz), 106.73, 106.33, 54.88, 52.25, 50.63, 40.34, 35.84, 28.99, 7.55. Anal. Calcd. for C27H27F4N5O5: C, 56.15; H, 4.71; N, 12.13. Found: C, 56.02; H, 4.70; N, 12.15.</p><!><p>The title compound was obtained starting from 0.177 g (0.31 mmol) of 6c and 25 mL of 2 M NaOH. Yellow solid (62%): mp 225–227 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.94 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 13.5 Hz, 1H), 7.47 (t, J = 5.5 Hz, 1H), 7.21–7.14 (m, 2H), 6.88 (dd, J = 9.5, 2.0 Hz, 1H), 4.58 (q, J = 7.0 Hz, 2H), 3.39–3.33 (m, 6H), 2.69–2.65 (m, 4H), 2.63 (t, J = 6.0 Hz, 2H), 1.41 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 176.12, 166.08, 153.84, 152.50 (d, JCF = 160.0 Hz), 148.47, 145.42 (d, JCF = 10.0 Hz), 137.18, 133.54, 129.71, 124.82 (d, JCF = 32.5 Hz), 123.63, 121.46, 119.19 (d, JCF = 7.5 Hz), 111.15 (d, JCF = 22.5 Hz), 107.06, 105.76, 55.93, 52.37, 49.45, 49.42, 49.03, 14.29. Anal. Calcd. for C25H25F4N5O5: C, 54.45; H, 4.57; N, 12.70. Found: C, 54.31; H, 4.56; N, 12.66.</p><!><p>The title compound was obtained starting from 0.077 g (0.13 mmol) of 6d and 25 mL of 2 M NaOH. Yellow solid (36%): mp 249–251 °C; 1H NMR (500 MHz, DMSO-d6): δ 8.94 (s, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 13.5 Hz, 1H), 7.59 (s, 1H), 7.17 (s, 1H), 7.06 (s, 1H), 6.84 (br m,1H), 4.59 (app s, 2H), 3.29–3.21 (br m, 6H), 2.62–2.53 (br m, 4H), 2.46 (t, J = 6.0 Hz, 2H), 1.81–1.73 (br m, 2H), 1.42 (t, J = 7.0 Hz, 3H); 13C NMR (125 MHz, DMSO-d6): δ 176.14, 166.04, 153.77, 152.44 (d, JCF = 163.8 Hz), 148.59, 137.16, 133.55, 129.76, 126.58, 125.47, 124.85 (d, JCF = 31.3 Hz), 123.60, 121.43, 119.26, 111.27 (d, JCF = 22.5 Hz), 107.12, 69.77, 49.07, 33.65, 31.26, 28.98, 22.06, 14.38. Anal. Calcd. for C26H27F4N5O5: C, 55.22; H, 4.81; N, 12.38. Found: C, 55.07; H, 4.82; N, 12.35.</p><!><p>The stock solution of the Griess reagent (purchased by Sigma Aldrich Srl) was obtained by dissolving 200 mg of the powder in 5 mL of H2O MilliQ. For nitrite quantification, each selected compound was dissolved in anhydrous DMSO to obtain a 1 mM solution (A). Subsequently, 160 μL of each solution A was diluted with 840 μL of H2O MilliQ to obtain solution B (160 μM). A total of 500 μL of B was then added to a quartz cuvette containing 500 μL of Griess reagent solution (80 μM). The resulting solutions were then irradiated with a 300 W tungsten lamp at a distance of 40 cm for 15 min, 1 h, and 2 h, recording the absorbance spectrum of each sample at each time point and reading the peak increase at 540 nm. The relationship of absorbance and concentrations of nitrite was constructed by drawing a standard curve using the known concentrations of NaNO2 (5, 10, 20, 30, 40, and 50 μM).</p><!><p>The cell lines DU145 (HTB-81, human prostate carcinoma), PC3 (CRL-1435, human prostate adenocarcinoma), MCF7 (HTB-22, human breast adenocarcinoma), MDA-MB231 (HTB-26, human breast adenocarcinoma), and HCT116 (CCL-247, human colorectal carcinoma) were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA); WH1 human fibroblasts were kindly provided by Dr. Guven.51 All the cells were maintained under standard culture conditions (37 °C; 5% CO2) in the RMPI1640 medium (PC3, MCF7, and MDA-MB231 cells), while DU145 and HCT116 cells were in the DMEM and Iscove's medium (WH1 cells) supplemented with 10% fetal calf serum, 1% glutamine, and 1% antibiotics mixture; for HCT116 and DU145 cells, 1% sodium pyruvate and 1% non-essential amino acids were also added to the culture medium.</p><!><p>The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed on all the cell lines tested as previously described52 with minor modifications. Briefly, according to the growth profiles previously defined for each cell line, adequate numbers of cells were plated in each well of a 96-well plate in 0.1 mL of complete culture medium. Cells were allowed to attach for 24 h before the treatment at 37 °C for 72 h with the compounds at concentrations ranging between 0.1 and 75 μM, bringing the final volume to 0.2 mL/well. Each experiment included eight replications per concentration tested; control samples were run with 0.2% DMSO. At the end of the incubation period, MTT (0.05 mL of a 2 mg/mL stock solution in PBS) was added to each well for 3 h at 37 °C. Cell supernatants were then carefully removed, the blue formazan crystals formed through MTT reduction by metabolically active cells were dissolved in 0.120 mL of DMSO, and the corresponding optical densities were measured at 570 nm using a Universal Microplate Reader EL800 (Bio-Tek, Winooski, VT).</p><p>To evaluate the contribution of NO release on cell viability, the MTT assay was performed on DU145 and PC3 cells following treatment with 7b and 7c with 1 h irradiation and 48 h incubation in a drug-free medium.</p><p>IC50 values were estimated from the resulting concentration–response curves by nonlinear regression analysis using GraphPad Prism software, v. 5.0 (GraphPad, San Diego, CA, USA). Differences between IC50 values were evaluated statistically by analysis of variance with a Bonferroni post-test for multiple comparisons.</p><!><p>P. aeruginosa PAO1 was chosen as a model microorganism53 and was grown overnight in a Luria Bertani (LB) medium at 37 °C on an orbital shaker at 200 rpm. Minimum inhibitory concentrations (MICs) of quinolone and quinolone derivatives were determined against P. aeruginosa PAO1 by a broth dilution method. Overnight cultures were diluted 100-fold to give a cellular concentration of 107 CFU/mL. Decreasing concentrations of compounds, from 200 to 0.1 μg/mL, were added to bacterial samples in a two-fold dilution series. Upon 24 h of incubation at 37 °C, the bacterial samples were observed for microbial growth, and MIC values were determined as minimal concentrations of drugs at which no turbidity was detectable. The assays were performed at least three times.</p><!><p>The relative efficiency of fluoroquinolone and derivatives in binding P. aeruginosa cells was determined by an indirect method.54P. aeruginosa PAO1 overnight cultures were centrifuged (5000 rpm for 10 min), and the supernatant was removed. Pellets were suspended and 10-fold diluted in PBS. Fluoroquinolone and fluoroquinolone derivatives were administered at 10 μM and incubated for 1 h at 37 °C in the dark to allow the interaction between the compounds and cells. After dark incubation, samples were centrifuged (10,000 rpm for 10 min), and the visible spectra of the supernatant were recorded (k = 380–700 nm) and compared with the corresponding visible spectrum of each compound.</p><!><p>Upon the overnight growth of P. aeruginosa PAO1 and S. aureus ATCC6538P (methicillin susceptible S. aureus, MSSA), cultures were diluted in phosphate buffer (PBS–KH2PO4/K2HPO4, 10 mM, pH 7.4) to reach a concentration of ∼106 CFU/mL. Compound 7c was added to a cell suspension at a final concentration of 10 μM. Cells were incubated in the dark for 10 min and then irradiated under light at 410 ± 10 nm (20 J/cm2) or incubated in the dark as a control. Soon after irradiation, the bacterial concentration was evaluated by the viability count technique. Briefly, an aliquot of each sample was 10-fold serially diluted in PBS and a volume of 10 μL of each diluted and undiluted sample was inoculated on LB agar. After overnight incubation at 37 °C, the corresponding cellular concentration was calculated and expressed as CFU/mL.</p><!><p>Flexible ligand docking experiments were performed employing AutoDock 4.2.6 software implemented in YASARA (v. 19.5.5, YASARA Biosciences GmbH, Vienna, Austria)55,56 using the crystal structure of the human Topo IIα (PDB ID: 5GWK), bacterial Topo IIA (PDB ID: 2XCT), and bacterial DNA gyrase (PDB ID: 6M1S) retrieved from the PDB Data Bank as a fully optimized one and the Lamarckian genetic algorithm (LGA). The maps were generated by the program AutoGrid (4.2.6) with a spacing of 0.375 Å and dimensions that encompass all atoms extending 5 Å from the surface of the structure of the crystallized ligands. All the parameters were inserted at their default settings as previously reported.57 In the docking tab, the macromolecule and ligand are selected, and GA parameters are set as ga_runs = 100, ga_pop_size = 150, ga_num_evals = 25,000,000, ga_num_generations = 27,000, ga_elitism = 1, ga_mutation_rate = 0.02, ga_crossover_rate = 0.8, ga_crossover_mode = two points, ga_cauchy_alpha = 0.0, ga_cauchy_beta = 1.0, and number of generations for picking worst individual = 10. Since no water molecules are directly involved in complex stabilization, they were not considered in the docking process (although in the crystallized structure of the bacterial DNA gyrase there are three structural water molecules that form hydrogen bonds between the ligand and some of the amino acids present on the enzymatic site, this network of H-bonds is intrinsic with the structure of the crystallized ligand. Docking calculations performed with the crystallized water molecules led to unsatisfactory results; therefore, the removal of water molecules in the peripheral regions of the binding site does not influence the calculated free binding energies in any way). All protein amino acid residues were kept rigid, whereas all single bonds of ligands were treated as fully flexible. The values of the energies of docking, in kcal/mol, have been calculated employing the "hybrid" force field implemented in AutoDock that contains terms based on molecular mechanics as well as empirical. Although the prediction of absolute binding energies may be less accurate compared to more computationally expensive, purely force field-based methods, this semi-empirical approach is considered as well-suited for the relative rankings.58</p><!><p>The semi-empirical calculations were performed using the parameterized model number 6 Hamiltonian59 as implemented in the MOPAC package (MOPAC2016 v. 18.151, Stewart Computational Chemistry, Colorado Springs, Colorado, USA). All molecules were fully optimized employing the eigenvector following the algorithm and a gradient minimization of 0.01 together with the precise and ddmin = 0 keywords.</p><!><p>The MD simulations of the human Topo IIα/DNA/1a ternary model system were performed with the YASARA Structure package (19.11.5).55 A periodic simulation cell with boundaries extending 8 Å from the surface of the complex was employed. The box was filled with water, with a maximum sum of all bump water of 1.0 Å and a density of 0.997 g/mL with an explicit solvent. YASARA's pKa utility was used to assign pKa values at pH 7.4,60 and system charges were neutralized with NaCl (0.9% by mass). Water molecules were deleted to readjust the solvent density to 0.997 g/mL. The final system dimensions were approximately 122 × 122 × 122 Å3. The ligand force-field parameters were generated with the AutoSMILES utility,57 which employs semi-empirical AM1 geometry optimization. Moreover, the assignment of charges, by the assignment of the AM1BCC atom and bond types with refinement was performed using the RESP charges, and finally the assignments of general AMBER force field atom types. Optimization of the hydrogen bond network of the various enzyme–ligand complexes was obtained using the method established by Hooft et al.61 This model allowed addressing ambiguities arising from multiple side-chain conformations and protonation states that are not well resolved in the electron density. The protein was treated with an AMBER ff14SB force field and62 the ligand with GAFF2,63 and the TIP3P model was used for water. The cutoff was 8 Å for van der Waals forces (the default used by AMBER),64 and no cutoff was applied to electrostatic forces (using the Particle Mesh Ewald algorithm).65 A 100 ps MD simulation was run on the solvent only. The entire system was then energy-minimized using first the steepest descent minimization to remove conformational stress followed by a simulated annealing minimization until convergence (<0.01 kcal/mol Å). The equations of motions were integrated with multiple timesteps of 1.25 fs for bonded interactions and 2.5 fs for nonbonded interactions using the NPT ensemble at a temperature of 298 K and a pressure of 1 atm. The temperature was controlled using the Berendsen thermostat,66 and the pressure was controlled using the solvent-probe pressure control mode barostat.67 The MD simulation was then initiated with an equilibration period of 10 ns for the assessment of the ligand's correct pose, and a classical production MD simulation of 100 ns was performed analogously to other experiments reported by us.68,69 The MD trajectories were recorded every 100 ps.</p><!><p>To this purpose, we used the iPBSA script, according to the procedure reported in detail in the original publication,70 with the algorithm for MM/PBSA implemented in the freely available AmberTools21 suite,71 to analyze the ligand/enzyme complex coordinates recorded during the MD simulation.</p><!><p>NMR spectra, fluorescence and absorption spectra, visible spectrum before and after cell binding, and IC50 values on DU145, PC3, MCF7, MDA-MB231, and HCT116 cell lines of compounds 1a, 1b, 3a, 3b, 6a–6d, and 7a–7d (PDF)</p><p>Molecular formula strings (CSV)</p><p>jm1c00917_si_001.pdf</p><p>jm1c00917_si_002.csv</p><!><p>All authors contributed to the present paper and have given approval to the final version of the manuscript.</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Protein N-Terminal Processing: Substrate Specificity of Escherichia coli and Human Methionine Aminopeptidases\xe2\x80\xa0

Methionine aminopeptidase (MetAP) catalyzes the hydrolytic cleavage of the N-terminal methionine from newly synthesized polypeptides. The extent of methionyl removal from a protein is dictated by its N-terminal peptide sequence. Earlier studies revealed that MetAPs require amino acids containing small side chains (e.g., Gly, Ala, Ser, Cys, Pro, Thr, and Val) as the P1\xe2\x80\x99 residue, but their specificity at positions P2\xe2\x80\x99 and beyond remains incompletely defined. In this work, the substrate specificities of Escherichia coli MetAP1, human MetAP1, and human MetAP2 were systematically profiled by screening against a combinatorial peptide library and kinetic analysis of individually synthesized peptide substrates. Our results show that although all three enzymes require small residues at the P1\xe2\x80\x99 position, they have differential tolerance for Val and Thr at this position. The catalytic activity of human MetAP2 toward Met-Val peptides is consistently two orders of magnitude higher than that of MetAP1, suggesting that MetAP2 is responsible for processing proteins containing N-terminal Met-Val and Met-Thr sequences in vivo. At positions P2\xe2\x80\x99 to P5\xe2\x80\x99, all three MetAPs have broad specificity, but are poorly active toward peptides containing a proline at the P2\xe2\x80\x99 position. In addition, the human MetAPs disfavor acidic residues at the P2\xe2\x80\x99 to P5\xe2\x80\x99 positions. The specificity data have allowed us to formulate a simple set of rules that can reliably predict the N-terminal processing of E. coli and human proteins.

protein_n-terminal_processing:_substrate_specificity_of_escherichia_coli_and_human_methionine_aminop

<!>Materials and General Methods<!>Synthesis of MetAP Library<!>N-Hydroxylsuccinimidyl 3-[4-(4-dimethylaminophenylazo)benzoylamino]propionate (Dabcyl-\xce\xb2-Ala-OSu)<!>4-(4-Dimethylaminophenylazo)benzoic acid hydrazide<!>MetAP Library Screening (Method A)<!>MetAP Library Screening (Method B)<!>Peptide Sequencing by Partial Edman Degradation-Mass Spectrometry (PED-MS)<!>Synthesis of Individual Peptides<!>MetAP Activity Assay<!>DNA Constructs and Cell Transfection<!>Initiator Methionine Release Assay<!>Design and Synthesis of MetAP Substrate Library<!>MetAP Library Screening<!>Substrate Specificity of E. coli MetAP1<!>Substrate Specificity of Human MetAP1<!>Substrate Specificity of Human MetAP2<!>Kinetic Properties of Selected MetAP Substrates<!>In Vivo Validation of MetAP1 and MetAP2 Substrate Specificity<!>Corroboration with Literature Data<!>Predicted N-Terminal Met Removal Pattern in E. coli and Human Proteome<!>Conclusion<!>

<p>Ribosomal protein synthesis is universally initiated with methionine (in eukaryotic cytoplasm) or N-formylmethionine (in prokaryotes, mitochondria, and chloroplasts). During protein maturation, the N-formyl group is removed by peptide deformylase, leaving methionine with a free NH2 group (1). Subsequently, the initiator methionine is removed from many but not all of the proteins by methionine aminopeptidase(s) (MetAPs). For example, in a cytosolic extract of Escherichia coli, only 40% of the polypeptides retain the initiator methionine, whereas the majority of the polypeptides display alanine, serine, or threonine at their N-termini (2). There are two types of MetAPs, MetAP1 and MetAP2. While eubacteria have only MetAP1 and archaea have only MetAP2, eukaryotes including yeast have both MetAP1 and MetAP2 in the cytoplasm and MetAP1D in the mitochondria (3). MetAP activities are essential for survival in both bacteria and yeast (4–6). As a result, there has been a renewed interest in designing specific inhibitors against bacterial MetAP1 as novel antibiotics (7–10). We and others have previously discovered that MetAP2 is the molecular target of fumagillin, a fungal metabolite and potent inhibitor of angiogenesis, validating MetAP2 as a novel target for anticancer treatment (11, 12). In fact, a semisynthetic analog of fumagillin, TNP-470, has entered Phase II clinical trial as an anticancer agent (13). TNP-470 preferentially inhibits endothelial cell growth in tumor vasculature by arresting the cell cycle in the late G1 phase (14, 15). Presumably, inhibition of MetAP2 by TNP-470 prevents the methionine removal from one or more critical proteins and therefore their maturation. However, the identity of these MetAP2-specific substrates is currently unknown.</p><p>N-terminal methionine removal is a co-translational process and occurs as soon as a polypeptide emerges from the ribosome (16). Previous studies indicate that the fate of the N-terminal methionine is dictated by the substrate specificities of MetAPs, which require Met as the P1 residue and amino acids with small side chains as the P1' residue (e.g., Ala, Gly, Pro, Ser, Thr, or Val) (17–24). Amino acid residues at positions P2' and beyond also play a role. For example, a hemoglobin variant with a proline at position 3 (P2') retains the initiator methionine in vivo, whereas mutation of the proline to Arg or Gln leads to complete cleavage of the methionine (20). Chang et al. reported a two-fold difference in activity of purified yeast MetAP1 toward Met-Ala-Ile-Pro-Glu vs Met-Ala-Ile-Pro-Ser, which differ only at the P4' position (21). These studies clearly demonstrate the existence of sequence specificity by MetAPs. However, because these studies employed the individual synthesis and activity assay of each peptide, the sequences that have been tested represent only a small fraction of the possible protein N-terminal sequence space and are generally limited to examine the specificity at the P1, P1' and occasionally P2' positions. One recent study by Frottin et al. has generated a more complete specificity profile of E. coli MetAP1 and Pyrococcus furiosus MetAP2, by synthesizing and assaying ∼120 short peptides (25). To our knowledge, no systematic study has been performed for any eukaryotic MetAPs. A potential approach to systematically determining the substrate specificity of a MetAP is to screen it against a combinatorial peptide library. This, however, has not been possible due to the technical difficulty in differentiating reaction products from unreacted substrates, which are both peptides with free N-termini. Herein, we report a novel peptide library screening strategy and its application to determine the sequence specificity of E. coli MetAP1, human MetAP1 and MetAP2. The specificity information should be useful in designing specific MetAP2 inhibitors as anticancer agents and compounds specific for the bacterial MetAP1 as antibacterial agents.</p><!><p>1H and 13C NMR spectra were recorded on a Bruker 400 DPX spectrometer at 300 and 75 MHz, respectively. Fmoc-protected L-amino acids were purchased from Advanced ChemTech (Louisville, KY), Peptides International (Louisville, KY), or NovaBiochem (La Jolla, CA). O-Benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole hydrate (HOBt) were from Peptides International. All solvents and other chemical reagents were obtained from Aldrich, Fisher Scientific (Pittsburgh, PA), or VWR (West Chester, PA) and were used without further purification unless noted otherwise. N-(9-Fluorenylmethoxycarbonyloxy) succinimide (Fmoc-OSu) was from Advanced ChemTech. Phenyl isothiocyanate (PITC) was purchased in 1-mL sealed ampoules from Sigma-Aldrich, and a freshly opened ampoule was used in each experiment. N-(4-[4'-(Dimethylamino)phenylazo]benzoyloxy)succinimide (Dabcyl-OSu) was from ABD Bioquest (Sunnyvale, CA). Amino polyethylene glycol (PEGA) resin (0.4 mmol/g, 150–300 µM in water) was from Peptide International. Clear amide resin (90 µm, 0.23 mmol/g) was from Advanced ChemTech. α-Cyano-4-hydroxycinnamic acid (α-CCA) was purchased from Sigma and recrystallized prior to use. E. coli MetAP1 was purified from an E. coli strain overproducing the enzyme as previously described (26). Recombinant human MetAP1 and MetAP2 were expressed and purified as previously described (11, 26). Anti-Flag, anti-PGM1, and anti-TXNL1 antibodies were purchased from Sigma or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).</p><!><p>The peptide library was synthesized on 2.0 g of PEGA resin (0.4 mmol/g, 150–300 µm in water). All of the manipulations were performed at room temperature unless otherwise noted. Synthesis of the N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker began with acylation of the resin bounded amine with glutaric anhydride (3.0 equiv) in 15 mL of CH2Cl2 containing diisopropylethylamine (1.1 equiv). After 3 h, the resin was washed with CH2Cl2 and the newly formed carboxylic acid was treated with 15 mL of CH2Cl2 containing 5,5-dimethylcyclohexanedione (4.0 equiv), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI, 2 equiv), and N,N-diaminopyridine (2 equiv) for 36 h. The excess reagents were removed by filtration and the beads were washed with CH2Cl2 (50 mL) and DMF (50 mL). Next, the resin was incubated with 1,5-diaminopentane (20 equiv) in 15 mL of CH2Cl2 to furnish the Dde linker. Coupling of the first amino acid and the rest of peptide synthesis followed standard Fmoc/HBTU chemistry using four equiv of Fmoc-amino acids. To construct the random region, the resin was split into the desired number of equal portions and each portion was coupled twice with 4 equiv of a different Fmoc-amino acid plus HBTU/HOBt/N-methylmorpholine (NMM) for 2 h at room temperature. To differentiate isobaric amino acids during MS analysis, 5% (mol/mol) CD3CO2D was added to the coupling reaction of Leu, whereas 5% CH3CD2CO2D was added to the coupling reaction of norleucine (27). After the coupling of the last random residue, the resin was pooled into three sub-libraries according to the identity of the last random residue. Sub-libraries I and II contained Ser and Thr as the N-terminal residues, respectively, while sub-library III contained an equimolar mixture of the other 16 L-α-amino acids excluding Ser and Thr. The three sub-libraries were separately coupled with 4 equiv of Fmoc-Met-OH and deprotected by treatment with the modified reagent K (TFA:phenole:H2O:thioanisole:ethanedithiol:anisole = 79:7.5:5:5:2.5:1). The resin-bound sub-libraries were washed exhaustively with CH2Cl2 and DMF, suspended in DMF, and stored under argon atmosphere at 4 °C.</p><!><p>To a 100 mL dry flask was added Dabcyl-OSu (0.366 mg, 1 mmol), DMF (25 mL), triethylamine (0.42 mL, 3.0 mmol), and β–alanine (0.091g, 1.02 mmol). The mixture was stirred at room temperature for 18 h. The solvent was removed under vacuum and the solid residue was washed with H2O and then dried via azeotrope with toluene to give 0.322 g (88% yield) of a crude product. The crude product (0.322 g, 0.946 mmol) was dissolved in DMF (15 mL), to which N-hydroxysuccinimide (0.163 g, 1.42 mmol) and EDCI (0.272 g, 1.42 mmol) were added. The mixture was stirred overnight and the solvent was removed under vacuum. The solid residue was dissolved in dichloromethane, washed with H2O, dried over Na2SO4, and concentrated to give a red solid (0.372 g, 90% yield). 1H NMR (250 MHz, CDCl3) δ 7.96-7.86 (m, 6H), 6.77 (d, 2H, J = 9.3), 3.92 (m, 2H), 3.11 (s, 6H), 3.00-2.95 (m, 2H), 2.89 (s, 4H). HRMS (ESI): Calcd for C22H24N5O5 (M++H) 438.1772, found 438.1764.</p><!><p>To a solution of Dabcyl-OSu (0.366 g, 1 mmol) in DMF was added dropwise anhydrous NH2NH2 (31.4 µL, 1.0 mmol) at room temperature. The mixture was stirred for 20 min. The solvent was removed under vacuum and the solid residue was washed with H2O and dried under vacuum to give 0.253 g (89.4%) of product as a red solid. 1H NMR (400 Hz, DMSO-d6): δ 9.86 (s, 1H), 7.97-7.93 (m, 2H), 7.82-7.77 (m, 4H), 6.86-6.82 (m 2H), 4.55 (brs, 2H), 3.08 (s, 6H). 13C NMR (100 Hz, DMSO-d6): δ 165.3, 154.0, 152.9, 142.7, 133.6, 128.1, 125.1, 121.6, 111.6, 39.9. HRMS (ESI): calcd for C15H17N5ONa (M+ + Na) 306.1331, found 306.1323.</p><!><p>A typical screening involved 20 mg of sub-library III (∼71,000 beads), which was transferred into a disposable Bio-spin column (2.0 mL). The resin was washed with DMF (5 × 1.8 mL), H2O (5 × 1.8 mL), and a MetAP screening buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, and 0.1 mM CoCl2). After incubation with the screening buffer for 10 min, the resin was treated with 75–500 nM MetAP at room temperature for 30–180 min. The reaction was terminated by removing the enzyme solution (via filtration) and washing the resin with a 0.2 M EDTA solution (5 × 1.8 mL). After 10 min incubation with 0.2 M EDTA, the resin was washed with 1 M NaCl (5 × 1.8 mL) and incubated with 1 M NaCl for 10 min. The resin was next washed with H2O (5 × 1.8 mL) and DMF (5 × 1.8 mL) and treated with 1.2 equiv of 39:1 (mol/mol) Fmoc-OSu and Dabcyl-β-Ala-OSu dissolved in a 9:1 (v/v) DMF: phosphate buffer (50 mM, pH 8.0) for 1.5 h. The resulting resin was washed with DMF (5 × 1.8 mL) and incubated with 20% piperidine in DMF for 5 and 15 min. The resin was washed with DMF (5 × 1.8 mL) and H2O (5 × 1.8 mL) and incubated overnight with 100 mg/mL CNBr in 70% formic acid at room temperature. After the overnight incubation, the resin was thoroughly washed with 70% HCOOH and transferred into a 60 × 15 mm Petri dish with H2O. After the addition of a drop of 6 M HCl into the Petri dish, the red colored beads were manually removed from the dish using a micropipette with the aid of a dissecting microscope. A control reaction without MetAP produced no colored beads.</p><!><p>Sub-library I or II (typically 4.3 mg) was washed and treated with MetAP as described above. After the MetAP reaction, the resin was washed exhaustively with H2O and then treated with 2 equiv of NaIO4 dissolved in 0.01 M sodium phosphate buffer (pH 7.0) for 5 min at room temperature. The reaction solution was removed and the resin was washed with H2O. The resin was suspended in 0.9 mL of 1% ethylene glycol in H2O and incubated for 10 min. The resin was washed with H2O (5 × 0.9 mL) and treated with 0.1 equiv of Dabcyl-NHNH2 in a 7:3 (v/v) mixture of 30 mM NaOAc buffer (30 mM, pH 4.5)/MeCN for 1.5 h at room temperature. The reaction solution was removed and the resin was washed with MeCN (5 × 0.9 mL) and DMF (5 × 0.9 mL). The resin was treated with 10 equiv of NaBH3CN in 0.9 mL of DMF containing 1% HOAc for 36 h. After that, the resin was washed with DMF (5 × 0.9 mL), H2O (5 × 0.9 mL) and transferred into a Petri dish with H2O. The solution was acidified with HCl and the red colored beads were manually removed from the library.</p><!><p>Positive beads derived from the same screening experiment were pooled into a single reaction vessel, suspended in 160 µL of 2:1 (v/v) pyridine/water containing 0.1% triethylamine, and mixed with an equal volume of 80:1 (mol/mol) PITC and Fmoc-OSu in pyridine (625 mM of PITC and 7.8 mM of Fmoc-OSu). The reaction was allowed to proceed for 6 min at room temperature, and the beads were washed sequentially with pyridine, CH2Cl2, and TFA. The beads were treated twice with 500 µL of TFA for 6 min each. The beads were washed with CH2Cl2 and pyridine and the PED cycle was repeated 5 (for hits from method A) or 6 times (for hits from method B). Finally, the beads were treated twice with 1 mL of 20% piperidine in DMF at room temperature (5 min each). The beads were washed exhaustively with water and transferred into individual microcentrifuge tubes (1 bead/tube). Each bead was treated with 10 µL of 1.5% hydrazine in THF/H2O (1:1) at room temperature for 15 min. The solution was acidified by the addition of 10 µL of 7% TFA in H2O. The solvent was removed under vacuum and the released sample was dissolved in 5 µL of 0.1% TFA in water. One µL of the peptide solution was mixed with 2 µL of saturated α-cyano-4-hydroxycinnamic acid in acetonitrile/0.1% TFA (1:1), and 1 µL of the resulting mixture was spotted onto a 384-well sample plate. Mass spectrometry was performed at Campus Chemical Instrument Center of The Ohio State University on a Bruker III MALDI-TOF instrument in an automated manner. The data obtained were analyzed by Moverz software (Proteometrics LLC, Winnipeg, Canada).</p><!><p>Individual peptides were synthesized using standard solid phase Fmoc/HBTU chemistry. Each peptide was synthesized on 100 mg of CLEAR-amide resin (0.49 mmol/g). Ninhydrin test was used to monitor the completion after each coupling reaction. After cleavage and deprotection with a modified reagent K (7.5% phenol, 5% water, 5% thioanisole, 2.5% ethanedithiol, 1% anisole in TFA), the crude peptides were precipitated in cold diethyl ether and purified by reversed-phase HPLC on a C18 column (Varian 120 Ǻ, 4.6 × 250 mm). The identity of the peptides was confirmed by MALDI-TOF mass spectrometric analyses.</p><!><p>A typical assay reaction (total volume of 100 µL) contained 1x MetAP reaction buffer (30 mM Hepes, pH 7.4, 150 mM NaCl, 0.1 mM CoCl2) and 0–200 µM peptide substrate. The reaction was initiated by the addition of MetAP (final concentration 5–1000 nM) and quenched after 10–90 min with the addition of three drops of TFA. The reaction mixture was centrifuged at 15,000 rpm in a microcentrifuge for 5 min and the clear supernatant was analyzed by reversed-phase HPLC on an analytical C18 column (Vydac C18 300 Å) eluted with a linear gradient of acetonitrile in water containing 0.05% TFA (10–40% acetonitrile in 18 min). The percentage of substrate-to-product conversion was determined from the peak areas of the remaining substrate and the reaction product (monitored at 214 nm) and kept at <20%. The initial rates were calculated from the conversion percentages and plotted against [S]. Data fitting against the Michaelis-Menten equation V = Vmax · [S]/(KM + [S]) or the simplified equation V = kcat[E][S]/KM (when KM >> [S]) gave the kinetic constants kcat, KM, and/or kcat/KM.</p><!><p>The coding sequences of BTF3L4, phosphoglucomutase (PGM1) and thioredoxin-like protein 1 (TXNL1) proteins were amplified by the polymerase chain reaction (PCR) from the cDNA pool of HEK293T cells with the introduction of EcoRI and BamHI restriction sites. The DNA primers used for PCR amplification of the BTF3L4 gene were 5'-CGCGAATTCAGCATGAATCAAGAAAAGTTAGCC-3' and 5'-CCGGGATCCGTTAGCTTCATTCTTTGATGC-3'. The primers used for the PGM1 gene were 5'-CGCGAATTCGCGATGGTGAAGATCGTGACAGTTAAG-3' and 5'-CGCGGATCCGGTGATGACAGTGGGTGC-3'. The primers used for the TXNL1 gene were 5'-GAGCGGCCGCCATGGTGGGGGTGAAG-3' and 5'-CGCGCGGATCCGTGGCTTTCTCCTTTTTTGC-3'. The PCR products were cloned into the p3xFLAG-CMV-14 vector (Sigma), resulting in the addition of a C-terminal 3xFlag tag. The final constructs were verified by DNA sequencing. The plasmids for transfection were isolated from E. coli using PureLink™ HiPure Plasmid Filter Purification Kit and PureLink™ HiPure Precipitator Module (Invitrogen).</p><p>HEK293T cells were grown in a humidified environment at 37 °C with 5% CO2 in low glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) unless otherwise stated. One day prior to transfection, the cells were seeded with a density of 2 × 106 cells per 10-cm petri dish containing 10 mL of media. Immediately before transfection, 10 µg of the appropriate plasmid DNA (p3xFLAG-CMV-14-BTF3L4, p3xFLAG-CMV-14-PGM1, or p3xFLAG-CMV-14-TXNL1) was added to 1 mL of serum-free DMEM, to which 25 µL of Superfect transfection reagent (Qiagen, 301305) was added. The mixture was incubated at room temperature for 10 min. The cell media was then changed and the transfection mixture was added in a dropwise fashion.</p><!><p>This assay was a modification of a previously described procedure (28, 29). At 19.5 h post-transfection, the cells were treated for 4.5 h with either vehicle (DMSO), MetAP1 inhibitor IV-43 (30) (10 µM), MetAP2 inhibitor TNP-470 (100 nM), or both in 4 mL of methionine-free DMEM supplemented with 4.5 g/L glucose and sodium pyruvate (Mediatech) and 10% dialyzed FBS (Gibco). For the final 4 h of treatment, the cells were incubated with 0.2 mCi of [35S]-methionine (PerkinElmer, NEG709A). The media was removed by aspiration and the cells were resuspended in 5 mL of ice cold PBS, collected by centrifugation at 225 × g, and resuspended using a Pasteur pipet in 1 mL of ice cold RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, pH 8.0) containing 1X protease inhibitor cocktail (Roche, 11872580001). Following a 10 min incubation (at 4 °C), the cell lysate was centrifuged at ∼16,000 × g for 20 min (4 °C), and the cleared supernatant was transferred to a fresh tube containing 30 µL of anti-Flag conjugated agarose resin (Sigma, A2220) that had been equilibrated by washing with RIPA buffer (3 × 1 mL). After incubation for 1.5 h on a rotating wheel (4 °C), the resin was collected by centrifugation at 500 × g for 30 s (4 °C) and washed five times with 0.9 mL of ice cold RIPA buffer (by inverting the tube 15 times between centrifugation steps). Finally, the resin was washed twice with 0.9 mL of ice cold MetAP reaction buffer (50 mM HEPES, 150 mM NaCl, 100 µM CoCl2, pH 7.5) before being resuspended in 250 µL of MetAP reaction buffer and split into two equal aliquots. Each aliquot was centrifuged at 500 × g and the supernatant was aspirated. The supernatant from one aliquot was discarded (the "no enzyme" tube) and 50 µL of MetAP reaction buffer was added to the resin. The supernatant from the other aliquot (the "enzyme reaction tube") was set aside as the "wash" sample and recombinant MetAP1 and MetAP2 (5 µM each) were added to the resin suspended in 50 µL of MetAP reaction buffer. After incubation overnight (∼16 h) at room temperature, 75 µL of MetAP reaction buffer was added to each tube, followed by pelleting the resin and transferring the supernatant to a fresh tube. The 35S content of each sample (including the "wash" sample) was determined by mixing with 1 mL of scintillation fluid (PerkinElmer, 1200-439) and scintillation counting on a 1450 MicroBeta apparatus (Wallac). The percentage of [35S]-methionine release was calculated from the ratio of counts in the reaction buffer to the sum of these counts and the counts from the corresponding resin sample.</p><!><p>A one-bead-one-compound (OBOC) MetAP substrate library in the form of NH2-MX1X2X3X4X5LNBBR-Dde-resin [where B is β-alanine and X1–X5 are 2-aminobutyrate (Abu or U) and norleucine (Nle or M), which were used as Cys and Met surrogates, respectively, or any of 16 proteinogenic amino acids except for Arg, Cys, Lys, and Met] was synthesized on PEGA resin (0.4 mmol/g, 150–300 µm in diameter when swollen in water) (Scheme 1). A methionine was placed at the N-termini of all sequences because it is already well established that all MetAPs have a stringent requirement of Met as the P1 residue (21, 31). Arg, Lys, Cys, and Met were excluded (or replaced) from the random region because they would interfere with the screening procedure (vide infra). The invariant sequence LNBBR was added to facilitate MALDI MS analysis, by providing a fixed positive charge (Arg) and shifting the mass of all peptides to >500 (to avoid overlap with MALDI matrix signals). The peptides were covalently linked to the PEGA resin via a Dde linker, which permits facile peptide release by treatment with hydrazine prior to MS analysis. To construct the Dde linker, the free amino group on PEGA resin was first acylated with glutaric anhydride and the resulting carboxylic acid (1) was activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and condensed with 5,5-dimethylcyclohexane-1,3-dione to give enol 2 (32). Subsequent reaction of enol 2 with excess 1,5-diaminopentane provided the desired Dde linker 3 containing a free amine as the anchor for peptide synthesis. After the addition of the LNBBR sequence using standard Fmoc/HBTU chemistry, the random region was synthesized via the split-and-pool method (33, 34). After the addition of the last random residue (X1), the library was split into three sub-libraries. Sub-libraries I and II contained Ser and Thr as the X1 residue, respectively, whereas in sub-library III, the X1 residue represented an equimolar mixture of the other 16 amino acids. The theoretical diversity of the library is 185 or 1.9 × 106.</p><!><p>Two different methods were devised to screen the MetAP substrate library. Method A (Scheme 2a) was designed for sub-library III, which was subjected to limited MetAP reaction, so that only those beads that carried the most efficient substrates of MetAP would undergo partial cleavage of the N-terminal methionine (usually a few percent), while the vast majority of beads (which displayed poorer substrates or inactive peptides) would have little or no reaction. The library was then treated with a 39:1 (mol/mol) mixture of Fmoc-OSu and Dabcyl-β-Ala-OSu, a red dye molecule. This resulted in the N-terminal acylation (or carbamoylation) of all library peptides (both MetAP reaction products and unreacted substrates) and all of the library beads became red colored (due to attachment of the Dabcyl moiety). Next, the library was treated with piperidine to remove the N-terminal Fmoc group, followed by CNBr to cleave any N-terminal methionine. For beads/peptides that did not undergo any MetAP reaction, CNBr would remove the N-terminal methionine, along with the Dabcyl group, rendering these beads colorless. On the other hand, any MetAP reaction products would not be affected by the CNBr treatment because they no longer contained any methionine in their sequences. Thus, those beads that had undergone significant MetAP reaction (positive beads) would retain the Dabcyl group and remain red colored. The positive beads were manually removed from the library using a micropipette and their peptide sequences were determined by PED-MS (27). It is worth noting that the use of 39:1 Fmoc-OSu/Dabcyl-β-Ala-OSu was critical for successful library screening, as it ensured that there were sufficient amounts of peptides with free N-termini (after Fmoc removal) for PED-MS analysis. It also improved the color contrast between positive and negative beads. When only Dabcyl- β-Ala-OSu was used for N-terminal acylation, all of the library beads retained significant red color after the CNBr treatment, because methionine removal by CNBr was not quantitative.</p><p>The above method was less effective for sub-libraries I and II (which contained Ser and Thr at the P1' position, respectively) because CNBr cleavage of methionine is inefficient when Ser or Thr is next to methionine (35). The β-hydroxyl group of Ser (or Thr) can intramolecularly react with the five-membered-ring intermediate, resulting in the conversion of the methionine into a homoserine instead of peptide bond cleavage. We therefore developed an alternative approach (method B) for those two sub-libraries (Scheme 2b). Enzymatic cleavage of the N-terminal methionine from the library peptides generated an N-terminal Ser or Thr, which was selectively oxidized by sodium periodate into a glyoxyl group. The newly formed aldehyde group was then selectively conjugated with Dabcyl-hydrazide and reduced with NaBH3CN. Again, the resulting positive (red colored) beads were readily identified from the library and sequenced by the PED-MS method.</p><!><p>A total of 105 mg of sub-library III (∼3.9 × 105 beads) was screened against E. coli MetAP1 in five separate experiments (25 or 25 mg of resin each). The most colored beads were isolated and sequenced by PED-MS to give 236 complete sequences, which should represent the most efficient substrates of E. coli MetAP1 (Table 1). Similar sequences were selected from each screening experiment, demonstrating the reproducibility of the screening method. E. coli MetAP1 has the most stringent specificity at the P1' position, with Ala being the most preferred amino acid (in 84% of all selected sequences), followed by glycine (10%) and 2-aminobutyrate (6%) (Figure 1a). At the P2' position, E. coli MetAP1 tolerates a wide variety of amino acids, but with preference for hydrophilic residues (Glu, Asp, Asn, Gln, Ser, and Thr) or small residues such as Ala and Abu. A few of the selected peptides also contained a Phe at the P2' position. Interestingly, out of the 236 selected peptides, none contained a proline, histidine, or tryptophan at this position. The enzyme also has broad specificity at the P3' position, where hydrophobic residues were frequently selected, whereas acidic residues (especially Asp), Gly, His, and Trp were underrepresented. There appears to be a negative correlation between the P2' and P3' positions with respect to acidic amino acids. When the P2' residue was acidic, the P3' residue was almost always hydrophobic; and vice versa, when Asp or Glu was present at the P3' position, the P2' residue was typically hydrophobic (Table 1). None of the most preferred substrates contained acidic residues at both P2' and P3' positions. No obvious selectivity was observed at the P4' or P5' position. Screening of sub-libraries I and II (9 mg each) by method B revealed a similar specificity profile at the P3'–P5' positions, although Ala and acidic residues (Asp and Glu) were more frequently selected at the P2' position when Ser or Thr was the P1' residue (Figure 1b and Table S1 in Supporting Information).</p><p>To identify the less efficient substrates and MetAP-resistant sequences, sub-library III (40 mg) was treated exhaustively with a high concentration of E. coli MetAP1 (1.0 µM) for an extended period of time (overnight). In addition to intensely colored beads, this screening experiment also produced many medium and lightly colored beads (∼15% of all library beads), which should represent the less efficient substrates of E. coli MetAP1. We randomly selected some of the medium colored beads for sequence analysis and obtained 232 complete sequences (Table S2 in Supporting Information). Our results showed that 40% of these less efficient substrates contained a Val as the P1' residue, while the rest of peptides had Gly (27%), Abu (23%), and Ala (10%) at this position. When the subset of sequences that contained Val as the P1' residue were analyzed, the specificity profile at the P2'–P5' positions was essentially identical to that observed for the most efficient substrates (Figure 1c). Similar analysis of the other subset of sequences (which contained Gly, Abu, or Ala as the P1' residue) showed that they usually contained less favorable amino acids at the P2' (e.g., Gly, Ile, Leu, and Pro) and/or P3' positions (e.g., Asp, Glu, and His) (Figure 1d). Sequence analysis of 171 randomly selected colorless beads from the library showed that most of the MetAP-resistant peptides contained amino acids with large side chains as the P1' residue, while none of them had Ala at the P1' position (Figure 1e and Table S3 in Supporting Information). Only six of the peptides contained Gly (MGDVFQ, MGPTET), Abu (MUHINS, MUPETF, MUILVE) or Val (MVLFHH) at the P1' position. These peptides either contained a disfavored residue at the P2' position (Pro or His) or were very hydrophobic (MUILVE and MVLFHH), making the beads poorly swelled in aqueous solution and thus poorly accessible to the enzyme during library screening. As expected from the large number of colorless beads (∼75% of all beads), there was no obvious "selectivity" at the P2'-P5' positions. These data indicate that all N-methionyl peptides containing a small P1' residue (Ala, Gly, Abu, Ser, Thr, and Val) are accepted as E. coli MetAP1 substrates, albeit with different catalytic efficiencies.</p><!><p>Human MetAP1 was similarly screened against sub-libraries I–III to identify peptide sequences that are most preferred or less preferred by the enzyme, and resistant to the enzyme. The most intensely colored beads were selected from sub-libraries III (60 mg) and I (9 mg) and sequenced to give 128 and 74 unambiguous sequences, respectively (Table S4 and Table S5). Similar sequences were selected from the two sub-libraries (Figure 2a, b). In general, human MetAP1 has a similar specificity profile to E. coli MetAP1, but also has some unique features. Like the E. coli enzyme, human MetAP1 prefers Ala at the P1' position, but this preference is stronger than that of E. coli MetAP1 (124 out of the 128 best substrates had Ala as the P1' residue). Only four peptides contained Gly (3 peptides) or Abu (1 peptide) as the P1' residue. The underrepresentation of Abu at this position suggests that amino acids with larger (than a methyl group) side chains are poorly tolerated by the human MetAP1 active site. This notion is further supported by the data from sub-library II (which contained Thr as the P1' residue) (Figure 2c and Table S6 in Supporting Information). Screening of sub-library II against human MetAP1 only produced lightly colored beads, despite the use of a higher enzyme concentration (800 nM) and longer reaction time (2 h), suggesting that any peptide containing a Thr at the P1' position is a relatively poor substrate of human MetAP1. Other features shared with the E. coli enzyme include the absence (or underrepresentation) of Pro at the P2' position and His and Trp at both P2' and P3' positions among the most active substrates. The most striking difference between the E. coli and human MetAP1 lies in their different tolerance to acidic residues. While E. coli MetAP1 prefers Asp and Glu at P2', P4', and P5' positions (Figure 1), the human enzyme strongly disfavors acidic residues at all positions. Out of the 245 most preferred substrates selected from sub-libraries I, II, and III, only one sequence [MA(Nle)DWE] contained acidic residues. Another difference is that while E. coli MetAP1 disfavors Gly at P2' and P3' positions, the human enzyme has no such discrimination.</p><p>The less optimal substrates and inactive peptides were identified by treating sub-library III (12 mg) with a high concentration of human MetAP1 (2.7 µM) for 24 h and randomly selecting a fraction of the medium colored and colorless beads for sequence analysis. The less optimal substrates contained Val (33%), Gly (28%), Abu (24%) and Ala (15%) as the P1' residue (Table S7 in Supporting Information). Out of the 48 Gly-, Abu-, and Ala-containing (at P1' position) peptides, 35 contained at least one acidic residue, often at the P2' and/or P3' position, while three other peptides had Pro as the P2' residue (Figure 2d). The Val-containing peptides (at position P1') contained no Pro at the P2' position and much fewer acidic residues in their sequences (Figure 2e). None of the MetAP1-resistant peptides contained Ala or Gly at the P1' position, while two peptides had Abu or Val as the P1' residue (Figure S1 in Supporting Information). These data provide further support for our conclusion that a larger P1' residue (e.g., Val and Thr), a Pro at the P2' position, and/or acidic residues at P2' to P5' positions can decrease the catalytic activity toward human MetAP1. They also suggest that human MetAP1, like the E. coli enzyme, will accept essentially all N-methionyl peptides containing Ala, Gly, Abu, Ser, Thr, or Val at the P1' position as substrates, although the Thr- and Val-containing peptides are generally inefficient substrates.</p><!><p>Sub-libraries I (4.3 mg), II (4.3 mg), and III (80 mg) were screened against human MetAP2 and the most intensely colored beads were sequenced to give 96, 65, and 632 complete sequences, respectively (Tables S8–10 in Supporting Information). In addition, 88 medium colored beads were isolated from sub-library III and sequenced (Table S11 in Supporting Information). In general, human MetAP2 has a very similar specificity profile to human MetAP1 (Figure 3). For example, both human MetAP1 and MetAP2 disfavor Pro at the P2' position, Trp at P2' and P3' positions, and acidic residues at positions P2' to P5'. The main difference between the two enzymes is their specificity at the P1' position. While only one out of the 128 most preferred substrates of human MetAP1 contained an Abu as the P1' residue, MetAP2 selected Abu with the second highest frequency (122 out of 632 sequences), more frequently than Gly (90 sequences). Moreover, 19 of the most preferred substrates had a Val as the P1' residue. This suggests that the S1' site of human MetAP2 is more capable of accommodating amino acids with larger side chains than does MetAP1. This notion is also supported by our data on sub-library II. We found that treatment of sub-library II with 250 nM human MetAP2 generated intensely colored beads in 2 h, while treatment with 800 nM human MetAP1 for 2 h only produced lightly colored beads. We noted that unlike E. coli and human MetAP1, MetAP2 selected a significant number of His at the P2' and P3' positions. The underlying reason is not yet clear.</p><!><p>Fifteen peptides were synthesized individually on a larger scale, purified by HPLC, and assayed against the three MetAPs to confirm the screening results as well as provide specificity data on amino acid residues excluded from the library (i.e., Arg and Lys) (Table 2, entries 1–15). Peptides 1 and 2 are two of the most preferred substrates of E. coli MetAP1, selected from sub-libraries III and I, respectively (Table 1 and Table S1). Peptide 12 is a preferred substrate of human MetAP2 selected from sub-library III (Table S8). The rest of the peptides are variants of peptides 1 and 12, designed to test the effect of the P1'–P3' residues on MetAP activity. A Tyr was added to the C-terminus of each peptide to facilitate their concentration determination. Since most of the peptides did not reach saturation at the highest concentration tested (1.0 mM), only their kcat/KM values are used for comparison.</p><p>As expected, peptides 1 and 2 were highly active toward the E. coli enzyme, having kcat/KM values of 14000 and 28000 M−1 s−1, respectively (Table 2). Replacement of the Ala at the P1' position with Gly, Pro, Thr, and Val decreased the activity by 3.4-, 4.7-, 9.3-, and 44-fold, respectively. This is largely consistent with the screening results, except for the proline-containing peptide. Previous studies have also shown that peptides containing Pro as the P1' residue are efficiently hydrolyzed by MetAPs (17–25). Thus, the absence of Pro from the P1' position among the selected substrates were likely caused by the bias of the screening procedure (method A). Proline, which bears a secondary amine, is significantly less reactive to activated esters such as Dabcyl-β-Ala-OSu than the other 19 proteinogenic amino acids (36). Presumably, MetAP reaction products that contained an N-terminal proline were not efficiently labeled by Dabcyl-β-Ala-OSu and became false negatives. Replacement of the P2' residue (Glu) with an arginine, which was excluded from the library for technical reasons, slightly increased the catalytic activity (by 1.3-fold). This suggests that the frequent selection of acidic residues at the P2' position was not due to the presence of favorable charge-charge interactions; the E. coli enzyme simply prefers a hydrophilic residue at this position. Indeed, other neutral, hydrophilic residues such as Asn, Gln, and Ser were also frequently selected at this position (Figure 1). Consistent with the absence of Pro from the P2' position among the most preferred substrates, peptide 9 (MAPIEIY) had an activity that was too low to be reliably determined by the HPLC assay. A surprising finding was that peptide 10, which contains a His at the P2' position, had excellent activity (kcat/KM = 14500 M−1 s−1), even though His was not at all selected at this position (Figure 1). Careful kinetic analysis showed that at higher concentrations (>100 µM), peptide 10 inhibited the MetAP activity (data not shown). During HPLC analysis of the MetAP reaction mixture, we observed that peptide 10 formed a complex with the Co2+ ion (used in MetAP assays). The peptide-metal complex had a different retention time from the free peptide and disappeared when a lower concentration of Co2+ ion was used in the assay buffer. Presumably, during library screening, the His-containing peptides inhibited the MetAP enzymes by binding to their active-site metal ion(s). Inhibition of MetAP by Met-X-His peptides had also been reported by other investigators (16).</p><p>Kinetic assays of human MetAP1 and MetAP2 against the above peptides also confirmed their specificity profiles revealed by library screening. Indeed, both human enzymes disfavor acidic residues. Replacement of a Glu at the P2' position by either Arg or His increased the catalytic activity by 2–22-fold (Table 2, compare peptides 1, 7, and 10). Likewise, a Glu at the P4' position reduced the activity of both enzymes by 7–12-fold (compare peptides 10 and 12). Human MetAP2 is more tolerant than MetAP1 to larger P1' residues. While all of the peptides containing Ala, Gly, or Pro at the P1' position had similar activities toward the two enzymes (MetAP2/1 ratio between 0.43 and 3.7), peptides 6 and 11, which contain a Val at the P1' position, were two orders of magnitude more active toward MetAP2 than MetAP1. MetAP2 also had a 16-fold higher activity than MetAP1 toward a Thr-containing peptide (peptide 5). To test whether this is a general property of MetAP2, we synthesized five additional Met-Val peptides, derived from the N-terminal sequences of human proteins sulfurtransferase, cyclophilin A, hemoglobin α chain, thioredoxin-like protein 1 (TXNL1), and attractin, which are known to undergo N-terminal methionine removal in vivo (Table 2, peptides 16–20) (37). All five peptides had kcat/KM values in the range of 2000–10000 M−1 s−1 toward MetAP2, but were only poorly active toward human MetAP1 (74–200-fold lower activity). Our data suggest that these five proteins, and likely all Met-Val- and Met-Thr-containing proteins in human, are mainly processed by MetAP2 in vivo. Previous X-ray crystal structural studies have shown that human MetAP1 has a narrower active-site cleft than MetAP2 (by ∼1 Å) and the reduced size restricts the access of fumagillin and related compounds to the active site of MetAP1 (26). Similarly, the smaller active site of MetAP1 would sterically clash with the larger side chains of Val and Thr at the P1' position. Finally, we tested two Trp-containing peptides (Table 2, peptides 14 and 15) to determine the effect of Trp on MetAP activities. Surprisingly, although none of the enzymes selected Trp among their most preferred substrates (Figures 1–3), both peptides (which contain a Trp at the P2' and P3' positions, respectively) were efficient substrates of human MetAP1 and MetAP2. A possible explanation for this discrepancy may be that beads displaying hydrophobic Trp-containing peptides swelled poorly in the aqueous screening buffer, rendering the peptides on these beads poorly accessible to the MetAP enzymes.</p><!><p>To validate the in vitro substrate specificity data, we examined the N-terminal Met removal from phosphoglucomutase 1 (PGM1) and TXNL1 in vivo. PGM1 and TXNL1 contain N-terminal sequences of MVKIVT and MVGVKP, respectively, and are expected to be specific substrates of MetAP2. The protein BTF3L4, which has an N-terminal sequence of MNQEKL and is not expected to undergo N-terminal Met removal, was used as a negative control. Human embryonic kidney epithelial cell line HEK293T overexpressing C-terminally Flag-tagged PGM1, TXNL1, or BTF3L4 proteins were treated with IV-43 [a specific MetAP1 inhibitor (38)] and/or TNP-470 [a specific MetAP2 inhibitor (13)] and then pulsed with [35S]Met to label any newly synthesized proteins. The putative substrate proteins (and control) were immunoprecipitated from the cell lysates with antibodies against the Flag tag. The partially purified proteins were then treated with recombinant MetAP1 and MetAP2 in vitro to cleave any retained N-terminal [35S]Met residue, which was caused by inhibition of the cellular MetAPs. Treatment of the cells expressing TXNL1 with either TNP-470 alone or TNP-470 and IV-43 in combination, but not IV-43 alone, resulted in a two-fold increase in the N-terminal Met retention over the DMSO control (Figure 4). No significant increase in Met retention was observed for cells expressing BTF3L4 or PGM1. These results demonstrate that TXNL1 is indeed a specific substrate of MetAP2. The result with PGM1 was unexpected and may be caused by one or both of the following factors. Inspection of the structure of PGM1 shows that the retained Met would be buried in the folded protein and thus inaccessible to MetAP action (39). Alternatively, the N-terminal Met might be N-acetylated upon forced retention in cells and protected against MetAP2 in vitro. In this regard, treatment of human and mouse cells with TNP-470 resulted in the retention and acetylation of the N-terminal Met of cyclophilin A, a well-established MetAP2-specific substrate (40).</p><!><p>Previous studies have led to the conclusion that both bacterial and eukaryotic MetAPs prefer small amino acids (e.g., Ala, Gly, Ser, Pro, Cys, Thr, and Val) as the P1' residue (17–25). Our results confirmed these earlier findings. Several studies demonstrated the importance of residues at positions P2' and beyond for catalytic activity (20, 21). The only systematic study of MetAP substrate specificity was carried out by Frottin et al. (25), who synthesized and assayed ∼120 short peptides against E. coli MetAP1 and P. furiosus MetAP2. Their data suggest that N-terminal methionine removal from proteins in vivo is dictated by the substrate specificity of MetAP(s). Our data on E. coli MetAP1 are largely consistent with the results of Frottin et al, but with one key difference. Frottin et al. reported that E. coli MetAP1 had poor activity against peptides containing acidic residues at P2'–P4' positions. In contrast, our library screening and kinetic analysis of individually synthesized peptides both showed that the E. coli enzyme prefers acidic residues at P2', P4', and P5' positions. This discrepancy is likely due to the fact that Frottin et al. employed very short peptides containing free C-termini (primarily tri- and tetrapeptides), which generally had poor activities toward the E. coli enzyme (kcat/KM ∼ 102–103 M−1 s−1). As described previously, our screening data revealed a negative correlation between the P2' and P3' positions with regard to acidic residues (i.e., the enzyme disfavors the presence of negative charges at both positions). Presumably, the negatively charged C-terminus of the peptides used in the Frottin work interfered with the binding of acidic peptides to the enzyme active site.</p><p>Proteomics analyses of TNP-470 treated cells have identified several mammalian proteins as MetAP2-specific substrates, including bovine cyclophilin A (N-terminal sequence MVNPTV) (41) and glyeraldehyde-3-phosphate dehydrogenase (MVKVGV) (41) and human thioredoxin (MVKQI) (40), SH3 binding glutamic acid rich-like protein (MVIRV) (40), and elongation factor-2 (MVNFT) (40). In vitro kinetic assays of the N-terminal peptides of these proteins confirmed that they all have two orders of magnitude higher activity toward MetAP2 than MetAP1. Thus, together with TXNL1 identified in this work, all six MetAP2 specific substrates contain a Val as the P1' residue, consistent with our library screening data. One known exception is human protein 14-3-3γ, which became incompletely processed when either normal or tumor epithelial cells were treated with siRNA specific for MetAP2 or MetAP1-specific inhibitor IV-43 (30, 42). The N-terminal sequence of this protein, MVDREQ, suggests that it is a poor substrate for both MetAP1 and MetAP2 (Table 2, entry 6). It is thus not surprising that an increase in retention of its N-terminal methionine was observed upon inhibition of either MetAP1 or MetAP2, suggesting that both MetAP1 and MetAP2 are necessary to completely process its N-terminal Met.</p><!><p>On the basis of the specificity data from the current work and in the literature (43), we make the following predictions about the N-terminal Met removal (Table 3). In E. coli, proteins containing small residues Ala, Cys, Gly, Pro, and Ser as the P1' residue and any amino acids other than Pro at the P2' position are expected to undergo complete Met cleavage. When the P1' residue is any amino acid other than Ala, Cys, Gly, Pro, Ser, Thr, and Val or if the P2' residue is Pro, the N-terminal Met is retained. When the P1' residue is Thr or Val and the P2' residue is not Pro, N-terminal Met cleavage is variable, depending on several factors such as the actual N-terminal sequence at P2'–P5' positions, the level of protein expression, and the growth condition of the cell. In mammalian cells, proteins containing Ala, Cys, Gly, Pro, or Ser at the P1' position generally undergo complete N-terminal processing, catalyzed by MetAP1, MetAP2, or both. When the P1' residue is Thr or Val, the N-terminal Met removal is primarily catalyzed by MetAP2 and the extent of cleavage depends on the sequence at P2'–P5' positions. When the P2' residue is not Asp, Glu, or Pro, the N-terminal processing is expected to be complete. When Asp, Glu, or Pro is the P2' residue, Met removal is either incomplete or does not occur. It should be noted that these rules are meant to be a general guideline and occasional exceptions have been observed.</p><!><p>We have developed a novel peptide library method to systematically profile the sequence specificity of bacterial and human MetAPs. The results show that the specificity of MetAPs is largely dictated by the P1' residue, however, the sequence at P2'–P5' positions does have a significant effect on the extent of Met removal, especially for proteins containing less optimal P1' residues (e.g., Thr and Val). Further, we show that the primary difference between human MetAP1 and MetAP2 is their differential tolerance for Thr and Val at the P1' position, suggesting that MetAP2 is primarily responsible for N-terminal processing of proteins that contain N-terminal Met-Val and Met-Thr sequences. This difference in substrate specificity between human MetAP1 and MetAP2 may be ultimately responsible for the unique effects of inhibitors of MetAP2 on angiogenesis. Finally, our data have permitted us to formulate a more complete set of rules for predicting the fate of N-terminal Met of bacterial and mammalian proteins.</p><!><p>This work was supported by grants from the National Institutes of Health (GM062820, CA132855, and CA078743). BN was supported by the NIH Medical Scientist Training Program Grant T32GM07309.</p><p>Supporting Information Available: Tables containing the peptides sequences selected against the three MetAP enzymes and additional figures. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>2-aminobutyrate</p><p>N-hydroxylsuccinimidyl 3-[4-(4-dimethylaminophenylazo)benzoylamino]propionate</p><p>N-(4-[4'-(dimethylamino)phenylazo]benzoyloxy)succinimide</p><p>N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl</p><p>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide</p><p>N-(9-fluorenylmethoxycarbonyloxy) succinimide</p><p>O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate</p><p>1-hydroxybenzotriazole hydrate</p><p>methionine aminopeptidase</p><p>norleucine</p><p>one-bead-one-compound</p><p>polymerase chain reaction</p><p>partial Edman degradation-mass spectrometry</p><p>amino polyethylene glycol</p><p>phosphoglucomutase</p><p>phenyl isothiocyanate</p><p>thioredoxin-like protein 1</p><p>Substrate specificity of E. coli MetAP1. (a) Most preferred substrates selected from sub-library III (total 236 sequences); (b) most preferred substrates from sub-library I and II (total 124 sequences); (c) Val-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 92 sequences); (d) Ala-, Abu-, and Gly-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 140 sequences); and (e) sequences derived from colorless beads in sub-library III (total 169 sequences). Displayed are the amino acids identified at each position (P1' to P5'). Occurrence on the y axis represents the number of selected sequences that contained a particular amino acid at a certain position.</p><p>Substrate specificity of human MetAP1. (a) Most preferred substrates selected from sub-library III (total 128 sequences); (b) most preferred substrates from sub-library I (total 74 sequences); (c) most preferred substrates selected from sub-library II (total 43 sequences); (d) Ala-, Abu-, and Gly-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 50 sequences); and (e) Val-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 24 sequences). Displayed are the amino acids identified at each position (P1' to P5'). Occurrence on the y axis represents the number of selected sequences that contained a particular amino acid at a certain position.</p><p>Substrate specificity of human MetAP2. (a) Most preferred substrates selected from sub-library III (total 632 sequences); (b) most preferred substrates from sub-libraries I and II (total 144 sequences); (c) Ala-, Abu-, and Gly-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 52 sequences); and (d) Val-containing sequences (at P1' position) derived from medium colored beads in sub-library III (total 16 sequences). Displayed are the amino acids identified at each position (P1' to P5'). Occurrence on the y axis represents the number of selected sequences that contained a particular amino acid at a certain position.</p><p>Retention of the initiator methionine of TXNL1 caused by inhibition of MetAP2 by TNP-470. (a) Immunoblotting (IB) of immunoprecipitated (IP) C-terminal 3xFlag-tagged BTF3L4 protein from HEK293T cell lysate; (b) Immunoblotting of immunoprecipitated C-terminal 3xFlag-tagged PGM1 protein from HEK293T cell lysate (3W, three times washes with RIPA buffer; 6W, six times washes with RIPA buffer); (c) Immunoblotting of immunoprecipitated C-terminal 3xFlag-tagged TXNL1 protein from HEK293T cell lysate; (d) and (e) Immunoprecipitated C-terminal 3xFlag-tagged BTF3L4, PGM1 and TXNL1 were aliquoted for [35S]-methionine scintillation counting after incubation with MetAP reaction buffer (d) or after in vitro processing by both MetAP1 and MetAP2 (e). IV-43 or IV (10 µM), TNP-470 or T (100 nM) or both were added for the last 4.5 h. The error bars are standard errors from two (BTF3L4 and PGM1 in (d)), three (TXNL1 in (d) and BTF3L4 and PGM1 in (e)) or five (TXNL1 in (e)) independent experiments. N-Terminally methionine unprocessed BTF3L4, PGM1 and TXNL1 have 3, 12 and 8 methionine residues per molecule, respectively. * p=0.02; *** p=0.0003.</p><p>Synthesis of MetAP Substrate Librarya</p><p>aReagents and Conditions: (a) glutaric anhydride (3 equiv), DIPEA, CH2Cl2; (b) 5,5-dimethylcyclohexane-1,3-dione (4 equiv), EDC (2 equiv), DMAP (2 equiv), CH2Cl2; (c) NH2-(CH2)5-NH2 (20 equiv), CH2Cl2; (d) solid-phase peptide synthesis using Fmoc chemistry, split-and-pool synthesis at random positions, and deprotection; (e) 1.5% NH2NH2 in THF/H2O (1:1).</p><p>Two Methods for Library Screening against MetAP</p><p>Most Preferred Substrates of E. coli MetAP1 Selected from Sub-library III (236 Total)a</p><p>Sequences were obtained from five screening experiments performed at 75–100 nM E. coli MetAP1. U, α-L-aminobutyric acid; M, norleucine.</p><p>Peptide selected for further kinetic analysis.</p><p>Kinetic Constants of E. coli and Human MetAPs against Selected Peptidesa</p><p>All peptides contained a free N-terminus and a C-terminal amide. NA, no detectable activity; ND, not determined.</p><p>Peptides selected from the combinatorial library.</p><p>Peptides derived from human proteins.</p><p>Prediction of N-Terminal Met Removal in E. coli and Mammalian Cytoplasm</p><p>"<M" indicates that the Met is at the N-terminus of a protein; "[ACGPS]" indicates that the P1' residue is Ala, Cys, Gly, Pro, or Ser; "[^P]" indicates that Pro is excluded from the P2' position.</p>

PubMed Author Manuscript

Side Chain Conformational Averaging in Human Dihydrofolate Reductase

The three-dimensional structures of the dihydrofolate reductase enzymes from Escherichia coli (ecDHFR, ecE) and Homo sapiens (hDHFR, hE) are very similar, despite a rather low sequence identity. Whereas the active site loops of ecDHFR undergo major conformational rearrangements during progression through the reaction cycle, hDHFR remains fixed in a closed loop conformation in all of its catalytic intermediates. To elucidate the structural and dynamic differences between the human and E. coli enzymes, we carried out a comprehensive analysis of side chain flexibility and dynamics in complexes of hDHFR that represent intermediates in the major catalytic cycle. NMR relaxation dispersion experiments show that, in marked contrast to the functionally important motions that feature prominently in the catalytic intermediates of ecDHFR, millisecond time scale fluctuations are not detectable for hDHFR side chains. Ligand flux in hDHFR is thought to be mediated by conformational changes between a hinge-open state when the substrate/product-binding pocket is vacant and a hinge-closed state when this pocket is occupied. Comparison of X-ray structures of hinge-open and hinge-closed states shows that the \xce\xb1F helix changes position by sliding between the two states. Analysis of \xcf\x871 rotamer populations derived from measurements of 3JC\xce\xb3Co and 3JC\xce\xb3N couplings indicates that many of the side chains that contact the \xce\xb1F helix exhibit rotamer averaging that may facilitate the conformational change. The \xcf\x871 rotamer adopted by the Phe31 side chain depends upon whether or not the active site contains substrate or product. In the hE:NADPH holenzyme, a combination of hinge-opening with a change in the Phe31 \xcf\x871 rotamer opens the active site to facilitate entry of substrate. Overall, the data suggest that, unlike ecDHFR, hDHFR requires minimal backbone conformational rearrangement as it proceeds through its enzymatic cycle, but that ligand flux is brokered by more subtle conformational changes that depend on the side-chain motions of critical residues.

side_chain_conformational_averaging_in_human_dihydrofolate_reductase

<!>Protein Purification and Sample Preparation<!>Methyl Side Chain Assignments<!>13C Methyl R2 Relaxation Dispersion Experiments<!>Measurement of 3J couplings and determination of \xcf\x871 rotamer populations<!>Chemical Shift Derived Rotamer Populations<!>Resonance Assignments<!>Millisecond Timescale Methyl Dynamics<!>3JC\xce\xb3CO and 3JC\xce\xb3N Couplings and Side Chain Rotamer Populations<!>DISCUSSION<!>Comparison of Rotamer Averaging in the hDHFR and ecDHFR Michaelis Model Complexes<!>Rotamer Averaging in the hDHFR Catalytic Intermediates<!>hE:FOL:NADP+ compared to hE:THF:NADP+<!>Average \xcf\x871 conformation in hE:NADPH<!>Average \xcf\x871 conformation in hE:THF<!>Phe31 is a substrate/product gate keeper<!>Role of side chain disorder in movement of the \xce\xb1F helix<!>CONCLUSIONS

<p>E. coli dihydrofolate reductase (ecDHFR, ecE)(1) has long been a paradigm for the study of the relationship between enzyme dynamics and function.1–13 Interestingly, its human counterpart (hDHFR, hE) achieves the same catalytic function, with arguably better efficiency,14 by utilizing quite different dynamic behavior.15 hDHFR and ecDHFR share the same major catalytic cycle (Figure 1). The flexible Met20 loop of ecDHFR samples distinct closed and occluded conformations as ecDHFR proceeds through its enzymatic cycle: the ground states of ecE:NADPH and ecE:FOL:NADP+ (a model for the Michaelis complex, ecE:DHF:NADPH) have a closed Met20 loop whereas the product complexes have an occluded Met20 loop.7,16 The equivalent "Met20 loop" region(2) of hDHFR has the sequence LPWPP instead of MPWN in ecDHFR and is locked in a closed conformation throughout the catalytic cycle.15 Ligand binding in hDHFR occurs through a hinge opening motion of the adenosine subdomain relative to the loop subdomain (Figure 2), rather than from the conformational sampling of the Met20 loop as in ecDHFR. The hinge motion in hDHFR is likely mediated by longer hinge loops than are present in ecDHFR: in hDHFR, hinge-1 is defined as residues Thr39 through Leu49 and hinge-2 as His127 through Leu131, compared to Thr36 through Pro39 and Pro105 through Gln108 in ecDHFR.15 Substrate complexes of hDHFR adopt the hinge-closed conformation, where the active site is tightly packed, thereby favoring the hydride transfer reaction. 15N R2 relaxation dispersion experiments reveal conformational fluctuations on a millisecond time scale in all of the intermediates of the major enzymatic cycle of ecDHFR.6,8 In contrast, millisecond timescale dynamics are not observed in the corresponding complexes of hDHFR but motions on a faster µs timescale are observed in the hE:FOL:NADP+ complex.15</p><p>We have recently demonstrated the exquisite sensitivity of 3JCγCO and 3JCγN couplings to the average χ1 dihedral angle conformation and rotamer averaging of side chains in the complexes of ecDHFR that constitute intermediates in the enzymatic cycle.12 Here, we extend this analysis to hDHFR, with the timescale independent side chain χ1 conformational information for γ-methyl and aromatic residues obtained from 3JCγCO and 3JCγN coupling measurements. This coupling-derived rotamer data is augmented by rotamer population estimates based on 13Cmethyl chemical shifts for leucine residues.</p><!><p>Human DHFR was expressed and purified as described previously.17 Briefly, hDHFR was expressed in BL21 (DE3)(DNAY) cells in M9 minimal media with 2 mM folate at 37 °C. Different isotope labeling patterns were used for assignment, dynamics, and rotamer experiments. U-[1H, 15N] samples were produced using 15N ammonium chloride (0.5 g/L) and 15N ammonium sulfate (0.5 g/L). All 3J coupling experiments were performed on U-[1H, 13C, 15N] samples grown in 100% H2O, with [U-13C] glucose (3 g/L) and 15N salts. A U-[13C, 15N], 1H/2H labeled sample (with a CHD2 dominant methyl isotopomer) was prepared by growth in 100% D2O media. Samples for 13C methyl CPMG relaxation dispersion experiments were prepared using 100% H2O, the 15N salts, and [1-13C] glucose to achieve the labeling pattern described by Lundström et al.18 Samples for stereochemical assignment of leucine and valine methyls were produced by the method of Neri et al.19 using 10% 13C glucose (0.4 g/L) and 90% 12C glucose (3.6 g/L). 1 L cultures were grown to an OD600 of ~0.9, induced with 1 mM IPTG at 15°C. Cells were harvested after ~24h and pellets were frozen until purification. Cells were lysed in the presence of folate. hDHFR was eluted from a Q-sepharose column with weak salt (<100mM NaCl) and further purified by HPLC purification, taking care to avoid an impurity which elutes at slightly higher percentage acetonitrile. Complexes were prepared by unfolding lyophilized protein in 8M urea containing 10 mM DTT, followed by rapid dilution into Tris buffer at pH 8.5 containing the desired ligands. After concentration of the solution, exchange into the final degassed buffer was performed in an argon-equilibrated glove box due to the highly oxygen-sensitive nature of THF and NADPH solutions. NMR buffer contained 10% D2O/90% H2O or 100% D2O, as applicable, with 50 mM KPi, 50 mM KCl, 1mM DTT, and 1mM EDTA at pH 6.5 for all samples, except for hE:NADPH which was prepared at pH 8.0 for increased stability. Final sample concentrations ranged from ~300 µM for binary complexes to 1 mM for ternary complexes. The complexes were formed by addition of 6-fold excess cofactor [NADP+ or NADPH] and 10-fold excess substrate or product [FOL or THF], as applicable. NMR samples were further degassed on a vacuum line, overlaid with argon, and flame sealed in an amberized tube. Under these conditions the hE:FOL:NADP+ sample is very stable, hE:THF:NADP+ and hE:THF are stable for up to a month, hE:NADPH is stable for ~1 week, and hE:THF:NADPH is extremely unstable, with spectra changing in a matter of hours. The quality of the ligands in the final samples was ascertained by comparison of 1D proton spectra of the samples to reference ligand spectra. High purity (6S)-THF was purchased from Schircks Laboratories and high purity NADPH and NADP+ were purchased from Sigma.</p><!><p>Backbone assignments (HN, N, Cα, and partial Cβ) for all hDHFR complexes were made using triple resonance methods, as reported elsewhere.15 Side chain assignments for hE:FOL:NADP+ were performed at 300 K utilizing standard 2D 15N- and 13C-HSQC experiments and the following 3D NMR experiments, with the labeling scheme, % D2O in the NMR buffer, and 1H spectrometer frequency indicated in brackets: HCCH-TOCSY & HCCH-COSY20 (U-[1H,13C,15N] in 100% D2O; 600 MHz), 120 ms mixing time 13C-NOESY21 (U-[1H,13C,15N] in 100% D2O; 900 MHz), 15N-TOCSY (U-[1H,15N] in 10% D2O; 600 MHz), and a CHD2-selective-TOCSY22 and HNCACB23 (U-[13C,15N], 1H/2H in 10% D2O; 800 MHz). Stereochemical assignments for the hE:FOL:NADP+, hE:THF, and hE:NADPH complexes were obtained from 10%-13C, 15N labeled protein using the CHD2-selective 13C-HSQC experiment described by Otten et al.22 in which Leu-δ2 and Val-γ2 resonances have the same phase as Met-ε resonances. Methyl assignments obtained for the hE:FOL:NADP+ complex were used as a starting point for assignments of 13C methyl resonances in the hE:THF and hE:NADPH complexes, which were finalized using 13C-NOESY and 15N-TOCSY experiments for each complex.</p><!><p>Relaxation dispersion experiments report on ms-µs timescale exchange processes. Methyl 13C CPMG relaxation dispersion experiments were performed on a [1-13C, 15N] hE:FOL:NADP+ sample at multiple temperatures (280 K, 300 K, and 310 K) using a Bruker DRX 800 MHz spectrometer and the pulse schemes described by Skrynnikov et al.24 and Lundström et al.25 Using [1-13C] glucose as the sole carbon source leads to isolated 13C enriched methyls for most methyl side chains; however, the presence of 13C-13Cmethyl labeling for Thr-γ2 and Ile-δ1 methyls can interfere with accurate spin-relaxation measurements due to the one bond C-C coupling.18 Experiments were performed using a total relaxation period TCPMG of 40 ms and refocusing delays 1/τcp of 100, 200, 400*, 600, 1000, 1400*, 1800, and 1900 s−1 where * denotes experiments completed in duplicate. Dispersion curves were fit using the program GLOVE.26</p><!><p>The 3JCγCO and 3JCγN coupling constants describe the average χ1 orientation of the side chain with respect to the backbone. There are established NMR experiments to measure 3JCγCO and 3JCγN coupling constants for Ile, Thr, and Val γ-methyls27,28 and for aromatic residues.29 These rely on 13C-HSQC and 15N-HSQC difference spectra, respectively. A reference spectrum has maximal intensity as coupling is not evolved, and a second spectrum, where the coupling of interest is allowed to evolve, results in peak intensities that are attenuated as a function of that coupling. For the methyl-containing residues, the couplings are calculated from the relationship (Ia−Ib)/Ia = 2 sin2 (πJCγXT), where Ia is the reference spectrum intensity, Ib is the intensity for either the 3JCγCO or 3JCγN experiment, and the delay T is 28.6 ms. Similarly, for aromatic residues the couplings are calculated from the relationship Ib/Ia = cos (2πJCγXT), where Ia is the reference spectrum intensity and Ib is the intensity for the amide of residue i+1 in the 3JCγCO experiment or for residue i in the 3JCγN experiment. The delay T was 25 ms for 3JCγCO experiments and 50 ms for 3JCγN experiments. All 3J measurements were made in triplicate or better to reduce the contribution of spectral noise to rotamer calculations.</p><p>The measured coupling constants allow for the calculation of χ1 rotamer populations assuming a 3-site jump model according to the following equations12,30: (1)Jmeas,CγN3=p180Jt,CγN3+p+60Jg,CγN3+p−60Jh,CγN3 (2)Jmeas,CγCO3=p180Jt,CγCO3+p+60Jg,CγCO3+p−60Jh,CγCO3 (3)p180+p+60+p−60=1 where 3Jmeas,CγN and 3Jmeas,CγCO are the experimentally measured coupling constants; p−60, p60, and p180 are the populations of the respective χ1 rotamer states; and Jt, Jg, and Jh for CγCO and CγN are the expected coupling values for the fully populated 180°, +60°, and −60° χ1 rotamers, respectively (Table 1 and Figure S1). Populations were fit by minimizing the squared difference between measured and calculated 3JCγN and 3JCγCO couplings for each residue: Σ(1/σJmeas)2 (Jcalc - Jmeas)2, where σ is the standard deviation based on the three or more measurements of 3J. Populations for Val residues represent a simultaneous fit of γ1 and γ2 couplings. The extent of rotamer averaging is related to the major rotamer population, pmajor ≡ max[p60, p180, p−60], where more rotamer averaging is associated with a smaller pmajor value. Population ranges were determined using 3J ± σ. Note that small values of 3J are prone to larger errors (by percent) when the primary contribution to error is from noise in the NMR spectra. Unique rotamer averaging is present when the 3J values of a residue in one or more complexes have non-overlapping error ranges.</p><p>While the interpretation of these 3J coupling values in terms of a 3-site rotamer hopping model is generally justified, there are clear cases where the predicted minor rotamer is not sterically feasible, as was observed also for ecDHFR.12 This is especially evident for some well-packed aromatic residues. In such cases it is more appropriate to interpret the 3J couplings in terms of local motions within a rotamer well, where these side chains undergo local averaging about the χ1 angle, or in terms of a relatively fixed but skewed rotamer (i.e. with non canonical χ1). Case and co-workers have demonstrated that local χ1 averaging can lead to a broadening of the expected Karplus curves such that the maximum values are reduced and the minimum values are increased.31 Consideration of these issues for individual residues can give insights into local conformational changes between complexes for residues that can clearly not accommodate the otherwise predicted minor rotamers; pmajor remains a proxy for these types of in-well rotamer averaging since it is a measure of the deviation of the 3J couplings from the expected values for the fully populated staggered rotamers.</p><!><p>There has been recent success in correlating 13C methyl chemical shifts with valine χ1 and leucine and isoleucine χ2 rotamer populations.32,33 The correlation between the chemical shift and dihedral angle has been largely attributed to a γ-substituent effect – the influence of an atom in the gauche position three bonds away – based upon early studies of small molecule 13C chemical shifts.34,35 For leucines, a simple relationship between the chemical shifts of the methyls has been reported (4)Δδ(C13)=Cδ113−Cδ213=−5+10·ptrans where ptrans is the population of the χ2 = 180° rotamer. Despite the nine possible χ1, χ2 pairs for leucine, only two of these are significantly sampled in structures in the PDB: (180°, +60°) and (−60°, 180°).36 Therefore, knowledge of the χ2 rotamer is largely predictive of the χ1 rotamer conformation. This approach was used to determine Leu χ1, χ2 rotamer populations in hDHFR based on the 13Cmethyl chemical shift assignments for hE:FOL:NADP+, hE:THF, and hE:NADPH. To account for contributions of neighboring aromatic rings to the 13C chemical shift, ring currents corrections were calculated using Shifts-4.337.</p><!><p>Methyl side chain assignments for several intermediates of the hDHFR major enzymatic cycle are summarized in Supplementary Table S1. The methyl and backbone assignments for all complexes have been deposited in the BioMagResBank. The 15N-HSQC15 and 13C-HSQC spectra of the hE:THF:NADP+ and hE:FOL:NADP+ complexes are virtually indistinguishable, whereas the spectrum of hE:THF:NADPH shows extensive broadening for resonances of residues near the active site and. complete assignments could not be obtained for this complex. There are substantial chemical shift changes in the hE:NADPH and hE:THF binary complexes for both backbone and side chain atoms relative to the ternary complexes. The average chemical shift differences from the ternary Michaelis model complex hE:FOL:NADP+ are plotted on the structures of hE:NADPH and hE:THF in Figure 3.</p><!><p>We have shown previously using 15N-CPMG R2 relaxation dispersion experiments that there are no detectable millisecond time scale backbone fluctuations in the hE:FOL:NADP+, hE:THF:NADP+, hE:THF, and hE:NADPH complexes.15 To determine whether hDHFR side chains experience µs-ms timescale conformational fluctuations, such as are observed for ecDHFR, 13Cmethyl CPMG relaxation dispersion experiments were performed on the hE:FOL:NADP+ complex over temperatures ranging from 280 K to 310 K. A representative set of methyl relaxation dispersion data is shown in Supplementary Figure S2. While some residues show hints of dispersion at 280 K, e.g. Ile114-Cδ and Ile151-Cγ2, none of the methyl-containing residues show clearly defined 13Cmethyl dispersion curves. This is contrary to what was observed for ecDHFR, which exhibits exchange on the µs-ms time scale for most methyls,38 but is consistent with 15N-CPMG experiments for hDHFR.15 Based on these results, and given that hE:FOL:NADP+ exhibits a single set of resonances in HSQC spectra, any conformational exchange processes must be occurring on a timescale faster than detectable by CPMG relaxation dispersion experiments, and/or involve very small changes in chemical shift.</p><!><p>χ1 rotamer populations for the complexes corresponding to the intermediates in the major enzymatic cycle of hDHFR have been determined from 3JCγCO and 3JCγN coupling constants for Ile, Thr, Val, and aromatic residues. Populations were determined by minimizing the difference between 3Jmeas and 3Jcalc as described in Methods. The rotamer populations at each residue are summarized graphically in Figures S3–S4. χ2 rotamer populations for Leu residues were estimated from 13C methyl chemical shift values according to Equation 4. The χ2 rotamer populations for the Leu residues imply a similar population of the corresponding χ1 rotamer since Leu rotamer pairs are predominantly (χ1, χ2) = (180°, +60°) or (−60°, 180°).36 The 3J coupling constants for methyl and aromatic residues are shown in Figure 4 and Figure 5, respectively, with complete tables of measured couplings and rotamer populations for each complex given in Supplementary Tables S2 through S11. The 13C chemical shifts values and calculated χ2 rotamer populations for Leu residues are given in Table S12 and the χ2 180° populations are shown in Figure 6.</p><p>As was the case for ecDHFR, many residues exhibit the same rotameric averaging in all complexes of hDHFR. These are shown as spheres in Figure 7, colored according to pmajor with a white (pmajor =1) to red (pmajor <0.5) gradient. Residues that exhibit rotamer populations that differ in one or more of the hDHFR complexes are shown in teal. These "variable population" residues cluster largely in the active site and in regions proximal to the ligand binding pockets. Some residues beyond these zones also exhibit variations in rotamer averaging: of particular note are three residues (Tyr156, His127, and Leu97) proximal to the αF helix, which slides ~2.5 Å between the hinge-closed and hinge-open states of the protein. Rotamer populations determined using the 3-site staggered rotamer assumption are indicated for all residues, but as mentioned in the Methods section, this interpretation may not be appropriate for some well-packed aromatic residues, for which the 3J couplings are better interpreted as reporting on local motions about the major χ1 rotamer instead of rotamer hopping.12</p><!><p>As has been shown by Dunbrack and co-workers, the backbone dihedral angles are predictive of the primary χ1 rotamer sampled by side chains.36 Indeed, the major rotamers for the hDHFR complexes in solution are consistent with the favored rotamer based on the X-ray backbone dihedrals for that complex (Figure S5). 3J couplings and chemical shift values can give information about the distributions of rotamer populations sampled in solution, both in terms of staggered rotamer hopping and in-well rotamer averaging. Analysis of these rotamer distributions in the intermediate complexes of the hDHFR enzymatic cycle gives insights into the structural variation hDHFR undergoes during catalysis.</p><!><p>Rotamer populations were previously determined for the complexes representing the intermediates in the E. coli DHFR catalytic cycle.12 Many residues in the active sites of hDHFR and ecDHFR are conserved. These are shown in green in Figure 8A if the side chains are identical, or in cyan if the residue is conserved (Leu, Ile, or Val in both ecDHFR and hDHFR, or aromatic in both). A comparison of the 3J couplings for the identical methyl and aromatic residues indicates that the average χ1 conformation is essentially the same in the human and E. coli enzymes (Figure 8B). The residues showing the greatest deviation are Val50 and Tyr121 (Val40 and Tyr100 in ecDHFR), which are sensitive to the bound ligands.</p><p>Differences in rotameric averaging are observed for those residues that are not conserved between hDHFR and ecDHFR (termed non-equivalent residues). In situations where an aromatic residue has been substituted for a methyl residue (red residues in Figure 8A), the aromatic shows a decrease in rotamer averaging relative to the methyl residue. An example of this is a residue at the interface of the Met20 and FG loops - Phe142 in hDHFR, which is replaced by Val119 in ecDHFR. Val119 displays a very high level of rotamer averaging in the occluded, but not the closed, complexes of ecDHFR. In hDHFR, which has a closed Met20 loop throughout the catalytic cycle, Phe142 is predominantly in the χ1 = −60° rotamer conformation in all complexes. The non-equivalent Ile, Thr, and Val residues of hDHFR show an enhanced degree of rotameric averaging compared to ecDHFR, with an average pmajor of 0.68 ± 0.18 in hDHFR versus 0.90 ± 0.07 in ecDHFR for the E:FOL:NADP+ complex (see Figure S6 for histograms of the pmajor distributions for hDHFR and ecDHFR E:FOL:NADP+). Many of the residues with enhanced rotamer averaging in hDHFR are located in loop regions that represent insertions into the core secondary structure shared by both the human and E. coli enzymes. This is particularly apparent in the hDHFR hinge regions, where increased rotamer averaging of small side chains along domain interfaces may facilitate the quasi-rigid body movements seen in the hinge-open versus hinge-closed states.</p><!><p>Residues showing variable rotamer averaging in one or more complexes of hDHFR (teal residues in Figure 7) are located predominantly in the adenosine binding subdomain (upper half of each structure). Unlike ecDHFR, which exhibits variable rotamer averaging for numerous residues in the loop subdomain,12 hDHFR, in which the "Met20" loop that remains closed throughout the catalytic cycle, shows similar levels of rotamer averaging in the loop subdomain for all complexes (Figure 7). The major exception is Phe31 on the αC helix, which has a different primary rotamer in hE:NADPH versus the substrate/analogue bound complexes. Many of the residues with variable rotamer averaging in the hDHFR complexes appear to be sensitive to variations in the position of the αF helix, which is adjacent to hinge-2 (Figure 2). In contrast, the methyl side chains in and around the hinge-1 region show similar, elevated levels of rotamer averaging in each of the hDHFR complexes.</p><!><p>The 15N-HSQC and 13C-HSQC spectra for hE:THF:NADP+ and hE:FOL:NADP+ are virtually identical, indicating that the bound FOL and THF interact in very similar manner with the protein backbone and side chains in the ternary complexes (Figure S7). This is in marked contrast to E. coli DHFR, where substantial changes in chemical shift are observed between FOL and THF complexes due to different interactions between the protein and the pterin ring; the protonated N8 atom of the pterin ring in THF (and DHF) forms a hydrogen bond with the carbonyl of Ile5, an interaction that cannot be made by FOL7,11. It is interesting that these differences between the human and E. coli enzymes are paralleled in the binding affinities. FOL, DHF, and THF all bind hDHFR with similar affinities.14 In contrast, ecDHFR binds FOL, DHF, and THF incrementally more tightly, with about an order of magnitude difference in affinity between each successive ligand.39</p><p>Methyl 13C chemical shifts are very sensitive to rotamer populations.32,40–42 The 13C HSQC spectra of hE:FOL:NADP+ and hE:THF:NADP+ are very similar, indicating that the distributions of rotamers for these complexes are virtually indistinguishable. This is also confirmed by comparison of 3JCγCO and 3JCγN coupling constants. Based on the 3J couplings, Val8 is an apparent exception; however, the large uncertainties in the small 3J values for this residue make the rotamer analysis unreliable and methyl 13C chemical sifts suggest that the rotamer population is similar (predominantly χ1 = +60°) in all complexes.</p><!><p>As is shown in Figure 3, many backbone and side chain resonances of the hE:NADPH complex, generally associated with residues in the substrate binding pocket and the cofactor binding site, exhibit different chemical shifts than in the ternary complexes. The changes in average χ1 conformation also span these regions. Unlike ecE:NADPH, which shows a widespread increase in χ1 rotameric averaging throughout the complex compared to the ecE:FOL:NADP+ complex, several residues in hE:NADPH show decreased rotamer averaging relative to the ternary complexes. This is the case for Tyr156 which has an average χ1 conformation that depends on the conformation of the αF helix as will be discussed in detail below. Val50 also shows decreased rotamer averaging in hE:NADPH compared to each of the other complexes. This is likely to be a consequence of the hinge-open conformation of hE:NADPH compared to the hinge-closed conformation of each of the other complexes. Phe31 adopts a unique χ1 conformation in hE:NADPH compared to the other complexes, as will be discussed below. Several residues do show increased rotamer averaging in the hE:NADPH complex compared to the other hDHFR complexes. The reduced 3JCγN and 3JCγCO coupling constants of Trp57 and Phe58, respectively, in hE:NADPH compared to the hinge-closed complexes (Tables S7–S11) suggests an increase in in-well χ1 rotamer averaging of these residues. Ile114 also shows an increase in rotamer averaging. Ile114/Val115 are analogous to Val93/Ile94 of ecDHFR. In ecDHFR, Ile94, with its side chain oriented towards substrate, was found to be especially sensitive to the nature of the bound ligand in the substrate binding pocket.12 Val115 which is oriented towards substrate in hDHFR does not appear to have the sensitivity to substrate that the larger Ile side chain in ecDHFR does. Instead, hDHFR Ile114 which faces away from the substrate binding pocket and towards the αF helix, shows elevated rotamer averaging in the binary complexes. It is not clear if this is a consequence of changes in the position of the αF helix, which appears to drive the rotamer averaging in numerous other residues, or if it is an effect propagated from the substrate binding site.</p><!><p>As in the hE:NADPH complex, several residues in hE:THF are sensitive to the conformation of the αF helix, discussed below. Unlike hE:NADPH, most of the chemical shift differences in hE:THF relative to the ternary complexes are associated with residues clustered near the vacant cofactor binding site (Figure 3). The absence of NADP/H leads to an increase in rotamer averaging for Leu75. Ile60, Ile114, and Val115 also show increased rotamer averaging relative to the ternary complexes. Ile114 shows elevated rotamer averaging that is similar to that of hE:NADPH. In hE:THF, the methyl groups of Ile60 and Val115 show significant chemical shift changes relative to hE:FOL:NADP+, which is consistent with a change in rotamer averaging. Increased averaging of these residues, which are both near the bound substrate, may be a consequence of their position along the trajectory taken by αF as it slides from one position to the other.</p><!><p>It has been suggested on the basis of X-ray structures and molecular dynamics simulations that Phe31, located on the αC helix following the 'Met20' loop, functions as a gate keeper residue that controls access to the active site.15,43,44 In solution, the 3J coupling constants show that the Phe31 side chain is fully in the χ1 = −60° rotamer when substrate or product is bound, and fully occupies the χ1 = 180° rotamer in the hE:NADPH complex, where the substrate/product binding pocket is empty (Figure 9). The combination of hinge-opening plus rotation of Phe31 into the 180° χ1 rotamer in the hE:NADPH holoenzyme opens the active site to facilitate entry of substrate. However, once bound, the para-aminobenzoylglutamate (pABG) moiety of the substrate would clash sterically with the phenyl ring,15,43 triggering the conformational transition to the −60° rotameric state of Phe31. In the −60° rotamer, the phenyl ring is packed tightly against the pterin ring of the substrate and may contribute to transition-state stabilization, since the hydride transfer rate of Phe31 mutants is significantly decreased.45 Indeed, when the equivalent residue at this site in ecDHFR, Leu28, is mutated to phenylalanine, the hydride transfer rate is greatly enhanced, emphasizing the importance of this residue in active site organization.46 Product release requires the reverse transition, in which the conformation of the Phe31 side chain changes from χ1 = −60° to the 180° rotamer of the holoenzyme. It is notable that the energetics are such that Phe31 fully populates either the −60° or the 180° rotamer in the various catalytic intermediates, and we see no evidence for rotamer averaging in any of the complexes at the level of uncertainty of our measurements.</p><!><p>The primary conformational change that hDHFR undergoes as it progresses through the catalytic cycle is between a hinge-closed state, in complexes where substrate or product is bound, and a hinge-open state formed when the substrate/product binding pocket is vacant, i.e. in the E:NADPH complex. One of the structural consequences of this is a change in location of the αF helix, which is adjacent to hinge-2. In transitioning from the hinge-open state to the hinge-closed state, the αF helix slides ~2.5 Å towards the active site (Figure 2B).15 In the hinge-closed state, helix αF is restrained by a pair of hydrogen bonds involving the side chains of His127 and Tyr156. Tyr156, which is on the β-sheet of the loop subdomain, forms a hydrogen bond from its hydroxyl group to the backbone CO of Met125 in the hinge-closed but not in the hinge-open complexes, since the shift in the αF helix in the hinge-open conformation increases the distance between these atoms from 2.6 to 4.3 Å (Figure 10A, B). In the hE:THF:NADP+ and hE:FOL:NADP+ complexes the 3JCγCO value is reduced relative to the binary complexes, suggesting that Tyr156 in the ternary complexes adopts a more skewed χ1 rotamer to form the hydrogen bond or that the side chain undergoes more in-well rotamer averaging while tethered to the Met125 backbone.</p><p>His127 is located in hinge-2, at the C-terminus of the αF helix. In the hinge-closed conformation, His127-Nε2 is close enough to Asp94-COδ to form a hydrogen bond, which cannot be formed in the hinge-open hE:NADPH complex (Figure 10A, B). The 3JCγN coupling constants for His127 in the ternary complexes and hE:NADPH indicate a fully populated 180° χ1 rotamer, in accord with the X-ray structures. The 3JCγN value is decreased in the E:THF complex, consistent with the skewed rotamer (χ1 = 209°) observed in the crystal structure of the E:FOL complex (1DHF). Leu97 is close in space to His127. In the hinge-closed complexes, the Leu97-Cδ1 methyl is next to the face of the His127 aromatic ring (Figure 10A) and is subject to a ring current shift; in the hinge-open hE:NADPH complex, the Leu97-Cδ1 moves away from the face of the histidine ring (Figure 10B). The 13Cmethyl chemical shifts of Leu97, corrected for the ring current contributions from His127, indicate increased χ2 rotamer averaging of the Leu97 side chain in the hE:NADPH complex than in either of the ternary complexes or the hE:THF complex (Table S12). In each of the hDHFR complexes, the space is available to Leu97 to sample multiple rotamer conformations and the backbone dihedrals would support either rotamer (Figure S5), but the chemical shifts suggest a very low level of rotamer averaging for the hinge-closed complexes. The χ2 +60° conformation would cause the hydrophobic methyl groups to be substantially solvent exposed, such that the better packed χ2 180° rotamer is substantially preferred. This suggests that in the hE:NADPH hinge-open state that the +60° rotamer of Leu97 is more protected from solvent than in the hinge-closed complexes, primarily as a consequence of the changed conformation of the adjacent αF helix.</p><p>The methyl-containing side chains that undergo rotamer averaging in the hinge-open hE:NADPH complex cluster in functionally important regions (Figure 10C). One such cluster (colored red in Figure 10C) includes Leu75, Leu93, Leu97, Ile114, and Leu133, all of which are in contact with the αF helix; Val120 on αF also displays rotamer disorder. Dynamic disorder in these side chains may well play a direct functional role by facilitating sliding of helix αF during the hinge-open/hinge-closed transition. A second cluster (Leu49, Leu99, Thr100, Leu105, Val109, and Val112; colored blue in Figure 10C) is located at the packing interface between the C-terminal end of helix αE and the β-sheet of the adenosine-binding subdomain, a region which contacts hinge-1 and undergoes structural rearrangement during opening or closing of the hinges. Several side chains in hinge-1 (colored pink) also exhibit rotamer averaging. Thus, it appears that the core of the adenosine-binding subdomain has intrinsic side chain flexibility that may function to "lubricate" the movement of secondary structure elements during hinge transitions. Most of these residues also undergo rotamer averaging in the hinge-closed complexes (hE:FOL:NADP+, hE:THF:NADP+, and hE:THF), showing that the flexibility is an intrinsic property of the adenosine-binding subdomain and is not influenced by the nature of the bound ligands. In contrast to the adenosine-binding domain, relatively few residues in the loop subdomain exhibit side chain disorder. Rotamer averaging is observed for Val135 and Ile151, which pack against Leu133 and may help accommodate its motions.</p><!><p>Conformational and dynamics data for a wide range of timescales have been collected for side chain residues of various complexes of human DHFR that represent intermediates in the catalytic cycle. Consistent with what has been found for the backbone,15 there is no evidence for millisecond timescale motions of the side chains in hDHFR complexes. This is either due to exchange processes that are too fast to detect with CPMG relaxation dispersion experiments, or because any exchange processes present do not cause significant changes in the chemical shift. Backbone R1ρ dispersion experiments show µs exchange processes in the hE:FOL:NADP+ complex,15 suggesting that similarly fast dynamics may be present for hDHFR side chains.</p><p>In contrast to ecDHFR, which undergoes transitions between closed and occluded active site conformations that influence side chain rotamer populations in the various catalytic intermediates12, hDHFR remains in the closed conformation in all complexes. As a consequence, differences in side chain χ1 and/or χ2 rotamer populations and averaging between the various hDHFR complexes are relatively small and are influenced more by the presence or absence of ligands, rather than conformational change. For hDHFR, the only substantial structural change is associated with a subdomain rotation that opens the active site cleft and causes helix αF to slide relative to neighboring secondary structural elements; most of the residues that pack against this helix exhibit rotamer disorder in all complexes studied and may "lubricate" the movement of the helix.</p><p>These data paint a picture of hDHFR as an enzyme that requires minimal conformational rearrangement as it proceeds through its enzymatic cycle. The hinge-open or hinge-closed state of the adenosine-binding subdomain and the side chain conformation of Phe31 appear to contribute in important ways to ligand flux and active site packing in human DHFR. However, we have thus far found no evidence for transient sampling of higher energy conformational substates in the catalytic intermediates of hDHFR that may facilitate progression through the reaction cycle. This is in marked contrast to the E. coli enzyme, in which millisecond time scale fluctuations play a major role in modulating the energy landscape and regulating ligand flux.8 Backbone motions may be present and necessary for hDHFR activity, but have been undetectable by the experiments thus far performed, which probe only millisecond time scale fluctuations. Based on our observation of pervasive backbone motions on the µs timescale in human DHFR,15 it seems highly likely that catalytically relevant motions in the human enzyme are much faster than in E. coli DHFR. Characterization of these motions will be the subject of future investigations of human DHFR complexes.</p>

PubMed Author Manuscript

Affinity capture mass spectrometry of biomarker proteins using peptide ligands from biopanning

Affinity capture mass spectrometry was used to isolate and ionize protein A from Staphylococcus aureus from both a commercial source and cell culture lysate using matrix assisted laser desorption/ionization mass spectrometry. Two surfaces are compared: gold surfaces with immunoglobulin G covalently immobilized and silica surfaces with a covalently bound small peptide discovered via biopanning. A detection limit of 2.22 bacterial cells/mL of culture fluid was determined using the immobilized peptide surfaces. This study emphasizes the ability to use peptide ligands to effectively capture a biomarker protein out of a complex mixture. This demonstrates the potential to use biopanning to generate capture ligands for a large variety of target proteins, and subsequently detect the captured protein using MALDI mass spectrometry.

affinity_capture_mass_spectrometry_of_biomarker_proteins_using_peptide_ligands_from_biopanning

Introduction<!>Materials<!>Antibody Plates<!>Peptide Plates<!>Cell Culture of Staphylococcus aureus<!>Capture of protein A from commercial product and cell lysate<!>MALDI TOF Mass Spectrometry<!>Surface Production for Affinity Capture Mass Spectrometry<!>Capture of Commercially Available Protein A<!>Capture of Protein A from Cell Lysate<!>Cell Concentration Gradient with Silica-Peptide Surfaces<!>Conclusions

<p>Detection of bacterial contamination, whether attributed to environmental outbreaks or biological warfare, has gained increasing importance over the last decade. In either situation, knowledge of the type and extent of bacterial contamination requires the use of a fast analytical technique with high sensitivity and selectivity. Traditional methods for pathogen detection require the collection and growth of microorganisms prior to biochemical assays, which is both time consuming and, dependent on growth media, may lead to biased results because of selective cell outgrowth.1,2 In recent years, the polymerase chain reaction (PCR) has increasingly been used to detect bacterial DNA.3,4 However, a standard sample is often a complex mixture containing several PCR inhibitors, particularly metal chelators, and DNA from many organisms may be present. In such cases, PCR results can be ambiguous, and requires the extraction of DNA, which can result in sample loss and can also be time consuming. Furthermore, a biological warfare agent may consist of only a single toxin protein, eliminating the presence of detectable DNA.5 It is in these situations that affinity capture mass spectrometry is ideally suited; it has the ability to extract biomarker proteins of interest and allows for rapid and sensitive detection.</p><p>Detection of bacterial contamination and strain typing using mass spectrometry is a well known technique.6-11 The current limitations of the application of mass spectrometry in biological studies lies in the great number of proteins and other biological molecules being ionized that may not be exclusive to one organism or strain. Additionally, some qualitative and quantitative variability is based on the media type used.12 The use of capture ligands to extract one protein of interest from this complex mixture offers the advantage of detecting a single biomarker that would be indicative of bacterial presence without a requirement for intact cells. Several studies have shown the successful use of antibodies bound to a solid surface for isolating a protein of interest from a complex mixture.13,14 While antibody capture is a viable technique, antibodies tend to be time consuming to generate, have storage and stability limitations such as proper buffering and temperature sensitivity which can prove difficult in some environmental and biological samples,15 and require additional chemistry to ensure proper orientation on a surface for solvent exposure to the epitope.13,16-18</p><p>Recently, a new technique involving biopanning with phage-displayed peptides offers the ability to identify small peptides that can be used in a similar manner to antibodies for on-target capture of biomarkers. This technique commonly uses a library of filamentous bacteriophages displaying short peptides fused to the pIII minor coat protein.19 Other variants utilize other microorganisms for surface display, and the displayed ligands can include small scaffold proteins, including the Z domain of Protein A and antibody fragments.20,21 By incubating this phage library with a surface coated with the biomarker protein and washing away unbound phage, it is possible to isolate and amplify a phage displaying a peptide that has high specificity for the protein of interest.22 This technique has been applied to the development of biosensors using dye labels15 and intrinsic fluorescence.23 The present report demonstrates the ability of a biopanning-generated peptide to capture protein A from complex mixtures. The peptide is covalently bound to a silica substrate via a linker and detection of the target protein is achieved after capture by directly ionizing from the surface with matrix-assisted laser desorption/ionization mass spectrometry. This technique has the added advantage of obtaining a m/z of the protein, whereas spectroscopic assays can merely tell whether a fluorescent probe is bound without determining the extent of non-specific adsorption.</p><p>The work presented here demonstrates a proof of concept that utilizes the protein A from Staphylococcus aureus, a cell wall-attached protein that traditionally binds to the Fc domain of immunoglobulins.31 A peptide ligand has also been generated for this protein using biopanning, allowing for clear comparison to antibody based affinity capture. The results from this work illustrate the potential to use biopanning-generated peptide ligands for affinity capture mass spectrometry to detect biomarker proteins of interest from a variety of biological and environmental samples.</p><!><p>Epichlorohydrin, diethylene glycol dimethyl ether, bromoacetic acid, ethanolamine hydrochloride, Immunoglobulin G from human serum, protein A from Staphylococcus aureus, bovine serum albumin, 3-aminopropyltri(ethoxy)silane, 25% glutaraldehyde, HPLC grade toluene, and 1,12-diaminododecane were purchased from Sigma-Aldrich (St. Louis, MO). Potassium hydroxide and microslides were purchased from VWR (West Chester, PA). NaOH was purchased from J.T. Baker (Phillipsburg, NJ), dextran T5000 was purchased from Amersham Biosciences (Pittsburgh, PA), 16-mercaptohexadecanol was purchased from Frontier Scientific (Logan, UT), N-ethyl-N-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and sinapic acid were purchased from Fluka Analytical (St. Louis, MO), and N-hydroxysuccinimide (NHS) was purchased from Acrös Organics (Morris Plains, NJ). Sodium cyanoborohydride was purchased from MP Biomedicals (Solon, OH), sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (Sulfo-SMCC) crosslinker was purchased from Pierce Biotechnology (Rockford, IL), the phage display selected peptide was synthesized by Sigma-Genosys, and the gold surfaces were produced by Evaporated Metal Films (Ithaca, NY).</p><!><p>The procedure for covalent immobilization of IgG on gold coated plates is performed using a carboxymethyldextran modified self-assembled monolayer as described by Löfås.24 Briefly, a 1.35mm × 1.75mm gold-coated glass chip was modified with a self assembled monolayer by immersion into 5mM 16-mercaptohexadecanol in 80:20 (v/v) ethanol:water for 48 hours at room temperature, utilizing the ability of thiols to adsorb on gold surfaces.25 This surface was then treated with 0.6M epichlorohydrin in 1:1 (v/v) 0.4M sodium hydroxide with diethylene glycol dimethyl ether for 4h at room temperature to form an epoxide. The epoxide was then modified with 0.3g/mL dextran T5000 in 0.1M sodium hydroxide for 20 hours at room temperature. The dextran layer was then modified to a free carboxylic acid by exposure to 1M bromoacetic acid in 2M sodium hydroxide for 16 hours at room temperature. The plates were washed with sonication 6 × 1 min between each step.</p><p>Immobilization of IgG on this surface was described by Johnsson.26 Briefly, the free carboxylic acid was reacted with 0.2M N-ethyl-N'-(dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.05M N-hydroxysuccinimide (NHS) for 15 minutes at room temperature. This allows for the nucleophilic displacement of the NHS ester with free amines to covalently bind the antibody. A 1ug/uL solution of IgG from human serum in phosphate buffered saline (PBS) was added to the plates and allowed to bind for 30 minutes at room temperature. After washing, unreacted NHS esters were capped with 1M ethanolamine hydrochloride pH = 8.5 for 10 minutes at room temperature.</p><!><p>Standard glass microscope slides were cut into 2.5mm × 2.5mm chips and cleaned for 1 hour in 10% (w/v) potassium hydroxide and dried for 1 hour at 100°C. A 2% (v/v) 3-aminopropyltri(ethoxy)silane solution in HPLC grade toluene was poured over the plates and reacted for 30 minutes with gentle shaking at room temperature. Excess silane was washed away with toluene, and a 2.5% solution of glutaraldehyde in pH 8 buffer was poured over the plates and reacted for 45 minutes. Excess glutaraldehyde was washed away with water, and the plates were reacted with 2% (w/v) 1,12-diaminododecane in toluene and was allowed to react with gentle shaking overnight. The plates were washed with copious amounts of toluene, and the surface was reacted with 0.1 mg/mL sodium cyanoborohydride for 1 hour with shaking. A 4.8 mg/mL solution of sulfo-SMCC crosslinker was prepared and reacted by spotting on the plates for 30 minutes, leaving a free maleimide group for reaction with the peptide ligand, which has a cysteine residue on the C-terminus. The peptide used for this capture surface was previously described,23 and its amino acid sequence, His-His-Lys-His-His-His, was modified at the C-terminus with Gly-Gly-Gly-Cys, where the glycine residues provide added flexibility and separation from the crosslinker, and the thiol of Cys allows for attachment to the maleimide crosslinker. A 1mg/mL solution of this peptide reduced with TCEP was reacted with the surface for 2 hours.</p><!><p>ATCC strain 12598 (Cowan Strain) was used to produce protein A for capture from lysed cell culture. The culture was maintained on blood agar plates (BAPs) until use. A single colony from a BAP was introduced to 25 mL of LB broth and the culture was allowed to grow overnight at 37°C with shaking. A 1mL aliquot from culture was pelleted with centrifugation, culture media was decanted and cells were resuspended in a 1 mg/mL solution of lysozyme. This suspension was then heated at 90°C for 1 hour to lyse the cells27. For the determination of the limit of detection, serial dilutions were prepared as follows. Cells were counted by light microscopy using a Petroff-Hauser counting chamber. A series of 9 consecutive 1:10 dilutions were prepared by taking aliquots from culture media, pelleting with centrifugation and lysing as discussed above.</p><!><p>Prepared plates, with antibody and peptide ligand, were treated with both commercial protein A (Strain 8325-4) and cell lysate (Cowan strain). It is important to note the difference in strains used for commercial and cell culture experiments. Commercial protein A was collected from the 8325-4 strain and the Cowan strain was grown in culture. These strains differ slightly in their protein A sequence, as well as in the number of IgG binding domains. A more thorough description of the strain differences will follow in the Results section. A 20μM solution of commercial protein A was prepared in PBS, and 50μL was spotted on each of the plates to cover. In the cell lysate experiment, 100μL was spotted to completely cover the plates. Protein A was allowed to react with both surfaces for 30 minutes prior to washing with water.</p><!><p>After capture by antibody or peptide, the chips were mounted on a MALDI plate (Microtitre plate (MTP) adapter for Prespotted AnchorChip Targets plate, Bruker Daltonics, Billerica, MA) with double sided tape. The surface of each plate was then spotted with 1μL of a saturated solution of sinapinic acid in 70:30:1 H2O : acetonitrile : trifluoroacetic acid, with three different spots prepared on each surface to allow for averaging of signal across the plates. Additionally, 1μL of 15 μM bovine serum albumin in saturated sinapinic acid solution was spotted on a blank plate to allow for external mass calibration. These spots were allowed to crystallize in ambient conditions. The plate was inserted into a Bruker Reflex III MALDI-TOF mass spectrometer (Bruker Daltonics) equipped with a nitrogen laser (λ = 337 nm) for ionization. Each spot was subjected to 300 laser shots, allowing for signal summing and averaging. Following ionization, ions are accelerated by a 20-keV electric field, travel down the drift tube and were detected in linear detection mode, allowing for the observation of high mass/charge ions. Spectra were exported as ASCII files and were processed using Microcal Origin 6.0 (Microcal Software Inc., Northampton, MA).</p><!><p>Two different surface constructions were used: gold surfaces with a modified self-assembled monolayer for antibody attachment, and silica surfaces modified with a hydrocarbon tether for peptide attachment. Each surface type was chosen based on tethering techniques used previously.23,24,26 Figure 1 illustrates schematically the chemistry involved in binding the ligand to the surface. There are advantages to using each surface. The gold surface utilizes a carboxymethyl-dextran hydrogel layer, allowing for both spatial separation of the attached antibody from the denaturing conditions of the gold surface, but also allows for the surface to be negatively charged, allowing for a "preconcentration" of protein by attraction to this surface, facilitating the kinetics of covalent attachment of the antibody.26 Using this hydrogel layer also minimizes the contribution of non-specific adsorption caused by interaction of solutes with the metal surface. Experiments were done without the use of this hydrogel layer with lysed cells, and results showed evidence of solute binding in the absence of a capture ligand (data not shown). While this binding was not at the same molecular weight of protein A, further experiments used this hydrogel layer to avoid any surface reactivity.</p><p>For tethering peptides, a less costly silica surface is used, and the binding of the peptide is straightforward, with the surface first silanized with a free amine exposed for attachment to glutaraldehyde. Interaction with a long chain amine at the free aldehyde end of glutaraldehyde provides the spatial distance necessary to reduce steric hindrance of the tethers by increasing the distance of the tethered peptide from the surface, assuming free rotation of the tether. Reacting this free amine with an amine-reactive crosslinker that is also thiol reactive on the exposed end allowing for the covalent bonding of a C-terminal cysteine containing peptide. Furthermore, binding at this C-terminal Cys allows for orientation of the peptide, leaving the N-terminal binding portion exposed.</p><p>The major advantage of using the peptide-linked surfaces over antibody-linked surfaces is stability and size. Antibodies, like proteins, have limitations in optimal temperature due to the necessity to maintain stable secondary and tertiary structure for activity. Additionally, the production of antibodies can result in a wide variety of structures. While the use of monoclonal antibodies minimizes heterogeneity, multiple isoforms are still produced due to differences in glycosylation and terminal processing which can result in antibodies with differences in binding efficiencies and specificity.28-30</p><!><p>After surface preparation, a solution of protein A was spotted and allowed to react for 30 minutes with both antibody and peptide linked plates, followed by several water washes to remove any unbound protein. Each surface was analyzed by MALDI-TOF MS in linear mode with external calibration using bovine serum albumin.</p><p>Protein A consist of 4 to 5 homologous IgG-binding regions displayed externally on the cell with a C-terminal portion that is responsible for binding to the peptidoglycan of the cell wall, where the number of IgG binding regions varies with strain.31 The Cowan strain, used for cell culture experiments, has 4 of these units (DABC), and strain 8325-4, used for commercial Protein A experiments has 5 (EDABC).32 Each of these regions is approximately 6.5-7 kDa in size33,34 and are each separated by an arginine residue that acts as a tryptic cleavage site. Protein A is bound to the cell wall via region X, which is approximately 20 kDa,35 and consists of many octapeptide repeats that allow the protein to bind to the peptidoglycan layer.</p><p>Additionally, each strain also produces slightly different protein A with varying amino acid sequence. Most of the differences seen in sequence can be attributed to point mutations.32 The expected molecular weights have been calculated from amino acid sequences derived from genetic information and Edman degredation. Table 1 shows these molecular weights, but also shows that different investigators have reported different sequences, further indicating the variability of protein A. Because the literature shows some inconsistency in the sequence, a spectrum of commercial protein A acquired for comparison using a standard MALDI plate is shown in Figure 2.</p><p>Figure 3 illustrates the ability of both the antibody and peptide surfaces to bind commercially available protein A isolated from strain 8325-4. Figures 3a and d show a mass spectrum of matrix ionized from the unmodified gold-antibody and silica-peptide plates, respectively as a negative control. Figures 3b and 3e show ionization from surfaces that had been fully modified with the antibody and peptide, respectively, in the absence of captured protein to demonstrate that the covalent attachment of the ligands were stable under ionizing conditions. Figures 3c and 3f show the mass spectrum of captured commercial protein A ionized from the two surface types. In these spectra, signal is produced from single IgG binding domains of protein A as well as 2, 3 and 4 intact domains, and region X with IgG binding units retained. A small peak is seen at approximately 55kDa, indicative of intact protein A, as well as a slight shoulder on the 27kDa peak, which represents region X bound to a single IgG binding unit, both of which are unlabelled due to low signal intensity. Because the purification of protein A from culture involves intact cells versus traditional expression in E. coli, it is likely that the truncated forms of protein A seen here, and subsequently later in culture based assay, is due to endogenous protease activity during cell lysis.</p><!><p>After successful capture of protein A from commercial sources, capture from cell lysate was initiated. Staphylococcus aureus Cowan strain (ATCC 12598) was grown in LB medium, and cells were lysed at 90°C with lysozyme as discussed above. This cell lysate was allowed to react with the plate surfaces for 30 minutes followed by several washes prior to ionization to remove any unbound or non-specifically bound protein. Both antibody-capture and peptide-capture surfaces were analyzed by MALDI-TOF MS in linear mode with external calibration with bovine serum albumin.</p><p>Figure 4 illustrates spectra obtained for protein A capture with gold-antibody plates (a) and silica-peptide plates (b) from cell lysate. Because cell lysis was performed with lysozyme, which should disrupt the peptidoglycan layer, it is expected that region X remains bound to the cell wall. This released DABC, the IgG binding units, from the cell into the media. These spectra illustrate capture of a single IgG binding unit, two IgG binding units, and all four IgG binding units intact. It is important to note the lack of non-specific adsorption or capture of other proteins from the lysate with these surfaces. If the ligands were cross-reactive with any other S. aureus proteins, those proteins would be detected. However, the spectra show signal corresponding to protein A only. Both gold-antibody and silica-peptide plates have high specificity for protein A, and show similar signal intensity. While it was expected that the signal intensity would be higher with peptide capture, it is possible that binding kinetics play a role in the strength of signal seen for antibody plates. The Kd of the protein A-IgG interaction has been reported as 10 nM36. Because the dissociation kinetics of the peptide with protein A are unknown, it is not possible to assess which has tighter binding affinity. However, it is hypothesized that the antibody possibly has a lower Kd, resulting in a higher concentration of protein A on the surface when compared to peptide plates. This assay demonstrates that the silica-peptide surface has comparable utility as previously reported antibody plates.</p><!><p>The data presented thus far demonstrated that the binding capabilities of the silica-peptide plates are comparable to traditional gold-antibody plates. Also, these plates have the added advantage of having minimal buffer requirements, greater thermal stability, lower cost, and ease of preparation23. Additionally, the peptide is significantly smaller than an antibody, allowing for a higher loading capacity on the surface. This theoretically increases the concentration of protein able to be captured on the plate, resulting in a lower detection limit, assuming that the tethers are not tightly packed.</p><p>To demonstrate the ability of silica-peptide surfaces to detect bacterial protein at low concentrations, a gradient was set up via serial dilutions of cells prior to cell lysis and protein A detection. For this analysis, aliquots of cell media of known concentration were diluted into fresh media, cells were pelleted and resuspended in 1 mL lysozyme. Cells were lysed at 90°C for 1 hour prior to surface capture. Each cell lysate was allowed to react with the silica-peptide surfaces for 30 minutes prior to MALDI-TOF analysis. The experiment was repeated in triplicate to ensure reproducibility. Spectra in Figure 5 illustrate an increase in signal intensity corresponding to an increase in cell concentration, and the minimum detectable concentration of cells was 2.22 cells/mL (S/N = 3). MALDI is inherently non-quantitative; signal intensity varies within an individual spot depending on crystallization of the matrix as the laser is rastred over the spot37. Therefore, the limit of detection has been defined here as the minimum cell concentration at which a detectable signal is present.</p><!><p>The ability to capture and analyze protein A from Staphylococcus aureus cell lysate has been demonstrated by using both gold-antibody and silica-peptide plates coupled with MALDI-TOF mass spectrometry. While both surfaces have shown utility in detecting a biomarker protein out of a complex mixture, silica-peptide plates have the added advantage of ease in preparation and stability. Furthermore, these peptide plates utilize the relatively new technology of biopanning for biosensor reagents, allow for the identification of a capture ligand for virtually any protein of interest and provide an affinity-capture method using a non-antibody-based ligand. More importantly, we have shown that low concentrations of Staphylococcus aureus cells, approaching PCR detection limits, can be detected. Biopanning for phage-display peptides allows for the development of affinity ligands from virtually any organism, permits ease in utilizing these peptides with affinity capture mass spectrometry, and holds great potential for application to developing a diagnostic for emerging pathogens.</p>

PubMed Author Manuscript

Hypervalent iodine promoted the synthesis of cycloheptatrienes and cyclopropanes†

A new strategy is reported for intramolecular Buchner-type reactions using PIDA as a promotor. Traditionally, the Buchner reaction is achieved via Rh-carbenoids derived from RhII catalysts with diazo compounds. Herein, the first metal-free Buchner-type reaction to construct highly strained cycloheptatriene- and cyclopropane-fused lactams is presented. The advantage of these transformations is in their mild reaction conditions, simple operation, broad functional group compatibility and rapid synthetic protocol. In addition, scaled-up experiments and a series of follow-up synthetic procedures were performed to clarify the flexibility and practicability of this method. DFT calculations were carried out to clarify the mechanism.

hypervalent_iodine_promoted_the_synthesis_of_cycloheptatrienes_and_cyclopropanes†

Introduction<!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!>Conclusion<!>Author contributions<!>Conflicts of interest

<p>Dearomatisation is an attractive synthetic strategy for the construction of polycyclic molecules as a variety of aromatic compounds are commercially available.1 Among the various de-aromatisations, the Buchner reaction has become an effective method for the preparation of cycloheptatrienes, which are important precursors for non-benzenoid aromatic tropylium ions and useful building blocks in synthetic methodology and total synthesis in the presence of transition metal catalysts.2</p><p>Traditionally, the Buchner reaction refers to the cyclopropanation of a benzenoid double bond with metal carbene to generate a norcaradiene, and subsequent electrocyclic ring opening leading to a cycloheptatriene (Scheme 1a).3 The reaction has been extensively studied by the Doyle, Merlic, Xu groups, and others, using mainly copper or rhodium complexes.4 Other metal catalysts, such as Ru, Co, Ag and Au salts were also screened.5 In these transformations, metal catalysts were essential to fulfill the control of reactivity and selectivity. Furthermore, diazocarbonyl compounds are required as the common surrogate of the carbene complexes in these reaction designs. Recently, the Wang group has described a novel Buchner reaction from N-tosylhydrazones through the treatment with base, based on in situ generation of diazo compounds (Scheme 1b).6 From the aspects of sustainability, environmental friendliness and safety, a metal-free promoted Buchner reaction, related to non-diazo compounds would be highly desirable.</p><p>Carbocation chemistry has been firmly established as one of the most important topics in organic synthesis, and cation intermediates have been thoroughly investigated in the past century, thus in turn leading to the development of new synthetic methodologies.7 Despite these achievements, a metal-free cation-induced [2 + 1] cyclization (CIC) to generate a norcaradiene or carry out a Buchner-type reaction has not yet been reported. We speculate such a protocol might be attractive as it enables the metal-free Buchner reaction without applying diazo compounds and expands the development of carbocation chemistry. Moreover, a CIC strategy might promote the construction of cyclopropanes via a new route not limited to the traditional name reactions such as the Simmons–Smith cyclopropanation, the Johnson–Corey–Chaykovsky reaction or the Kulinkovich reaction.8 Hypervalent iodine(iii) reagents have attracted great attention due to their high reactivity, especially in the construction of C–N bonds.9 For example, nitrenoid species resulting from hypervalent iodine(iii) reagents, combined with metal catalysts may undergo intramolecular C–H amination, representing a common strategy for the synthesis of highly complex nitrogen heterocyclic compounds.10 Recently, the Shi group has published a metal-free synthesis of γ-lactams using hypervalent iodine,11 in which nitrenium ions were generated through the treatment of amides with iodine(iii) reagents.12 Based on our previous research on hypervalent iodine and the synthesis of nitrogen heterocyclic compounds,13 herein we report such nitrenium ions could be trapped by internal alkynes, thus generating alkenyl cations, which might undergo CIC reaction for the construction of Buchner-type cycloheptatrienes or cyclopropanes (Scheme 1c). Through density functional theory (DFT) calculations, we found that stabilised cationic species would be generated and the formation of the C–N bond proceeds as the key step in the transformations.</p><!><p>Initial exploration was focused on the intramolecular amination of N-((5-phenylpent-4-yn-1-yl)oxy)benzamide I-1, which has been previously studied with respect to Rh(iii)-catalysed intramolecular annulation through C–H activation, and Cu-catalysed annulations via a radical-mediated cascade process by groups of Park, Han and others.14 Various common oxidants (2) including TBHP, H2O2, CAN, NIS, NBS, I2 and m-CPBA were initially employed in HFIP at room temperature, but no reaction occurred (Table 1, entries 1 and 2; for details see the ESI†). When PhI(OAc)2 (PIDA) was used as the transformation promotor, the product 3 was obtained in 41% yield (Table 1, entry 3). The reaction could be accomplished even within 1 min with a yield of 69% (Table 1, entry 4). Switching to other I(iii) reagents dramatically affected the efficiency of the reaction, particularly the use of PhI(OCOCF3)2 which gave an inferior yield (Table 1, entry 5). The fluorinated solvents were crucial for the transformation and we found TFE could efficiently promote the reaction well within 1 minute, while other solvents did not work well (Table 1, entries 6–9). Moreover, prolonging the reaction time to 12 h, 3 was slightly decomposed to give a yield of 83% (Table 1, entry 10). Of note, the use of three-carbon atom tether was vital to the reaction as the substrates with other tether lengths between the oxygen atom and the alkyne moiety did not afford the corresponding products A, B or C.</p><!><p>Reaction conditions: I-1 (0.2 mmol, 1.0 equiv.), oxidant 2 (1.2 equiv.), solvent (4.0 mL, 0.05 M), open in air, room temperature.</p><p>Isolated yield. TBHP = tert-butyl hydroperoxide, PIDA = PhI(OAc)2, PIFA = PhI(OCOCF3)2, HFIP = hexafluoro-2-propanol, TFE = trifluoroethanol, DMF = N,N-dimethylformamide.</p><!><p>With the optimal reaction conditions in hand, the scope of this intramolecular Buchner reaction was examined. The effect of substitutions on the aromatic ring of the benzamides was first investigated. As shown in Table 2, a variety of substituents including methyl-, methoxy-, isopropyl-, tert-butyl-, phenyl-, trifluoromethyl-, nitro-, halogen-, cyano-, ester-, and sulfonyl-groups were compatible with the reaction system. With regard to the para-substituted benzamides, electron-withdrawing substituents afforded higher yields (9–16) compared to those bearing electron-donating groups (4–8), particularly the sulfonyl group gave an almost quantitative yield of 16. The structure of 11 was further confirmed by X-ray crystallography (CCDC: 1874173). For the meta-substituted aromatic amides, the enantiomeric annulation products were obtained in a 1 : 1 ratio, indicating that sterics have a negligible influence on the transformation (17 and 18). Reactions with ortho-substituted benzamides proceeded smoothly to give the corresponding products (19–21) in good yields. The reaction also proceeded effectively for disubstituted phenyl amides and the target products (22 and 23) were obtained in 96% and 99% yield, respectively. This reaction system was also extended to a trimethyl-substituted benzamide proved successful, albeit with a relatively lower yield for 24. However, the direct annulation of the heterocyclic amide did not give the product 25, along with significant decomposition of the starting material. Next, a wide range of substituents on the phenyl group of alkyne moiety were also investigated. It was found that electron-donating and electron-withdrawing substituents were well tolerated in the reaction and the yields of the products (26–45) ranged from 40% to 96%. In general, substrates possessing the electron-withdrawing groups such as fluoro- (30), trifluoromethyl- (33) and cyano- (34) at the para-position of the phenyl ring led to a better performance compared to those with electron-donating groups, including methoxy- (27), phenyl- (28) and tert-butyl- (29). Reactions with chloro- and bromo-substituted benzamides also smoothly proceeded, to yield the corresponding products (31 and 32). Intriguingly, alkyne moieties bearing meta-substituted arenes were more effective than those having same substituents at the ortho-position of aromatic rings (36vs.35; 38vs.37 and 40vs.39), giving in each case higher product yields. Extension of the reaction to disubstituted reagents (41 and 42) at the 3,5-positions of the phenyl ring also proved to be successful. Furthermore, the reactions also worked well with heterocyclic substrates; benzamides bearing pyridinyl- and thiophenyl-groups reacted smoothly under the optimised conditions, affording the corresponding heterocycle-fused lactams (43 and 44) in 81% and 83% yields, respectively. Of note, halogen-containing compounds (31, 32, 39 and 40) could be applied as potential substrates for further functionalisation. To further highlight the attractiveness of this approach, an estrone-containing substrate was subjected to this transformation and the sterically crowded tricyclic framework (45) was afforded in a moderate yield.</p><!><p>Reaction conditions: I (0.2 mmol), PIDA (0.24 mmol, 1.2 equiv.), TFE (4.0 mL, 0.05 M), open in air, room temperature, 1 min, isolated yield.</p><p>The ratio of the unseparated isomers was determined by 1H-NMR analysis.</p><p>The diastereomer was derived from the axial chirality and the ratio was determined by 1H-NMR analysis.</p><!><p>After investigating the scope of aromatic alkyne moieties, other conjugated substituents on the alkyne moiety were tested in the intramolecular Buchner-type reaction and the results are presented in Table 3. The reactions with conjugated ene-yne-benzamides,15 which could be easily prepared through Sonogashira cross-coupling reactions, proceeded successfully to furnish the corresponding products (46–60) in 64–92% yields. It is notable that fluorinated alkenyl-substituted groups (54 and 55) could be employed efficiently in the reaction system. Moreover, the sensitive alkene moiety remained intact under the oxidative reaction conditions, representing a remarkable feature of the reaction protocol.16 Interestingly, in the case of a diyne-containing benzamide, the corresponding product was unstable and could not be isolated, while the 1,2-oxazinane-fused isoquinolin-1(2H)-one product (61) was finally obtained after a period of time through a retro-Buchner-type reaction process. However, the benzamides containing alkynyl iodine and alkynyl bromide moieties were unfortunately not viable via this transformation (62 and 63), due to the rapid decomposition of the products during workup.</p><!><p>Reaction conditions: I (0.2 mmol), PIDA (0.24 mmol, 1.2 equiv.), TFE (4.0 mL, 0.05 M), open in air, room temperature, 1 min, isolated yield.</p><!><p>The cyclopropane subunit is found in more than 4000 natural isolates and 100 therapeutic agents and studies on their synthetic strategies have been further propelled17 The synthetic preparation of cyclopropanes generally requires Simmons–Smith cyclopropanation or transition-metal-catalysed diazo decomposition/alkene insertion.8 Alternative preparative pathways have also been reported including Michael-initiated ring closure (MIRC) reactions and metal-catalysed cycloisomerisation of 1,6-enynes.18 A few examples of metal-catalysis systems involving in C–H activation have been developed.19 However, to the best of our knowledge, a synthetic route based on cation-induced [2 + 1] cyclisation reactions has not yet been reported. Therefore, it is particularly noteworthy that when alkenyl amides were applied as the substrates in this reaction, the above synthetic strategy could be expanded to the synthesis of cyclopropane-fused lactams under mild reaction conditions, which constitute key pharmacophores in many pharmaceuticals.20</p><p>The scope and generality of alkenyl amides of the type II were investigated (Table 4). A variety of functional groups including methyl-, propyl-, alkenyl-, phenyl-, heterocycles, trifluoromethyl-, nitro-, halo- and ester groups were compatible with the reaction system. The products 66–71 were obtained in moderate to high yields. As for substituted cinnamamides, various functional groups were well tolerated, furnishing the corresponding products 72–80 in 55–93% yield. Particularly, the substrates containing strong electron-withdrawing groups such as nitro- and trifluoromethyl-substituents produced 79 and 80 in good yields. Next, a wide range of substituents on the phenyl group of alkyne moiety were also investigated; the results showed that substrates possessing electron-withdrawing groups (84, 85, 87, and 89) have a better performance compared to those with electron-donating groups (81–83, 86, and 88), and the yields of the products ranged from 61% to 93%.</p><!><p>Reaction conditions: II (0.2 mmol), PIDA (0.24 mmol), TFE (2 mL), RT, air, 3 min, isolated yield.</p><!><p>Moreover, the structure of 85 was further confirmed by X-ray crystallography (CCDC: 1883915). This synthetic method is also compatible with diverse substrates decorated with various groups (90–95), for example, the heterocycle-containing product 90 was isolated in a 86% yield. It is worth mentioning that using 3-bromoacrylic amide led to the synthesis of 91 in 83% yield, which is not easily accessed through traditional cyclopropane synthesis strategies and leaves room for further functionalization of cyclopropane. In the cases of the enyne substrates, the transformation proceeded smoothly, thus affording the desired products 92 and 93 in 58% and 64% yields, respectively. The feasibility of extending this methodology to a dimethyl fumarate derivative was also demonstrated in a 74% yield (94). Impressively, the coumarin substrate gave an almost quantitative yield of pentacyclo-lactam 95. Furthermore, the substrate derived from fucidate also provided the desired polycyclic system 96 in moderate yield.</p><p>To gain some insight into the reaction mechanism, a control experiment was performed. A substituent intermolecular competition experiment with I-4 and I-12 was carried out and the results showed that the reaction is nucleophilic in nature (Scheme 2).</p><p>DFT calculations were carried out to better understand the mechanism using the M06-2X functional in Gaussian 09 program (Fig. 1–3).21 More details may be found in the ESI.† As shown in Fig. 1, benzamide 1 was chosen as the model substrate, which coordinates to PhI(OAc)2 to give intermediate Int1, followed by a rapid deprotonation process to afford the slightly endergonic species Int2.</p><p>All the attempts to locate the transition state of this process failed, indicating that this process should take place with low energy barrier. This is quite similar to the observations by the Shi group.11 From Int2, the release of the acetate ligand and following C–N bond formation viaTS1-2 leads the reaction to a stable vinyl cation, Int3. A reaction energy barrier of 22.1 kcal mol−1 is needed for this process, which is able to be overcome under the current reaction conditions studied. Other possible transition states corresponding to the C–H bond activation processes from Int2 were also investigated, with all the possible transition states possessing high energy barriers, thus are not be further discussed here (see more details for the C–H bond activation transition states in the ESI, Fig. S2 and S3†). From Int3, the cation-induced [2 + 1] cyclisation (CIC) reaction occurs via transition state TS3-4 to give rise to a strongly stabilised cationic species Int4, as shown in Fig. 2. This is a rapid process, with a low energy barrier of 0.4 kcal mol−1. Another possible CIC transition state, TS3-4′, achieved in the presence of CH3COO− was also located, which has an energy barrier of 8.0 kcal mol−1, and thus cannot compete with formation of TS3-4.</p><p>Finally, two possible ring re-arrangement pathways, leading to the formation of either [6,6,6]heterocycle-fused rings or [6,5,7]heterocycle-fused rings, were calculated as shown in Fig. 3. In transition state TS4-5, the Buchner-type ring expansion drives the reaction to the seven-membered ring intermediate Int5. The free energy barrier for this process is 6.5 kcal mol−1, which is obviously lower than that for the formation of the [6,6,6]heterocycle-fused rings intermediate Int8viaTS4-8. From Int5, the deprotonation process take place by transition state TS6-7 leading to the formation of the [6,5,7]heterocycle-fused lactam. The overall energy barrier for this reaction process is 22.1 kcal mol−1, indicating that the rate-determining transition state should be involved in the C–N bond formation process with the transition state TS1-2.</p><p>A gram-scale reaction (5.0 mmol) was also performed under the standard reaction conditions to investigate the applicability of the protocol introduced. Impressively, the cycloheptatriene product 3 and cyclopropane product 77 were obtained in 83% and 80% yields, respectively; similar to the yield of the model reaction (Scheme 3).</p><p>Moreover, to make the protocol more practical, iodobenzene was in situ oxidized to the corresponding hypervalent iodine reagent under electrochemical oxidation conditions, which promoted the annulation process smoothly. The results showed the efficiency and reliability of the electrochemical hypervalent iodine reagent generation (Scheme 4).</p><p>A series of follow-up synthetic transformations were also performed, focusing on product 3. 1,3-Dienes are ubiquitousin a wide variety of natural products and serve as versatile building blocks to rapidly increase molecular complexity.22 Various methods have been well-established to synthesise such versatile units.23 In this case, 1,3-diene 64 could be easily obtained from 3 with a good yield by simply increasing the reaction temperature to 80 °C in TFE under a metal-free reaction conditions, thus providing a unique reaction procedure among the well-established protocols. The structure of 1,3-diene 64 was further confirmed by X-ray crystallography (CCDC: 1911414). Moreover, the product 3 could be selectively reduced to the subsaturated carbocycle 65 in an 85% yield and thoroughly reduced to cycloheptane 65′ in a 90% yield under a constant atmosphere of 3 MPa H2. An electrochemical oxidative clean bromination of 3 using NaBr was also performed, producing the corresponding product 3′ in a 69% yield (Scheme 5).</p><p>Furthermore, several organic transformations were performed on compound 77. For example, 77 was hydrolysed in sodium hydroxide solution, giving a functional cyclopropanecarboxylic acid 97 in high yield, providing an effective route to such compounds (Scheme 6, path a). Meanwhile, compound 77 was reduced in the presence of Pd/C to produce 98 in a 90% yield (Scheme 6, path b). Furthermore, compound 77 reacted with NBS and MeOH to form 99 in a 99% yield (Scheme 6, path c). More importantly, it is nontrivial for three azo-groups to be introduced directly using eletrochemical diazidation methods, based on cyclopropane ring-opening in the construction of two six-membered ring systems (100), which could not be easily achieved through traditional synthetic methods (Scheme 6, path d).</p><!><p>In summary, we have developed, to the best of our knowledge, the first metal-free synthesis of heterocycle-fused lactams through Buchner-type reactions and the use of PIDA as promotor, applying three-carbon-atom tethered N-alkoxyamide as effective substrates,24 thus meaning a wide range of useful seven/three-membered carbocycles can be conveniently obtained. This protocol is attractive due to the mild conditions, simple operation and extensive functional group compatibility. Moreover, the reaction is completed in a short time under an air atmosphere at room temperature, proving a rapid alternative synthetic strategy to name reactions such as RhII-catalyzed-Buchner reactions and Michael-initiated ring closures for the construction of cycloheptatrienes and cyclopropanes. DFT calculations show that cation-induced [2 + 1] cyclization (CIC) is involved in the process and the formation of C–N bond determines the critical step. Further exploration on the use of the protocol to synthesise other highly-strained fused heterocyclic compounds is ongoing in our laboratory.</p><!><p>D.-F. Y. and Z.-C. W. conceived and performed the majority experiments. R.-S. G. and G.-Y. R. performed the part of the research work. S.-F. N. and M. L. performed DFT calculations. L.-R. W. and L.-B. Z. conceived and directed the project and wrote the paper. J. S. W polished the manuscript. All the authors discussed the results and commented on the manuscript.</p><!><p>There are no conflicts to declare.</p>

PubMed Open Access

Co-crystal structures of primed side-extending \xce\xb1-ketoamide inhibitors reveal novel calpain-inhibitor aromatic interactions

Calpains are intracellular cysteine proteases that catalyze the cleavage of target proteins in response to Ca2+ signaling. When Ca2+ homeostasis is disrupted, calpain over-activation causes unregulated proteolysis, which can contribute to diseases such as post-ischemic injury and cataract formation. Potent calpain inhibitors exist, but of these many cross-react with other cysteine proteases and will need modification to specifically target calpain. Here, we present crystal structures of rat calpain 1 protease core (\xce\xbcI\xe2\x80\x93II) bound to two \xce\xb1-ketoamide-based calpain inhibitors containing adenyl and piperazyl primed-side extensions. An unexpected aromatic-stacking interaction is observed between the primed-side adenine moiety and the Trp298 side chain. This interaction increased the potency of the inhibitor towards \xce\xbcI\xe2\x80\x93II and heterodimeric m-calpain. Moreover, stacking orients the adenine such that it can be used as a scaffold for designing novel primed-side address regions, which could be incorporated into future inhibitors to enhance their calpain specificity.

co-crystal_structures_of_primed_side-extending_\xce\xb1-ketoamide_inhibitors_reveal_novel_calpain-in

<!>Evaluating the inhibitory potency of 1 and 2 on \xce\xbcI\xe2\x80\x93II and m-calpain<!>X-ray crystal structures of the complexes of \xce\xbcI\xe2\x80\x93II with 1 and 2<!>Discussion<!>Materials<!>3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(6-amino-9H-purin-9-yl)propyl)-2-oxopentanamide (1, Z-Leu-Abu-CONH-(CH2)3-adenin-9-yl)<!>3-(Benzyloxycarbonyl-L-leucylamino)-N-(3-(4-methylpiperazin-1-yl)propyl)-2-oxopentanamide (2, Z-Leu-Abu-CONH-(CH2)3-(4-methylpiperazin-1-yl)<!>Fluorimetric analysis of \xce\xbcI\xe2\x80\x93II and m-calpain inhibition by 1 and 2<!>Determination of \xce\xbcI\xe2\x80\x93II-1 and \xce\xbcI\xe2\x80\x93II-2 crystal structures<!>Supporting Information<!>

<p>In response to Ca2+ signaling, the calpain family of intracellular cysteine proteases catalyzes the limited cleavage of target proteins, resulting in changes to processes such as gene expression, cytoskeleton remodeling and apoptosis.1 Problems arise following ischemic or cerebral injury, when cells lose their ability to regulate Ca2+ influx to the cytoplasm. The elevated Ca2+ concentration leads to calpain hyperactivation, which causes uncontrolled proteolysis and irreversible cell damage. Since their overactivation has been linked to the development of pathological conditions such as stroke, Alzheimer disease, Duchenne muscular dystrophy and cataractogenesis, calpains represent an important class of targets for pharmacological inhibition.2,3</p><p>To date, all known calpain isoforms are multidomain enzymes,4 with a catalytic cleft located at the interface between domains I and II.5 These two domains, which encompass the enzyme's proteolytic core, must each bind one Ca2+ ion to facilitate the rearrangement of the catalytic triad and substrate binding pocket into an active conformation.6 Although the various other domains also contribute somewhat to calpain activation, the susceptibility of full-length calpain to autolysis, subunit dissociation and aggregation following Ca2+ activation has complicated its study in the full-length form.7 The protease core though, remains resistant to autolysis and maintains its Ca2+-dependent activity, albeit, at a significantly reduced level.8 In addition, because of the relative ease with which they can be expressed in E. coli and crystallized, these protease cores have become an invaluable tool for the structure-based design of calpain inhibitors.9 While two structures have been reported for the Ca2+-activated human protease core,10,11 in our hands, the rat protease core has been much easier to purify and crystallize. The sequences for the protease cores of rat and human calpains 1 and 2 show a high degree of identity (87% between rat and human calpains 1 and 70% between rat calpain 1 and human calpain 2). Furthermore, because the active site clefts are particularly well conserved, the rat calpain 1 structure remains a suitable model for designing and studying inhibitors of calpain.</p><p>Of the reversible inhibitors that have been developed to target calpains, most are peptide analogues containing an electrophilic warhead group to covalently modify calpain's active site thiol.9,12,13 Although aldehyde and α-ketoamide functional groups have been widely used as warheads, the latter has emerged as the superior form with respect to both metabolic stability and cell permeability.12 However, the poor specificity of α-ketoamide inhibitors continues to limit their applicability as potential therapeutic agents.2 Consequently, there has been an increasing focus on designing peptidyl "address regions" flanking the warhead to target the inhibitor to the calpain active site. To improve specificity, these "address regions" are designed to correspond with calpain's residue preferences at each position in a peptide substrate. For instance, μ-calpain's protease core (μI–II) demonstrates a preference for hydrophobic residues on the N-terminal (unprimed) side of the scissile bond,14 specifically phenylalanine and leucine at the P1 and P2 positions, respectively. The crystal structure of μI–II in complex with 3 (SNJ-1945),15 a peptidyl α-ketoamide containing this optimized selection, shows each of the two side chains interacting with the substrate binding cleft, thus showing how this unprimed "address region" can target the warhead to calpain's active site. By itself though, this unprimed "address region" is insufficient to confer specificity towards calpain since the P2 leucyl side chain is also accommodated by a hydrophobic pocket in other cysteine proteases.16 Hence, there is an advantage to developing an additional optimal "address region" on the C-terminal (primed) side of the warhead. If the "address regions" on both the unprimed and primed sides can be incorporated into a single inhibitor, it would possess a substantially improved ability to specifically target calpain.</p><p>Previous studies on calpain inhibitors have shown that the extension of an inhibitor into the primed region can increase the inhibitor potency. For instance, Li et al17. showed that the inclusion of an arylalkyl primed-side substituent often improved the potency towards both calpains 1 and 2 as well as cathepsin B. In particular, the best primed-side substituent on the α-keto amide was a (CH2)3-Phe group. In another study, Chattergee et al.18 examined the effect of different primed-side extending biaryl sulfonamides and found that the inclusion of a spacer between the two aryl motifs reduced the inhibitory activity. Donkor et al.19 studied a series of inhibitors containing polar substituents in the P1′ position within the linker to a phenyl group. They modeled the polar group as interacting with Glu261, which we later showed to reside in a flexible loop.15 The common feature amongst these reports though, is that an aromatic moiety within the primed side was potentially beneficial to inhibitory activity. However, without crystal structures of these inhibitors bound to calpain, it is difficult to identify the interactions that these primed-side substituents might make with the active site.</p><p>Here, we present the crystal structures of μI–II bound to two α-ketoamide inhibitors 1,2 (Figure 1), each possessing substituents that extend deeper into the μI–II primed side cleft than previously observed. These structures also show the movement of a flexible gating loop out of the binding pocket in the presence of a ligand moiety stretching into the S-primed subsites. More importantly, the structure of μI–II bound to 1 demonstrates a novel non-covalent interaction between μI–II and the inhibitor, where the aromatic adenine moiety of 1 stacks against a conserved tryptophan in the catalytic cleft of μI–II and forms a hydrogen bond to the side chain of Glu300. These interactions may explain why 1 inhibits μI–II activity more potently than the nearly identical compound 2, which possesses a non-aromatic primed side substituent. The information gained from these structures should provide valuable insights for developing more specific inhibitors that can simultaneously target both the unprimed and primed subsites of calpains.</p><!><p>Upon calcium-activation, the rate of cleavage of the FRET substrate by m-calpain (Figure 2A) was approximately four times higher than that catalyzed by μI–II (Figure 2B). In the absence of either inhibitor, the autolytic inactivation of m-calpain caused the fluorescence intensity to plateau after just a few minutes of reaction. However, this autolytic inactivation was not observed with μI–II. Upon addition of either inhibitor, the increase in fluorescence was immediately attenuated, albeit more noticeably with m-calpain. For both m-calpain and μI–II, 1 caused a more distinct attenuation of fluorescence when compared to 2, indicating that the former is a more potent calpain inhibitor.</p><!><p>Owing to its stability and resistance to autolytic inactivation,8 μI–II was used to form calpain-inhibitor complexes with 1 and 2. A summary of crystallographic data collection and refinement statistics is presented in Table 1. Within the catalytic cleft of μI–II, the electron density of each inhibitor was clearly observed in the inner S-unprimed and S-primed subsites (Figure 3A and B). Electron density was weakest around the P3 phenyl ring of both inhibitors, suggesting that this group was more flexible and formed limited interactions with the S3-unprimed region. Nevertheless, when compared to the previously solved structures of μI–II bound to leupeptin or the α-ketoamide inhibitor 3,15 the position of the P3 phenyl ring in 1 and 2 was consistent with that of the P3 diethylene glycol in 3 and the P3 leucyl group in leupeptin; all four P3 groups overlap closely along the surface of domain I (Figure 4A).</p><p>Both inhibitors 1 and 2 are stabilized within the active site through interactions that are conserved in the structures of μI–II bound to leupeptin or 3. On the unprimed side, hydrogen bonds are formed between the inhibitor's peptide backbone atoms (amide nitrogen and carbonyl oxygen of the P2 residue and amide nitrogen of P1 residue) and Gly208 and Gly271 in the catalytic cleft (Figure 3C and D). At the α-ketoamide warhead of 1, 2 and 3, the newly formed hydroxyl group, which arises from nucleophilic attack by the active site thiol, forms a hydrogen bond with the imidazole ring of His272, while the carbonyl oxygen of the carboxyamide provides further stabilization via two polar contacts with Gln109 and Cys115.</p><p>As would be expected from the highly conserved hydrogen bonding interactions, a high degree of structural alignment was observed at the P2 leucine of each of the inhibitors 1, 2, 3 and leupeptin (Figure 4A). In fact, the backbone and side chains adopt nearly identical conformations. This strong overlap continued into the backbone of the P1 residue and its respective side chain β- and γ-carbons. In addition to the conserved hydrogen bonds, another contributing factor to the high degree of structural alignment is the narrowness of the binding pocket around the P1 residue. As described by Cuerrier et al.,15 this narrow pocket imposes restrictions on the ability of the P1 residue to rotate around the Cα-Cβ bond.</p><p>While the structures of leupeptin and 3 are terminated at, or shortly after the warhead, 1 and 2 possess much longer primed side extensions. Interestingly, the greatest degree of structural divergence between these two inhibitors is concentrated within this region. Structural alignment deteriorates substantially in the 3-carbon chain that lies on the C-terminal side of the warhead and is ultimately transmitted to the terminal ring groups (Figure 4A). It appears that the primed-side 3-carbon chain in 1 may adopt a different conformation from that of 2 in order to facilitate an aromatic stacking interaction between the adenine moiety of 1 and the side chain of Trp298. As shown in Figure 4B, it stacks in a coplanar conformation with respect to tryptophan's indole group, leaving a gap of only 3.5 Å between the two and allowing the formation of a hydrogen bond to Glu300. Neither this coplanar stacking nor any polar contacts were observed between the non-aromatic piperazyl ring of 2 and the primed side pocket (Figure 4C). The absence of this stacking interaction and a hydrogen bond to Glu300 therefore likely accounts for the weaker electron density around the piperazyl ring when compared to that of the adenine moiety (Figure 3A and B).</p><p>For both inhibitor-bound structures, although the electron density of μI–II was strong in most areas, the density was observed to be considerably weaker between residues 251–261, which comprise a known flexible loop region. Fitting these residues to the available density resulted in a loop that adopted an "open" conformation, with Glu261 displaced away from the S1′ subsite. This "open" conformation closely resembles the one reported in the structure of 3 bound to μI–II, but contrasts with the "closed" conformation observed in the leupeptin-bound structure (Figure 5).</p><!><p>As noted in previously solved μI–II structures, residues 251–261 in domain II comprise a flexible loop that possesses weak electron density and is considered to act as a gate for the catalytic cleft.11,15,20, When μI–II is bound to an inhibitor that extends only into the unprimed side, such as leupeptin, the flexible "gating" loop adopts the "closed" conformation, with Glu261 obstructing the S1′ subsite (Figure 5). However, in the presence of an inhibitor such as 1 or 2, which possesses a primed side group that extends into the S2′-subsite, the loop is oriented away from the active site in a much more "open" manner. This "open" conformation was first observed in the structure of μI–II bound to 3, where the inhibitor's P1′ cyclopropyl group was thought to displace Glu261 from the S1′ site.15 Although the purpose of the "open" and "closed" loop conformations has yet to be determined, it is possible that they could be signaling different catalytic stages, possibly via interactions with domains (e.g. domain III) that are absent in the isolated proteolytic core. The open loop conformation for instance, might be signaling the initial substrate-binding event where the polypeptide chain would stretch across the cleft. However, once catalysis has yielded the acyl-enzyme intermediate, which no longer possesses a primed side extension, the loop may close to signify the near completion of catalysis.</p><p>In addition to providing insight about the flexible gating loop, the crystal structures produced from this study also revealed the importance of the newly discovered aromatic stacking interaction between the primed-side adenine moiety of 1 and the indole ring of Trp298. This class of non-covalent interactions, commonly referred to as π-π stacking, has been widely documented in protein-ligand complexes, especially when the ligand is comprised of nucleic acids.21–26 One study in particular examined the stacking interactions between adenine and aromatic residues in the active site of DNA glycosylases.27 Since these enzymes remove damaged bases during the first step of base excision repair28 and have aromatic residues lining their active sites, it was thought that π-π stacking was responsible for substrate recognition and stabilization. By examining the available crystal structures of DNA glycosylases and performing computational analyses of the aromatic interactions, it was determined that out of the four aromatic amino acids (His, Phe, Tyr and Trp), the strongest stacking with adenine occurred in the presence of tryptophan, an apparent consequence of its side chain's large surface area and dipole moment.27 Furthermore, the optimal perpendicular separation between tryptophan and adenine was determined to range from 3.5 Å to 3.6 Å, which matches the distance observed in the complex of μI–II with 1.</p><p>Based on the energetic consequences of π-π stacking and considering that compounds 1 and 2 differ only with respect to their terminal primed side ring groups, the aromatic interaction therefore may explain the greater inhibitory potency of 1 over 2 observed in the fluorimetry assays (Figure 2). Through this stacking, the terminal primed side end of 1 is held more rigidly in place and likely works in conjunction with the unprimed side polar contacts to properly orient the α-keto carbonyl relative to the active site thiol (Figure 3). In contrast, the absence of this aromatic interaction in the structure of μI–II with inhibitor 2 causes the piperazyl ring to exhibit greater flexibility, which could be transmitted towards the warhead group, thereby impairing the α-keto carbonyl from achieving the necessary orientation required for potent inhibition.</p><p>Incidentally, the residue that facilitates the π-π stacking interaction, Trp298, is also involved in the later stage of active site assembly, where its side chain undergoes a radical change in position.6 Upon binding of the second and final Ca2+ ion to the μ-calpain protease core, the indole ring of Trp298 moves from its initial wedge-like position between the catalytic domains into the orientation observed in the inhibitor-bound structures. In this position, it helps shield two of the three catalytic triad residues (His272 and Asn296) from solvent exposure and thus facilitates catalysis. Therefore, the π-π stacking is done by Trp298 in its catalytically active conformation, suggesting that inhibitor 1 binds preferentially to the Ca2+-bound, active form of calpain.</p><p>Although the π-π stacking interaction on the primed side cleft is promising from the perspective of structure-guided drug design, it alone is expected to be insufficient to specifically target calpains since other cysteine proteases also possess a tryptophan residue that resides in a similar position to that in the catalytic conformation of calpain.6. As shown in Figure 6, when the structures of the leupeptin-bound papain, human liver cathepsin B and E-64-bound cathepsin K are aligned with that of the μI–II-1 complex, the analogous tryptophan residue in these other cysteine proteases could still interact with the adenine ring of 1, albeit perhaps less optimally when compared to Trp298. What the other papain-like cysteine proteases lack, though, is a residue equivalent to Glu300, so they would be unable to form a hydrogen bond to the amino group of the adenyl moiety. Along these lines, the stacked adenine ring could still be used as a scaffold for other functional groups, which could form contacts with structural features that are unique to calpain's primed side cleft. For instance, the shallow hydrophobic pocket formed by Ala262, Ile263 and Val269 (Figure 4A) is not observed in the primed side cleft of papain, cathepsin B or cathepsin K. Therefore, addition of an aliphatic extension to the adenine ring could facilitate interactions with the hydrophobic pocket. In addition, the replacement of the primary amino group with a positively charged substituent on the adenine moiety of 1 could enhance the electrostatic contacts with the Glu300 side chain. Modification of compound 1 to exploit these calpain-exclusive interactions holds promise for the development of new potent and selective inhibitors.</p><p>In conclusion, we have demonstrated that interactions with calpain's S-primed subsites represent an important aspect of inhibitor design that should not be ignored. As was highlighted in the two crystal structures, the aromatic stacking interaction and hydrogen bond between the primed side adenine ring of 1 and Trp298 and Glu300, respectively, of μI–II, provided the inhibitor with a decisive potency advantage over an equivalent inhibitor lacking a terminal aromatic group. Being the first non-covalent interactions reported on the primed side of a calpain-inhibitor complex, the two high-resolution structures described in this study should contribute greatly to the field of structure-guided drug design and in particular, to the development of potent and specific inhibitors of calpains.</p><!><p>The rat μI–II construct is composed of Gly27-Asp356 from rat μ-calpain and contains an N-terminal Met and C-terminal Leu-Glu-(His)6 tag. The full-length rat m-calpain, comprised of the 80 and 21 kDa subunits, contains the C-terminal Lys-Leu-Ala-Ala-Leu-Glu-(His)6 tag. Both constructs were recombinantly expressed in E. coli and purified as previously described.8,29. The calpain substrate (EDANS)-EPLFAERK-(DABCYL) was synthesized at the Alberta Peptide Institute (Edmonton, Alberta, Canada) and in the Peptide Synthesis Laboratory of the Protein Function Discovery Facility at Queen's University (Kingston, Ontario, Canada). All other reagents were obtained from common sources.</p><!><p>A mixture of adenine (4.05 g, 30 mM), 1-bromo-3-chloropropane (21.3 g, 13.4 mM), and potassium carbonate (10.4 g, 75 mM) in DMF (200 mL) was stirred at room temperature under argon for 4 days, then filtrated and evaporated to dryness. The crude product was washed with water and dried. Recrystallization from ethanol gave 9-(3-chloropropyl)adenine in 59% yield.</p><p>The 9-(3-chloropropyl)adenine (1.9 g, 9 mM) and sodium azide (1.75 g, 27 mM) in DMF was stirred at 80°C for 24 h, cooled to room temperature and filtered. The solid was washed with CH2Cl2. The solvent was removed from the combined filtrates and the residue was taken up in water with sonication. The aqueous layer was extracted with CH2Cl2 (3 × 60 mL). After removing solvent the crude product was recrystallized from ethanol to give 9-(3-azidopropyl)adenine as a white crystalline solid in 81% yield.</p><p>The 9-(3-azidopropyl)adenine(0.5 g, 2.3 mM) and 5 % palladium on carbon (0.5 g) in methanol was reacted with hydrogen gas at room temperature for 22 h. The catalyst was removed by filtration, and the solvent removed to give 9-(3-aminopropyl)adenine as a white solid in 76% yield. 1H NMR (DMSO-d6): 1.80 (m, 2H, CH2), 2.45 (m, 2H, CH2), 3.35 (s, 2H, NH2), 4.20 (m, 2H, CH2), 7.20 (s, 2H, NH2), 8.10 (s, 2H, CH). MS (ED+): 193.0.</p><p>The ketoamide product Z-Leu-Abu-CONH-(CH2)3-adenin-9-yl was obtained from 9-(3-aminopropyl)adenine and the ketoacid Z-Leu-Abu-COOH30 using the EDC/HOBt coupling method, purified by column chromatography on silica gel with 85:15 CH2Cl2:MeOH as the eluant, then recrystallized from CH3COOEt/hexane to give a white solid (27% yield). 1H NMR (CDCl3): 0.91 (m, 9H, CH3 of Val and Abu), 1.60–1.80 (m, 5H, CH2 and CH of Leu and Abu), 2.00 (m, 2H, CH2), 3.20 (2H, CH2), 4.24 (m, 3H, CH2 and α-H), 5.11 (s, 2H, Z), 5.20 (m, 1H, α-H), 6.20 (s, 1H, NH), 6.80 (b, 1H, NH), 7.20–7.40 (m, 6H, Ph and NH), 7.85 (d, 1H, CH of adenine), 8.36 (d, 1H, CH of adenine).</p><!><p>A solution of N-(3-bromopropyl)phthalimide (8.04 g, 30 mM) in xylene (60 mL) was added dropwise to a solution of 1-methylpiperazine (6.61 g, 66 mM) in xylene (90 mL) at 70 °C. After the addition was complete, the mixture was heated under reflux for 20 h. A precipitate was removed by filtration and the filtrate was concentrated. The crude product was purified by silica gel chromatography with 9:1 CH2Cl2:MeOH to give the product N-(3-(4-methylpiperazin-1-yl)propyl)phthalimide as an oil in 72% yield.</p><p>A solution of N-(3-(4-methylpiperazin-1-yl)propyl)phthalimide (6.2 g, 21.6 mM) and hydrazine monohydrate (1.13 g, 26 mM) in ethanol (60 mL) and methanol (60 mL) was refluxed for 4 h. After cooling to room temperature, concentrated HCl (2.4 mL) was added and the mixture heated under reflux for another 1 h. After removing the solvent, water (100 mL) was added, the mixture stirred and insoluble material removed by filtration. Solid K2CO3 (1.2 eq) and CH2Cl2 (100 mL) was added to the aqueous layer, the mixture stirred, and filtered. The organic layer was washed with water (3 × 20 mL). The combined aqueous layers were washed with Et2O. Water was removed from the organic layers, they were then dried and evaporated to give 1-(3-aminopropyl)-4-methylpiperazine as a oil in 39% yield. 1H NMR (CDCl3): 1.55 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.34 (t, 2H, CH2), 2.67 (t, 2H, CH2). MS (ES+): 157.9.</p><p>The ketoamide Z-Leu-Abu-CONH-(CH2)3-(4-methylpiperazin-1-yl) was synthesized from 1-(3-aminopropyl)-4-methylpiperazine and Z-Leu-Abu-COOH30 using the EDC/HOBt coupling method, and purified twice by column chromatography on silica gel using 80:20 CH2Cl2:MeOH and 85:15 CH2Cl2:MeOH as the eluant to give a yellow semi-solid in 16% yield. 1H NMR (CDCl3): 0.91 (m, 9H, CH3 of Val and Abu), 1.60–1.80 (m, 5H, CH2 and CH), 2.00 (m, 2H, CH2), 2.44 (s, 3H, CH3 of piperazine), 2.50–2.65 (m, 8H, CH2 of piperazine), 3.30 (m, 2H, CH2), 4.20 (m, 3H, CH2 and α-H), 5.10 (s, 2H, Z), 5.15 (m, 1H, α-H), 6.70 (b, 1H, NH), 7.20–7.30 (m, 6H, Ph and NH), 8.60 (b, 1H, NH).</p><!><p>Enzymatic activity was monitored in real-time using a 1.5 mL quartz cuvette and a Perkin-Elmer LS50-B Luminescence Spectrophotometer. Duplicate assays were performed with 1.3 μM (EDANS)-EPLFAERK-(DABCYL) (12) and 125 nM μI–II or m-calpain in a buffer solution containing 50 mM HEPES-HCl (pH 7.7) and 10 mM DTT. The reaction was initiated at the 420 s time mark using 4 mM CaCl2 and monitored at excitation and emission wavelengths of 335 nm and 500 nm, respectively. After a waiting period of 100 s, the inhibitor solution (either 1 or 2 dissolved in DMSO) was added at a concentration of 1 μM (for the m-calpain assays) or 50 μM (for the μI–II assays). Control assays were performed by adding an equivalent volume of DMSO after the 100 s delay.</p><!><p>A solution of 325 μM μI–II in 10 mM HEPES-HCl (pH 7.7) and 10 mM DTT was first incubated with 2 mM 1 or 2 at room temperature for 30 min. This solution was then mixed with an equal volume of a precipitant solution to cover conditions that expanded around 1.5–2.0 M NaCl, 10 mM CaCl2, and 0.1 M MES (pH 5.75–6.5). Using the hanging drop vapor diffusion method, crystals were obtained and subsequently soaked in a solution of the mother liquor supplemented with 20% glycerol, prior to data collection. Crystallographic data was collected at beamline X6A at the National Synchrotron Light Source, Brookhaven National Laboratory. The processing of data was performed using Mosflm31 and Scala32 from the CCP4 program suite.33 The inhibitor-bound structure was solved using the Ca2+-bound μI–II (PDB entry 1KXR) as the model for molecular replacement in MOLREP.34 The molecular topology descriptions of the inhibitors 1 and 2 were generated with the Dundee PRODRG2 server.35 Model refinement and building were performed using REFMAC536 and Coot,37 respectively. The structures shown in Figures 3 through 6 were prepared with PyMOL.38</p><!><p>Analytical data (high resolution mass determination and HPLC tracings) for target compounds. This material is available free of charge via the Internet at http://pubs.acs.org.</p><!><p>Chemical structures of 1 and 2, the two α-ketoamide calpain inhibitors used in this study. Both compounds contain a P1 α-aminobutanoic acid and P2 leucine residue, but the primed side extension of 1 ends with adenine ring, while that of 2 is terminated by a piperazine ring. The structure of the commercially available calpain inhibitor AK-295 is also shown for comparison.</p><p>Inhibitory effect of 1 and 2 on m-calpain (A) and μI–II (B). Duplicate assays were performed by adding 1.3 μM (EDANS)-EPLFAERK-(DABCYL) (1), 125 nM calpain (2) and 4 mM CaCl2 (3), then averaged to yield the plots shown. Autolytic inactivation of m-calpain, but not μI–II, was observed when no inhibitor was added (X). Immediately following addition of each inhibitor (4), the increase in fluorescence intensity was attenuated. 1(Y) caused a more distinct attenuation of fluorescence in both the m-calpain and μI–II assays, indicating that it is a more potent inhibitor when compared to 2 (Z).</p><p>Crystal structures of 1 and 2 bound to the active site of μI–II. The electron density of 1 (calculated with coefficients 2mFobs – DFcalc, φcalc and contoured at 1σ) and the inhibitor's polar contacts with μI–II are shown in panels A & C, respectively, while those for 2 are shown in panels B & D. The carve feature of PyMOL33 was used to limit the display of electron density to a distance of 2 Å from the inhibitors. Bonds are colored by atom type: carbon=gray (μI–II), carbon = green (1), carbon = cyan (2), nitrogen = blue, oxygen = red, sulfur = orange. The peptide subsites are labelled below panel C.</p><p>(A) Structural overlap of calpain inhibitors, achieved by aligning the polypeptide chain of each structure. Inhibitors are shown as sticks and accompanied by a surface representation of μI–II from the μI–II-1 complex. Atoms are colored as in Figure 3. The overlap of 1, 2, 3 and leupeptin shows a high degree of structural alignment from the P3 to the P1 residue. The hydrophobic pocket formed by Ala262, Ile263 and Val269 is highlighted in light green. (B & C). Orientation of terminal primed side ring groups of 1 and 2 relative to Trp298 in μI–II. A transparent surface representation of μI–II is also shown. (B) Primed side aromatic stacking interaction between the adenine moiety of 1 and W298. (C) This stacking interaction is absent for the non-aromatic piperazyl group of 2. Atoms are colored as for Figure 3.</p><p>Overlay of four μI–II structures, showing the two possible conformations of the gating loop (residues 256–261). Glu261 in μI–II and the bound inhibitor 1 are shown as sticks. The primed side extensions of 1 and 2 cause Glu261 in the gating loop to be displaced, resulting in an "open" conformation. This "open" conformation closely resembles that of the structure of μI–II bound to 3, where a P1′-cyclopropyl group causes Glu261 to be displaced. When bound to leupeptin, which lacks a primed side extension, the gating loop is in a "closed" position, with Glu261 blocking the S1' subsite. The polypeptides are colored as follows: green = μI–II-1 complex, cyan = μI–II-2 complex, magenta = μI–II-3 complex, yellow = μI–II-leupeptin complex.</p><p>Overlay of Trp298 in the μI–II-1 complex (μI–II in white, 1 in green) with the corresponding tryptophan in papain (magenta), cathepsin B (yellow) and cathepsin K (cyan). This overlay was achieved by first aligning three catalytic site residues (cysteine, histidine, tryptophan) of papain, cathepsin B and cathepsin K with those of the μI–II-1 structure. Inhibitor 1 and the overlaid cysteine, histidine and tryptophan residues are shown as sticks. The relatively similar alignments between the tryptophans suggests that aromatic stacking, by itself, will be insufficient to specifically target an inhibitor into the calpain active site. The PDB entries for the cysteine proteases are as follows: papain bound to leupeptin (1POP), human liver cathepsin B (1HUC) and cathepsin K bound to E-64 (1ATK). Nitrogens are colored blue, while oxygens are red.</p><p>Summary from Data Collection and Structure Refinement</p>

PubMed Author Manuscript

Actinic Wavelength Action Spectroscopy of the IO -Reaction Intermediate

Iodinate anions are important in the chemistry of the atmosphere where they are implicated in ozone depletion and particle formation. The atmospheric chemistry of iodine is a complex overlay of neutral-neutral, ion-neutral and photochemical processes, where many of the reactions and intermediates remain poorly characterised. This study targets the visible spectroscopy and photostability of the gas-phase hypoiodite anion (IO − ), the initial product of the I − + O3 reaction, by mass spectrometry equipped with resonance-enhanced photodissociation and total ion-loss action spectroscopies. It is shown that IO − undergoes photodissociation to I − + O ( 3 P) over 637 -459 nm (15700 -21800 cm −1 ) due to excitation to the bound first singlet excited state. Electron photodetachment competes with photodissociation above the electron detachment threshold of IO − at 521 nm (19200 cm −1 ) with peaks corresponding to resonant autodetachment involving the singlet excited state and the ground state of neutral IO possibly mediated by a dipole-bound state.

actinic_wavelength_action_spectroscopy_of_the_io_-reaction_intermediate

Introduction<!>Experimental Results<!>Computational Results<!>Photodepletion and photodissociation<!>Total ion-loss spectrum<!>Experimental<!>Theoretical

<p>In the atmosphere, iodide (I -) and its oxides, hypoiodite (IO -) and iodite (IO2 -) are relevant due to their reactivity towards ozone while iodate (IO3 -) anions, alongside its conjugate acid (HNO3) , have been implicated in particle formation [1][2][3][4] . Both Iand IOx -(x =1, 2, 3) are detected in ground-based atmospheric gas measurements where IO3concentrations exhibit a diurnal response, with lowest values detected during daytime, suggesting a link to photochemistry and night-time halogen chemistry 5 . In the lower stratosphere, both Iand IO3ions are present in detectable quantities and their fates in the presence of persistent ozone concentrations are under investigation 6 .</p><p>The relevance of iodine oxoacids (both neutral and anions) as aerosol nucleation agents is apparent 4 but details on their photostability under actinic radiation is lacking.</p><p>In the gas phase, Ireacts with O3 in a stepwise process first producing IO -, with subsequent reaction steps with O3 to form IO2 -+ O2 and ultimately IO3 -+ O2 [7][8][9][10] . The overall rate limiting step in the formation of IO3is the first step, I -+ O3 , while the subsequent steps involving IOand IO2occur with high efficiency (91% and 84% of the collision rate, respectively). Interestingly, the slow reversible reactions of IOand IO2 -(but not IO3 -) with dioxygen have also been studied with the latter case regenerating IO -10 . Although the reaction kinetics and nucleation processes of these anion intermediates are becoming better characterised, there remains a lack of information on the photochemistry of these atmospheric anions, including the simplest iodine oxide anion, IO -.</p><p>The electron binding energies of the iodine oxides have been measured and suggest that IO -(eBE = 2.3805 eV, 520 nm) [11][12][13] , IO2 -(eBE = 2.575 eV, 481.5 nm) 12,14 but not IO3 -(eBE = 4.70 eV, 263.8 nm) 14 may undergo photodetachment within the actinic window presenting a pathway to the corresponding neutral radicals. Recently, we demonstrated that photodissociation of the iodo-oxides anions, IO1-2occurs within the visible wavelength range (at 500 nm) and thus that these pathways are competitive with photodetachment at wavelengths relevant to the Earth's atmosphere. To explore this assertion, we present the first comprehensive investigation of the simplest iodine oxide, IO -, using a combination of resonance enhanced photodissociation and ion-loss spectroscopy and demonstrate that IOundergoes both photodissociation to I -+ O( 3 P) together with electron photodetachment to the neutral IO • radical under visible wavelength irradiation.</p><!><p>Visible light absorption by IOwas investigated using tunable-laser irradiation of m/z selected IOions confined within a room temperature linear quadrupole ion trap 15 . Action spectra were measured by monitoring both the generation of Iphotoproduct ions and the decrease in total ion count resulting from electron detachment. The thermodynamic limits for these pathways, and other plausible ones, are shown in Figure 1 with the four lowestenergy destruction pathways for IOranked by their relative energy (as determined from NIST data) [16][17][18][19] .</p><p>Assuming single-photon absorption, over the visible range, only I -( 1 S) + O ( 3 P) photodissociation with an onset at 13729 cm -1 (728 nm) and IO + ephotodetachment with an onset at 19200 cm -1 (521 nm) are accessible. Notably the I -( 1 S) + O ( 3 P) product pathway, the only photodissociation channel accessible in the visible wavelength range, generates O ( 3 P), which can react with molecular oxygen to form ozone as per the Chapman ozone-oxygen cycle 20 .</p><p>Figure 1: Schematic of the thermodynamic limits for the three lowest energy dissociation pathways of IO − (from NIST data) [16][17][18][19] . The electron photodetachment energy is also included 13 . All energies are relative to the IOelectronic ground state energy.</p><p>A representative single wavelength photodissociation mass spectrum of IO -(m/z 143) obtained at 19231 cm -1 (520 nm) is shown in Figure S1. The sole product ion observed is m/z 127, assigned as Iand assumed to form with O ( 3 P). Photofragment Oions (m/z 16) are not expected given insufficient photon energy (cf. Figure 1) but such ions lie below the low m/z cut-off of the instrument and, consequently, we cannot rule out their formation from multiphoton processes. A plot of Iphotoproduct yield as a function of laser power at 520 nm (Figure S1 inset) displays a linear trend, consistent with a single photon photodissociation process.</p><p>The photodepletion spectrum of the IOsignal (Figure 2A, obtained by plotting the difference in the total IOion count with the laser on and the laser off) exhibits a structured band system spanning the 15700 -22000 cm -1 range (636.9 -454.5 nm). The resonance-enhanced photodissociation (REPD) spectrum (Figure 2B) appears similar to the IOphotodepletion spectrum but with better signal to noise and exhibits a broad structured band centred at 19000 cm -1 with an onset at 15700 cm -1 , which tails off to baseline at 21800 cm -1 . A series of peaks spaced by ~230 cm −1 is present within this broad band which, as will be shown, arise from a vibrational progression in an electronically excited state of IO -. Peak locations are listed in Table S1. The presence of this extended vibrational progression implies that the anion excited-state is bound (at odds with some previous studies 21 but in accord with more recent reports 13 ) and therefore suggests that the photodissociation of IOis mediated by electronic state curve crossing. It is notable that peaks expected to appear at 19410 and 20060 cm −1 appear to be supressed relative to neighbouring peaks and this will be discussed later.</p><p>The total ion-loss spectrum (Figure 2C), which presumably results from electron photodetachment from IO -, is obtained by plotting the difference between the photodepletion and the photodissociation spectra, and is measured with an ion-trap storage time of 500 ms (allowing four laser pulses to irradiate the ion-packet per MS cycle). Since the Iphotoproduct is transparent in the visible region, the multi-shot experiment does not suffer additional complications arising from electron photodetachment from product ions. Over the spectrum, the total ion-loss signal rises sharply at photon energies greater than the EA of IO -(2.3805 eV, 19198 cm -1 ) 13 . The discernible peaks in the ion-loss spectrum are attributed to resonant autodetachment and two of these peaks align with the supressed peaks in the REPD spectrum. At the higher energy end of the broad band, the signal does not return to zero (unlike the REPD spectrum) presumably due to electron photodetachment into the continuum. The weak signal between 18100-19200 cm -1 below the detachment threshold is likely due to absorption by hot ions. Overall, the S/N of Figure 2B is superior to Figures 2A and 2C as the detection of photoproduct ions is essentially a background free measurement.</p><p>The normalised ratio of photodissociation signal to the total ion-loss signal can be represented as a bias spectrum, which is shown in Figure 2D. At the photon energy corresponding to the EA, the two processes have equal yields, with photodissociation dominating below the EA. On the high energy side of the EA, electron loss dominates although the bias returns to zero in a few instances between the resonance peaks of the photodetachment. At photon energies greater than 20 000 cm -1 the photodepletion is dominated by electron loss.</p><p>The same analysis was performed with shorter (Figure S3) and longer (Figure S4) ion trap storage times, allowing for either 1 or 9 laser pulses, respectively, to irradiate the ions before they are scanned out of the ion trap. For the longer (9 pulse) case, the S/N of the total ion-loss spectrum is significantly improved with at least five peaks clearly apparent (labelled I-V). Under these conditions however, these spectra may be affected by saturation, this is particularly obvious for the REPD spectrum. The sharp features labelled I-V in Figure 2 generally align with peaks in the total ion-loss spectrum, and this will be discussed below. (Spectra corresponding to storage times of 220 ms (1 laser pulse) and 1000 ms (9 laser pulses) are included in the supporting information: Figures S3 and S4, respectively).</p><!><p>The calculated potential energy curves for the first seventeen electronic states of IOassociated with the three lowest energy dissociation limits are plotted in Figure S5. Of these molecular states, five are within the energy range of the experiment (<22000 cm -1 ) and were subjected to higher level (icMRCI+Q/aug-cc-pwCV5Z-PP) treatment. These results are presented in Figure 3 with the calculated neutral ( 2 P3/2) potential energy curve also shown. The two spin-orbit states for the ground state of neutral IO are split by 2093(5) cm -1 such that the higher spin-orbit state ( 2 P1/2) resides at 21293 cm -1 relative to the IOground state (based on combining the experimental EA with computed spin-orbit values) 13 , and thus this state is at the higher energy edge of the experimental range and is probably not relevant to the experiments discussed here.</p><p>The ground state of the anion (X < l a t e x i t s h a 1 _ b a s e 6 4 = " q 5 2 X a G 4</p><p>L O e r B f r 3 f q Y t i 5 Y x c w e + A P r 8 w f h K J c q < / l a t e x i t ></p><p>N m 6 t x 6 t F + t 1 X p q z F j 3 7 4 A e s t 0 / r / p q S < / l a t e x i t ></p><p>Bond length (Å)</p><p>o H e w G y p Q P + E P 9 v h F T T + u B 5 5 h J j 0 J P / / a G 4 l 9 e I w J 3 t x U L P 4 y A + + z z I T e S G A I 8</p><!><p>We now return to discuss the photodepletion and REPD spectra (Figure 2A and B) considering now the calculated potential energy curves and molecular parameters. Calculated molecular constants for the 1 1 P excited state of IO − state are Te = 15040 cm −1 with an equilibrium bond length re = 2.34 Å (see SI for more information). Using spline fits to the icMRCI single-point energies, the Duo program 23 provided vibrational constants: we = 289.4 cm -1 and wece = 1.72 cm -1 . The experimentally derived ground electronic state parameters for IO − , reported by Gilles et al., are re = 1.929 Å, we = 581 cm -1 and wece = 4.37 cm − 1 11,12 . The FC factors based on these parameters were calculated using the PGOPHER program 24 for the 1 1 P -X 1 S + transition and the results are plotted in Figure S6A.</p><p>To better match the simulated intensity profile with the experimental photodepletion spectrum the Te value was raised from 15040 cm −1 to 16440 cm −1 (Figure S6B) and in this case the most intense transition corresponds to v' = 10. Alternatively, extending the excited state bond-length to 2.45 Å similarly shifts the FC simulation (Figure S6C). In this case, the most intense transition corresponds to v' = 16. In both cases the origin transition is predicted to be very weak, making it difficult to observe and assign. Ultimately, the correct vibrational numbering for the vibronic transitions requires an accurate value for Te and because this calculation has an uncertainly on the order of ±1500 cm −1 , confident vibronic assignments are not possible. The simulation should be ultimately assessed against a direct absorption spectrum. An alternate strategy could be to probe these vibronic transitions with raregas tagging pre-dissociation spectroscopy. These spectra are also likely to be perturbed by mixing of states, as will be discuss in the next section, further complicating the analysis. Nevertheless, the similarities between the measured and simulated spectra suggest the calculations provide useful insights into the excited state probed in the experiments.</p><!><p>Contained within total ion-loss spectrum in Figure 2(C) are five peaks labelled I -V. The two strongest, I and III, are located at 19410 cm -1 and 20020 cm -1 and spaced by 610 cm -1 . The next peak, labelled V, is centred at 20710 cm -1 and spaced 690 cm -1 from III. The positions I, III, and V align with supressed peaks in the REPD spectrum (Figure 2B), as indicated with the vertical dotted lines. The two smaller peaks labelled II (19810 cm -1 ) and IV (20450 cm -1 ) are spaced by 640 cm -1 . These two peaks also align with peaks in the REPD spectrum but the corresponding REPD peaks look comparatively sharper as if the higher energy side of the peak is missing. The spacing of ca. 650 cm -1 in both sets is close to the known vibrational frequency for the ground state of the IO neutral: 682 cm -113 . Figure 4 shows an expanded section of the key FC region from Figure 3 along with the REPD and total ion-loss spectra (from Figures S3 and S4) showing the position of peaks I-V and their relation to the known vibrational levels of the neutral IO electronic ground state. The neutral IO vibrational levels are located using known experimental values 13 . Peaks I-V do not align exactly with the vibrational energy levels of the X of the DBS and the anion 1 1 P valence state. Higher DBSs associated with IO X 2 P3/2 excited vibrational states can decay through a vibrational autodetachment process. Within the diabatic formulism, the DBSs and valence 1 1 P IOstates interact, leading to mixed vibronic states with both DBS and VS character. The situation is illustrated in Figure S7.</p><p>In this region of the spectrum, the vibrational energy level spacing for the 1 1 P state of IOis around a third that of neutral IO X 2 P3/2 state. From Figure 4 it appears that vibrational levels associated with the 1 1 P state IOlie ≈100 cm −1 below and above the three lowest X 2 P3/2 neutral vibrational levels. Neighbouring zero-order states associated with the 1 1 P state IOvibrational levels (shown in blue Figure S7) and the DBSs (shown in red Figure S7) presumably interact leading to mixed states having valence and DBS character. Relative rates for dissociation and electron detachment will depend on the respective rates for autodetachment and dissociation from the zeroorder DBS and valence state and the mixing coefficients of each state. It is likely that transitions to the zero-order valence states are significantly stronger than those to the zero-order DBSs such that the intensity of the transition will depend on the coefficient for the valence state in the mixed state.</p><p>The fact that strong transitions give rise to detachment (peaks I and III) suggests that, when energetically allowed, the rate of electron detachment from the zero-order DBSs is more rapid than dissociation from the zero-order valence states. It is possible that the lower rotational levels of IO can support dipole bound states whereas the higher rotational levels cannot. This means that lower rotational states of some vibrational levels may detach whereas higher levels dissociate. There are hints that this happens in some of the asymmetric photodissociation peak shapes (e.g., I and II).</p><p>A recent photoelectron study of IOusing high-resolution velocity map imaging by Wang et al. 13 reported electron photodetachment (ePD) peaks at 19432 cm -1 , 20104 cm -1 , and 20721 cm −1 . These peaks align with peaks I, III, and V measured here. Peak I was attributed to the origin transition to the electronic excited 1 1 P state (230 cm −1 above the EA). Our experimental and computational results show that peak I is not the origin transition and that vibrational levels associated with the 1 1 P state reside below the EA. Also, Wang et al. located an ePD peak at 19829 cm -1 and surmised that it may be associated with an excited triplet anion state. This peak aligns with II and we conclude that it is a transition to a vibrational level associated with the 1 with peaks in the total ion-loss spectrum attributable to electron loss via autodetachment facilitated by mixing of first singlet excited state (1 1 P) of IOand DBSs associated with vibrational levels of neutral IO in its ground electronic state (X 2 P3/2).</p><p>These results provide further considerations for the formation and fate of IOin the atmosphere. That is, while electron photodetachment from IO -(above the EA) feeds into accepted (IO • ) radical-driven ozone depletion pathways 30 , the observation of IOphotofragmentation yielding I -+ O ( 3 P) represents a possible pathway for ozone regeneration via the Chapman cycle 20 . The wavelength dependence of these competing photochemistries may play a role in the reported diurnal behaviour of IOin the atmosphere 5 . Future investigation of the relative branching fraction between photo-detachment and -dissociation will be central to atmospheric models of iodooxide anions and flow-on implications for ozone concentrations and particle formation.</p><!><p>Experiments were performed using a modified linear quadrupole ion trap mass spectrometer (Thermo Fisher Scientific LTQ XL) coupled with a tuneable, nanosecond pulsed, OPO laser (GWU-Lasertechnik flexiScan)</p><p>pumped by a Spectra-Physics QuantaRay INDI, which is explained in detail elsewhere [31][32][33] . IOwas created by first dissolving potassium iodate (KIO3) in HPLC grade methanol (>99.7%) and subjected to electrospray ionisation to generate IO2via source fragmentation. IO2was then isolated in the ion-trap and dissociated via collision induced dissociation (CID) into IOthat was isolated and interrogated via the laser in an MS 3 experiment.</p><p>Photodissociation experiments involved isolating and storing IOallowing irradiation with laser pulses, which was timed with a mechanical shutter synchronised with each individual isolation cycle. Photodissociation action spectra were recorded by a laser ON/OFF acquisition procedure. The ion signal with the laser off was subtracted from the ion signal with the laser on, to give the photoinduced signal, which was normalised by the total ion count when the laser is off. The accumulated raw data was processed via an in-house python script. Photodissociation action spectra were constructed by plotting the peak area of the only detected photoproduct, I -(127 m/z), and normalised to the total ion count, against the photon energy. These experiments were performed using three different ion-trap isolation cycles: 220 ms, 500 ms, and 1000 ms which correspond to one, four, and nine laser shots, respectively. Different isolation times were used to increase the signal-to-noise ratios of the photodepletion spectra. Spectra with isolation periods of 500 ms are reported in the main text while spectra with isolation periods of 220 and 1000 ms are included in the supporting information.</p><!><p>Using the MOLPRO 2019.2 program 34 potential energy curves (PEC) were constructed by scanning the IObondlength with the internally contracted multireference configuration interaction method with Davidson correction (icMRCI+Q) [35][36][37][38] . The state-averaged complete active space self-consistent field (CASSCF) method [39][40][41][42] was deployed to generate the wavefunction used in the icMRCI+Q calculation. CASSCF calculations were carried out for the seventeen states correlating with the three lowest energy dissociation limits 22 , which are I - . With this the seventeen states are labelled as follows: 1 3 Sand 1 3 P for the I -( 1 Sg) + O( 3 Pg) channel, X 1 S + , 1 1 S + , 1 1 S -, 1 1 P, 2 1 P, 1 1 Δ, 1 3 S + , 2 3 S + , 2 3 S -, 2 3 P, 3 3 P, 1 3 Δ for the I( 2 Pu) + O -( 2 Pu) limit, and 2 1 S + , 3 1 P, 2 1 Δ for the I -( 1 Sg) + O( 1 Dg) limit 22 . The X 1 S + , 1 1 P, 1 3 S -, and 1 3 P states are necessary to understand our results and thus for these four states the icMRCI+Q method was deployed to yield accurate energies. The X 2 P3/2 ground state of IO was also treated similarly. For both CASSCF and icMRCI+Q methods the oxygen atom was treated with the aug-cc-pwCV5Z basis set and for the iodine atom the pseudopotential approximation (-PP) extension was used [43][44][45][46][47] . The C2v symmetry point group was used with the active space consisting of all 14 valence electrons distributed among the 8 valence molecular orbitals (4a1, 2b1, 2b2). Spectroscopic constants of the 1 1 P state of IOwere calculated using the Duo program 23 by fitting a spline to the icMRCI+Q PEC points with vibrational energies of the 1 1 P excited state determined using an RKR procedure. Experimental values from Gilles et al. were used for the X 1 S + state 11,12 .</p>

ChemRxiv

Porous Organic Polymers as Fire‐Resistant Additives and Precursors for Hyperporous Carbon towards Oxygen Reduction Reactions

Cyclotriphosphazene (CP) based porous organic polymers (POPs) have been designed and prepared. The introduction of CP into the porous skeleton endowed special thermal stability and outstanding flame retardancy to prepared polymers. The nonflammable level of PNK-CMP fabricated via the condensation of 2,2'-(1,4-phenylene)diacetonitrile (DAN) and hexakis(4acetylphenoxy)cyclotriphosphazene (HACTP) through Knoevenagel reaction, in vertical burning tests reached V-2 class (UL-94) and the limiting oxygen index (LOI) reached 20.8 %. When used as additive, PNK-CMP could suppress the dissolving out of PEPA effectively, reducing environment pollution and improv-ing the flame retardant efficiency. The POP and PEPA co-added PU (m POP %: m PEPA % = 5.0 %: 5.0 %) could not be ignited under simulated real-scale fire conditions. The nonflammable level of POP/PEPA/PU in vertical burning tests (UL-94) reached V-0 class with a LOI as high as 23.2 %. The smoke emission could also be suppressed, thus reducing the potential for flame spread and fire hazards. Furthermore, carbonization of PNK-CMP under the activation of KOH yield a hyperporous carbon (PNKA-800) with ultrahigh BET surface area (3001 m 2 g À 1 ) and ultramicropore size showing excellent ORR activity in alkaline conditions.

porous_organic_polymers_as_fire‐resistant_additives_and_precursors_for_hyperporous_carbon_towards_ox

Introduction<!>Results and Discussion<!>Conclusions

<p>Porous organic polymers are a burgeoning family of sustainable materials utilizing natural, abundant and renewable precursors. These materials have gained increased attention in recent years. [1] Different to other porous materials, POPs are constructed by pure organic units via covalent bond through various synthetic methods and reactions. These inherent features, such as simple synthetic routes, wellcontrolled porosity, pre-designable structure and functionality make POPs applicable to various fields, including gas uptake and separation, energy and environment, organic photovoltaic, catalytic and other important areas. [2] In many cases, the combination of functional monomers and porous properties endowed outstanding performance to the targeted POPs excelling the pure monomers and overcoming the drawbacks existed in the monomers. [3] Inspired by these results, we anticipated to achieve a series of fireresistant POPs via the introduction of a flame retardant monomer (cyclotriphosphazene). By changing the starting composition of building units and reaction types, two novel CP-based POPs (conjugated PNK-CMP and PNS-CMP) were prepared. Compared with PNS-CMP, PNK-CMP exhibits higher thermal stability and used as the additive or co-additive to the commercial materials studying the flame retardancy performance. Furthermore, the multiple heteroatoms doping structure make these materials ideal precursors for the preparation of porous carbons with controllable element composition which are the most popular materials applied in the renewable energy and environmental related fields, e. g. rechargeable batteries, metal-air batteries, [2] supercapacitors [4] (SCs) and many other new technologies. [5]</p><!><p>As depicted in Figure 1, PNS-CMP and PNK-CMP were prepared according to the previous reported protocols. Briefly, PNS-CMP was fabricated via the self-polymerization of hexaphenoxycyclotriphosphazene (HPCTP) according to Scholl reaction under the catalytic of AlCl 3 in chloroform. [14] PNK-CMP is synthesized via the copolymerization of 2,2'-(1,4phenylene)diacetonitrile (DAN) and HACTP referred to Knoevenagel reaction under the catalytic of sodium methylate in methyl alcohol/THF mixtures. [15] The detailed procedures are given in the electronic supporting information (ESI). The targeted porous carbons denoted as PNSA-800 and PNKA-800 were fabricated via simple carbonization of prepared CMPs at 800 °C under the activation of KOH in a mass ratio of M polymer : M KOH = 1 : 2. [16] The control samples, i. e., PNS-800 and PNK-800 were prepared under the identical conditions but without the activation of KOH. And the detail was given the electronic supporting information.</p><p>Figure S1 exhibits the Fourier transform infrared (FT-IR) spectroscopy of prepared samples. Characteristic vibrations bands located at 1220 and 1420 cm À 1 belonging to the CP ring could be clearly observed for both polymers. [17] Furthermore, feature peaks ascribed to the stretching vibration of newly formed C=C bonds (1596 cm À 1 ) and the inherent C � N bonds (2218 cm À 1 ) could also be detected from the FTIR of the PNK-CMP. [18] And most important of all, the stretch vibration bands attributed to the carbonyl group around 1690 cm À 1 is almost disappeared, [19] further validating the successful built-up of the porous skeletons. Figure S2a presents the solid state 13 C NMR of as-synthesized polymers, from which strong carbon signals attributed to the phenyl units distributed from 70 to 145 ppm could be found for both polymers. Meanwhile, characteristic peaks of cyano group appeared at 117.3 ppm could be observed from the 13 C NMR of CPK-CMP. [18] The solid-state 31 P NMR spectroscopy of PNK-CMP were shown in the Figure S2b, [15] typical signals ranged from 0 to 25 ppm assigned to the CP ring could be detected. [20] The corresponding elemental analysis evidenced the coexistence of N, O, C and H for both prepared polymers.</p><p>Low temperature N 2 uptake measurements were performed to evaluate the porous properties of prepared materials. As shown in Figure 2a, almost vertical uptake could be observed in the low-pressure region (P/P 0 < 0.01), indicating the existence of micropore for all these prepared materials. [21] With the increasing of pressure, continue increase could be found for all these materials again. However, except the activated samples, obvious hysteresis loop in the medium pressure range and fast-adsorption in the pressure beyond 0.95 could be clearly detected, suggesting significant mesoporosity and the widely existence of macropore for other materials. [22] Meanwhile, the calculated BET surface area for PNK-CMP was only 138 m 2 g À 1 , but it increased to 513 and 3001 m 2 g À 1 for PNK-800 and PNKA-800, respectively. Similar to PNK-CMP, the BET surface areas of PNS-CMP were changed from 173 m 2 g À 1 to 479 (PNS-800) and 1812 m 2 g À 1 (PNSA-800). Besides, both the micro and meso pore volume was also increased, demonstrating large amount of smaller pores were generated during carbonization. All these demonstrate the activation of KOH could further improve the pore properties of POPs, significantly enhancing the surface areas. And that strategy is transferable across other POPs, especially the cyano containing materials to minimize the pronounced swelling effect in the porous adsorbed species. Figure 2b presents the pore size distributions curves of prepared samples. PNK-CMP shows a main peak at 2.18 nm with secondary peaks at 1.60, 2.74 and 3.79 nm, respectively. PNS-CMP exhibits a main peak at 2.10 nm with small peaks at 1.34, 2.48 and 3.89 nm, respectively, indicating hierarchical pore structure for prepared polymers. However, the pore size is centered at micropore ranges for the carbonized samples, e. g., PNKA-800 are located at 0.57 nm and PNSA-800 are situated at 1.43 nm, implying the substantially increased micropore. [4] And the detail about the porosity was listed in Table S1.</p><p>Field emission scanning electron microscope (FE-SEM) and TEM were performed to investigate the microstructure of as-synthesized samples. Figure 3a and Figure 3d showed the SEM of prepared polymers, from which, bulk stacked by aggregated spherical nanoparticles could be observed for both polymers. As presented in Figure 3g, 3j and Figure S3, the morphology could be maintained at a great extent after the activation. Widely distributed pore, ascribed to the inherent skeleton structure or the space generated by the pile up of particles, could be detected from the TEM given in Figure 3b-c, 3e-f, 3h-i, 3k-l, and Figure S3 by the light and shade contrast.</p><p>Like in previous report, the powder XRD patterns validate amorphous structure of prepared polymers (Figure S4a). And still, no clear peaks could be observed from the XRD of annealing samples (Figure S4b), indicative the amorphous features of prepared porous carbon. TGA were performed to examine the thermal stability of prepared polymers. According to Figure S5, owing to the high polarity of prepared polymers, dramatically weight loss attributed to the evaporation and desorption of adsorbed water (16.3 % for PNK-CMP and 6.8 % for PNS-CMP) could be observed at the low temperature range. Even at the temperature of 800 °C, the remaining carbon is beyond 55 wt % for PNK-CMP (vs 32 % of PNS-CMP), suggesting the super thermal stability of CP-based CMPs. Raman spectra of pyrolysis samples all displayed two intensive peaks around 1347 and 1586 cm À 1 , assigned to the D band and G band, respectively (Figure S6). [23] All these samples present high graphitic degree with a high intensity ratio of G band to D band (I D /I G ). For example, the intensity ratio of PNKA-800 is 0.98, and it reaches 0.95 for PNSA-800.</p><p>Figure 4 and Figure S7 displayed the X-ray photoelectron spectroscopy (XPS) of PNKA-800 and PNSA-800, respectively. The XPS survey spectrum of PNKA-800 (Figure 4a), shows obvious signals belonging to the C, N, O, and P, validating the existence of afore mentioned element in prepared carbon samples. Figure 4b shows the high-resolution C1s spectra, a dominant peak at 284.8 eV, combined with two small peaks distributed at 285.9 eV and 287.6 eV could be observed. [24] As presented in the Figure 4c, the N 1s spectra could be deconvoluted into three peaks located at 398.8, 399.8, and 401.1 eV, corresponding to the pyridine, pyrrole, and graphitic N species, respectively. [25] High resolution P 2p peaks could be divided into two peaks, situated at 133.2 and 133.9 eV (Figure 4e), attributing to PÀ C bond and PÀ O bond, respectively. [26] In accordance with the previous results, two obvious peaks at 530.9 and 532.4 eV, assignable to the CÀ O and CÀ P bond, could be obviously observed.</p><p>The special features of PNKA-800, especially the ample N, P content, stimulate us to study electrocatalytic property towards oxygen reduction in alkaline conditions (O 2 -saturated 0.1 M KOH solution). Figure 5a presents the LSV curves of PNKA-800 and Pt/C at 1600 rpm. PNKA-800 shows a onset potential (E onset ) of 0.935 V as well as a half-wave potential (E 1/ Furthermore, PNKA-800 exhibits a limited current density of 4.75 mA cm À 2 at 0.2 V (vs. 5.51 mA cm À 2 at 0.2 V). Figure S6 displayed the CV curves of PNKA-800. In contrast to the featureless curve in Ar-saturated solution, obvious cathodic peak could be clearly detected in the O 2 saturated solution. Figure 6b exhibited the polarization curves of PNKA-800 measured from 400 rpm to 2500 rpm, and the insert part is the corresponding Koutecky-Levich (KÀ L) plots calculated according the Equation S1. The electron-transfer number (n) obtained from the slopes of KÀ L plots are 3.75 at the potential of 0.2 V, suggesting a four-electron pathway for ORR. And that could also be validated by the RRDE measurements shown in in Figure 5c and 5d. The calculated electron transfer number according to the Equation S3 and S4 are ranged from 3.51 to 3.96, close to that of the commercial Pt/ C with a low H 2 O 2 yield below 17.1 % in the region from 0.2 to 0.8 V and consistent well with the result obtained from the KÀ L plots, further determining a high selectivity towards the four-electron reduction pathway of oxygen. For the real application of as-synthesized sample, an excellent cycle stability is desired. Hence, i-t test was conducted at static potential of 0.6 V (vs. RHE). Figure 5e presented the time dependent current density curves of PNKA-800. After a continuous running of 20000 s, only a negligible loss of 3.1 % of the initial current was detected for the synthesized sample, much better than that of commercial Pt/C catalyst (37.2 %), indicative PNKA-800 possessing superior durability. Figure 5f shows the polarization curves measured before and after the methanol-crossover test, almost identical curves were found with a negative shift of 10 mV in E 1/2 (vs 37 mV for Pt/C given in Figure S8). All these results evidenced PNKA-800 can act as a remarkable oxygen reduction electrocatalyst in alkaline solution applied in the metal-air battery.</p><p>For the unique structure, cyclotriphosphazene (CP) and CP-derived materials are widely investigated as the flame retardant additive, lowering smoke emission and heat release rate. Pentaerythritol octahydrogen tetraphosphate (PEPA) is a commercial flame retardant commonly used as additive in thermoplastic polyurethane elastomer (TPU), improving the flame retardant efficiency. A minimum addition of 10 % can play a better flame retardant effect. But PEPA could migrate out from the TPU which are harmful to the environment.</p><p>The similarity of structure and elemental composition for the PEPA and POP, make PEPA easily get into the hole of PNK-CMP. Inspired by this, we anticipated to inhibit the releasing of PEPA from the TTU via introduction of porous CMP. It is the first time that the porous PNK-CMP was applied as additive or co-additive with PEPA to commercially available TPU improving the flame retardant efficiency. As shown in Figure 6, CMP/TPU was fabricated via simple physical processing in different mass ratios of POP to TPU. One could observe clearly that the color of composites darkens with the increasing of mass ratio. While, CMP/PEPA/ TPU composites was prepared stepwise. PEPA was initially absorbed in the porous skeleton of CMP, then the composites was mixed with the TPU (mPOP% : mPEPA% = 5.0 % : 5.0 %).</p><p>To evaluate the flammability, vertical burning tests (UL-94) and limiting oxygen index (LOI) measurements were conducted. In UL-94 tests, the TPU/POP/PEPA composites possessed the highest level (V-0), similar to the TPU/PEPA, but higher than that of TPU/CMP composites (V-2). As shown in Table 1 and Video S1, S2, even directly exposed to an igniter for 10 s, the TPU/POP/PEPA sample could not be ignited. When the igniter was removed, the flame was extinguished instantly, and no obvious flame was observed on the surface of samples. After that test, compared with the TPU/POP composites, TPU/POP/PEPA maintained the original shape better, suggesting certain flame retardancy. Furthermore, as delivered in Figure 5c and 5d, large amount of nonflammable gas (N 2 ) was generated during the combustion process, released by the decomposition of composites, which are further transformed into protective carbon-layer coated on the material surface (Figure 5e). Meanwhile, the lOI value reaches 25.2 % for the TPU/POP/PEPA composites higher than the TPU/POP composites (23.0 %). The POP and PEPA coadded TPU (m POP % : m PEPA % : m TPU % = 5.0 % : 5.0 % : 90 %) could not be ignited under simulated real-scale fire conditions. And as observed form Figure 5c, 5d and 5e, a large amount of nonflammable gas was emitted from materials preventing the burning, after combustion carbon-layer was formed which to prevent further transfer heat to composites. This process conforms to the mechanism of intumescent flame retardant. Furthermore, when used as additive, PNK-CMP could suppress the dissolving out of commercial PEPA effectively, reducing environment pollution and improving the flame retardant efficiency. In contrast, TPU polymer insulation materials are easily ignited. Furthermore, the fire retardancy could also be validated by the TGA given in the Figure S9. Compared with the pure polymer, the thermal stability of composites enhanced greatly. And the ternary complex (m POP % : m PEPA % : m TPU % = 5.0 % : 5.0 % : 90 %) presents a TG similar to the binary complex (m PEPA % : m TPU % = 10 % : 90 %).</p><!><p>In summary, cyclotriphosphazene based CMPs were welldesigned and facilely prepared. The special elemental composition and special thermal stability endowed these polymers ideal candidates for multiple atoms doped porous carbon applicable for the energy and environment-related fields. Under the activation of KOH, hyperporous carbon with ultrahigh BET surface area of 3001 m 2 g À 1 and ultra uniform pore size distribution were obtained which could further be used as the carbon-based catalysts for ORR with excellent performance. The introduction of CP into the porous skeletons give outstanding flame retardancy to prepared polymers. And the porous features could suppress the dissolving out of commercial PEPA effectively, reducing the pollution triggered by traditional flame retardant. For example, the nonflammable level of POP and PEPA co-added TPU (m POP % : m PEPA % = 5.0 % : 5.0 %) reached V-0 class with a LOI as high as 23.2 % that could not be ignited under simulated real-scale fire conditions.</p>

Chemistry Open

ExoFiT trial at the Atacama Desert (Chile): Raman detection of biomarkers by representative prototypes of the ExoMars/Raman Laser Spectrometer

In this work, the analytical research performed by the Raman Laser Spectrometer (RLS) team during the ExoFiT trial is presented. During this test, an emulator of the Rosalind Franklin rover was remotely operated at the Atacama Desert in a Mars-like sequence of scientific operations that ended with the collection and the analysis of two drilled cores. The in-situ Raman characterization of the samples was performed through a portable technology demonstrator of RLS (RAD1 system). The results were later complemented in the laboratory using a bench top RLS operation simulator and a X-Ray diffractometer (XRD). By simulating the operational and analytical constraints of the ExoMars mission, the two RLS representative instruments effectively disclosed the mineralogical composition of the drilled cores (k-feldspar, plagioclase, quartz, muscovite and rutile as main components), reaching the detection of minor phases (e.g., additional phyllosilicate and calcite) whose concentration was below the detection limit of XRD. Furthermore, Raman systems detected many organic functional groups (-C≡N, -NH 2 and C-(NO 2 )), suggesting the presence of nitrogenfixing microorganisms in the samples. The Raman detection of organic material in the subsurface of a Martian analogue site presenting representative environmental conditions (high UV radiation, extreme aridity), supports the idea that the RLS could play a key role in the fulfilment of the ExoMars main mission objective: to search for signs of life on Mars.Led by ESA with the collaboration of Roscosmos, the ExoMars 2022 rover mission will pursue the detection of signs of present or past life on Mars 1,2 . To achieve this goal, the designed payload of the Rosalind Franklin rover will employ a set of panoramic instruments (PANCAM 3 and ISEM 4 ) to explore the surrounding environment, thus providing crucial data to be used in the navigation of the rover and in the identification of areas of high scientific interest. A ground-penetrating RADAR (WISDOM 5 ) and a passive neutron spectrometer (ADRON-RM 6 ) will investigate the subsurface, helping in the selection of potential drilling places. The ExoMars Drill Unit 7 (hosting the MA_MISS visible and near infrared spectrometer 8 ) will collect geologic samples down to a depth of 2 m, thus accessing material that have been sheltered from UV Radiation and further alteration processes. CLUPI 9 will provide textural information from the sampled materials through the collection of high-resolution images, while the sample preparation and distribution system (SPDS) will crush the materials and deliver the powders to the analytical laboratory of the rover 10 . Here, the visible/near-infrared spectrometer (MicrOmega 11 ) and the Raman Laser Spectrometer (RLS 12 ) will perform coordinated analyses 13 to identify the mineralogical composition of the samples and to reveal the potential presence of biomarkers. Spectroscopic results will be used to select the optimal scientific targets to be delivered to MOMA (Mars Organic Molecule Analyzer system), that will extract and analyse the organic molecules potentially preserved within the mineralogical matrix 14 .

exofit_trial_at_the_atacama_desert_(chile):_raman_detection_of_biomarkers_by_representative_prototyp

<!>Atacama desert (Chile).<!>Rover activity and samples collection.<!>Results<!>RLS ExoMars simulator.<!>Considerations for the ExoMars mission.<!>Conclusions

<p>Apart from the technical and engineering challenges that meant the development of the mentioned instruments, the success of the mission also relies on the complex coordination work required for their remote control and synergic management Recognizing the need for training the ExoMars teams and enhancing collaboration practices between instrument working groups, ESA organized the ExoMars-like Field Testing (ExoFiT) trials 15 , the second of which was carried out at the Atacama Desert (Chile) in February 2019. In addition to presenting a Martian-like desertic landscape, the presence of extremophile microorganisms populating Atacama's subsurface made this the ideal location to test the ability of the rover's payload to detect biomarkers in this kind of environments 16 .</p><p>During the trial, an emulator of the Rosalind Franklin rover (Charlie) was used to perform a complex sequence of scientific and engineering operations (from descending the landing platform to collecting drill cores) following the ExoMars Reference Surface Mission (RSM) 17 . During the mission simulation, the LCC team (Local Control Centre, located at the Atacama Desert, near the ESA Paranal Observatory) manoeuvred the rover and managed the acquisition and upload of the collected data. From 11,000 km of distance, the RCC team (Remote Control Centre, located at the European Centre for Space Applications and Telecommunications, UK) simulated the operations on Mars, planning the different activities for the next sol by only relying on the data returned by the rover 18 .</p><p>As part of the LCC team, science and engineering roles were covered by personnel from the University of Valladolid (UVa) and the National Institute for Aerospace Technology (INTA), who carried out the Raman characterization of the subsoil cores drilled by the rover. The Raman characterization was done using two spectrometers. A first mineralogical evaluation of the samples was performed using the RAD1 system (RAman Demonstrator 1), which is a portable RLS technology demonstrator assembled by the RLS team to carry out in-situ analyses in terrestrial analogue sites 19 . The RAD1 spectrometer has similar range of analysis (70-4200 cm −1 ), laser wavelength (532 nm) and power output (7 mW on the sample), spot of analysis (≈ 50 µm) and spectral resolution (6-10 cm −1 ) to the RLS, providing spectra qualitatively comparable to those soon gathered on Mars. Afterwards, more detailed spectroscopic analyses were carried out in the laboratory by means of the RLS ExoMars Simulator, which characteristics have been described elsewhere 20 . As detailed in previous works, this is the optimal instrument to predict the potential scientific outcome of the RLS flying model 21,22 . Indeed, in addition to the RLS-like optical spectral characteristics (as the RAD1), the spectrometer is coupled to a replicate of the ExoMars/ SPDS, and integrates the same algorithms developed for the RLS to perform the automatic multi-point analysis of Martian samples (e.g. Signal to Noise Ratio optimization, florescence quenching and acquisition parameters selection 23 ). Raman spectra from the ExoFiT exercise were obtained under the same operational constraints of the rover, and were finally compared to XRD data, being this the reference instrument for the mineralogical study of geological samples.</p><p>Recognizing the scientific and logistic value of this mission simulation, the present work aims to (1) summarize the preliminary analytical results obtained by the RLS team from the study of Atacama Desert samples, (2) evaluate advantages and disadvantages provided by the use of the RLS representative prototypes in ExoMarsrelated studies, and (3) extrapolate valuable information about the potential role the RLS could play in the fulfilment of the ExoMars mission objectives.</p><!><p>The Atacama Desert is a high plain covering an area of more than 100,000 km 2 between northern Chile and southern Peru. The hyper arid climate of this region, persisting unchanged for the last 10 million years, is due to the concurrence of the foehn effect (triggered by the Andean Mountains), the Humboldt current and high-pressure atmospheric conditions (caused by Pacific anticyclones) 24 . The ExoFiT trial was carried out in the region of Antofagasta, about 11 km west of the ESO Paranal observatory (altitude of 2200 m). According to previous studies, three kinds of rocks dominate the mineralogy of this area: granodiorites (white-pink colour) and andesite (dark green) are composed of plagioclase and quartz in different concentration ratio, while gabbros (dark-gray color) contain feldspar and amphibole/pyroxene minerals 25 . In addition to these primary minerals, alteration products such as phyllosilicates and oxides (e.g. hematite) can be found in the area together with evaporites (nitrates, sulphates and chlorides).</p><p>Based on the data collected at the ESO Paranal observatory, this area presents high temperature oscillations (from − 8 to + 25 ºC), extremely low humidity values (5-20%) and an average annual rainfall below 10 mm 26 . In addition to the mentioned parameters, the extremely high levels of surface ultraviolet (UV) irradiance (> 1100 W/ m 2 ) 27 , make this area the perfect terrestrial analogue site to investigate the suitability of microbial life in extreme environments, similar to those that can be found on Mars and other planets 16 . Despite the harsh environmental conditions, extremophile microorganisms populate the subsurface of Atacama by relying on metabolic mechanisms that may have analogies with those that could be adopted in the shallow subsurface of Mars [28][29][30] . In light of the forthcoming deployment of Raman spectrometers on Mars (beside the RLS, Sherloc 31 and SuperCam 32,33 instruments onboard the NASA/Mars 2020 rover also need to be mentioned), Vitek et al. published several works using Atacama rocks and soil samples to assess the capability of this technique to detect biomarkers, gathering encouraging results [34][35][36] .</p><!><p>As can be seen in Fig. 1, the Martian-like landscape of the area selected by the LCC team presents a reddish desertic pavement made of gravel, boulders and interspersed sand patches. The area also features small clay deposits and salt crusts, being these units of great astrobiological interest. Besides site selection, the LCC team took care of manoeuvring the rover and operating the whole set of ExoMars instruments accordingly to the commands received from mission control. As mission coordinator, the RCC made an assessment of the landing site, planning the descent from the landing platform and the driv-ing through a safe route to reach an area of scientific interest. The subsurface stratigraphy of the selected site was then analysed by WISDOM (scan grid of approximately 5 * 5 m). Based on subsurface radar results, RCC selected the optimal drilling site. After drilling, the extracted soil core was imaged by CLUPI, separated in two samples (upper part UP and lower part LP) and sent for Raman analysis. During the ExoFiT trial, two experiment cycles were conducted, giving a total of two cores and four subsamples in total. Since the granulometry of the sample affects the quality of Raman results 37 core samples were crushed and sieved to obtain a grain size distribution resembling the one prepared by the ExoMars/SPDS. The resulting samples were then placed into a replicate of the ExoMars sample holder and analysed by Raman.</p><p>Instruments. For the in-situ characterization of drill cores, Raman analyses were performed directly at the analogue site and by following the time constraint imposed by the mission simulation. To do so, the RAD1 spectrometer was used. Assembled by the ERICA group, this portable instrument is composed of a commercial excitation laser source of 532 nm, a high resolution Thermo Electrically (TE) Cooled CCD Array spectrometer (2168 × 512 pixels) and a high line density diffraction grating (1800 lines per mm). The instrument was optically harnessed by optical fibers to a microscope with a 50 × objective, reproducing the analytical footprint of RLS (50 µm). The time constrains applicable to these analyses during ExoFiT test limited the time per sample to 1-1.5 h, a timeframe shorter than the nominal ExoMars rover operations.</p><p>Complementary analyses were done at the laboratory using the RLS ExoMars Simulator, a system with similar spectroscopic performance to RAD1, but incorporating the automatic operation capabilities of RLS. The instrument includes a continuous green excitation laser (532 nm), a high resolution TE Cooled CCD Array spectrometer and an optical head with a long working distance objective of 50x. The instrument is coupled to three axis micrometric positioning system with a refillable container (emulating the ExoMars sample holder) that allows the definition of analysis rasters on the sample. Software-wise, the RLS ExoMars Simulator implements the same algorithms developed for the RLS 23 , allowing the automatic analysis of the samples auto adjusting the acquisition parameters. For both spectrometers, spectra acquisition was performed through a custom developed software based on LabVIEW 2013 (National Instruments), while the IDAT/SpectPro software was used for data processing and interpretation 38 . Knowing that the quantum efficiency of CCD detectors varies with the wavelength, the intensity of all spectra was corrected by following the method presented by Sanz Arranz et al. 39 Besides Raman analyses, the mineralogical characterization of powdered materials was complemented by XRD data. For this purpose, a laboratory Discover D8 XRD (Bruker) was used. The diffractometer is composed of a Cu X-ray excitation source (wavelength 1.54 Å) and a LynxEye detector. Fine-powdered rocks (granulometry ≤ 150 µm) were analysed by setting a scan range between 5 and 70° 2θ, a step increment in 2θ of 0.01 and a count time of 0.5 s per step. The collected diffractograms were interpreted using the BRUKER DIFFRAC.EVA software.</p><!><p>RAD 1. Drilled cores were analysed in-situ by using the portable RAD1 spectrometer. For this purpose, powdered samples were placed in a replicate of the ExoMars sample holder and, after flattening, a raster of measurements was performed by moving the X positioner at regular intervals of ≈ 300 µm. For each spot of analysis, the acquisition parameters were optimized manually. It must be noted that in-situ analyses were hampered by meteorological conditions, since the strong wind blowing during the trial produced vibrations to the spectrometer, compromising the acquisition of many Raman spectra. For this reason, a very limited number of spectra per sample (between 4 and 6) could be collected within the time constraints imposed by the mission simulation.</p><p>Despite the limited amount of Raman data, different mineral phases were successfully detected. Starting from ADC2 drill core, quartz (SiO 2 , 142, 204 and 464 cm −1 , Fig. 2a) was detected in both UP (upper part) and LP (lower On the other hand, Raman analysis of sample ACD2-LP displayed peaks at 276, 475 and 514 cm −1 , revealing the presence of feldspar (Fig. 2c). However, the mineral phase within this group was not clearly identified due to the low Signal to Noise Ratio (SNR) of the obtained spectra. Besides feldspar, the same spectra displayed a broad band at 3600 cm −1 (vibration of -OH group), together with an additional minor signal at 695 cm −1 . According to the work published by Wang et al., 2015 42 , these signals are characteristic of phyllosilicates within the mica subgroup (probably muscovite).</p><p>In-situ Raman analysis of ADC1 core were quite inconsistent since the two subsamples were highly fluorescent, which is a side-effect from electronic excitation that increases the background signal in the spectra, masking mineral Raman bands. Although the long exposure of the spot to the excitation laser helps quenching the florescence, this operation could not be performed due to the abovementioned vibrations induced by the wind. Despite this limitation, feldspar minerals were effectively detected in both LP and UP subsamples.</p><!><p>After the automatic adjustment of the spectra acquisition parameter to the constraints established for the ExoFiT trial, the number of Raman spectra automatically collected from each sample www.nature.com/scientificreports/ with this instrument varied between 9 and 12, which is below the minimum number of analysis per core that are expected to be carried out on Mars (20). This can be explained by the fact that the time dedicated to the in-situ study of drilled cores was narrowed due to logistic reasons (two RSM measurement cycles needed to be compressed within a time frame of 10 sols). However, additional spots were analysed to reach a total of 39 spectra per sample, being this the maximum number of analysis to be nominally performed on regular operations on Mars. The results described below are based on the complete set of Raman data gathered from each sample, although the summary provided in Table 1 allows one to distinguish the minerals detected within the ExoFiT-constrained time frame (black cross) from those additionally detected using nominal ExoMars mission parameters (red cross).</p><p>Starting from the ADC2 core, both UP and LP subsamples showed Raman features from quartz (main peak at 464 cm −1 and secondary signals at 124, 202, 263, 354, 805 and 1159 cm −1 , Fig. 3a), anatase (TiO 2 , main peaks at 142, 394, 510 and 634 cm −1 , Fig. 3b) and feldspar. By comparing the vibrational profile of feldspar spectra, different mineral phases were identified. For example, the positions of the peaks detected in the spectrum shown in Fig. 3c (main signals at 478 and 508, together with minor peaks at 167, 286, 409, 566, 763, 809 and 1100 cm −1 ) matched perfectly with the Raman features from albite 43 , being this mineral the Na-rich end member of the plagioclase subgroup (NaAlSi 3 O 8 ). As displayed in Fig. 3d, further spectra matched with anorthite reference spectrum (peaks at 150, 276, 401, 473, 515, 756, 799 and 1120 cm −1 , confirming the additional presence of K-feldspars in both subsamples. Albite and anorthite spectra were found to be often associated with additional peaks at 264, 407, 702 and 3628 cm −1 , which are consistent with the muscovite reference spectrum (KAl 2 (Si 3 Al) O 10 (OH) 2 , Fig. 3e). In addition to the mentioned mineral phases, calcium carbonate was additionally detected (main peaks at 149, 275, 709 and 1085 cm −1 , Fig. 3f) in both subsamples.</p><p>Raman results from ADC2-UP showed a higher and more complex mineralogical heterogeneity of this subsample when compared to AC2-LP. As shown in Fig. 3g, additional Raman peaks were found at 220 and 670 cm −1 , matching the characteristic signals of amphibole minerals (probably actinolite, Ca 2 (Mg,Fe) 5 Si 8 O 22 (OH) 2 ). As can be seen in Fig. 3h, the detection of clear peaks at 411, 490, 620, 1008 and 1135 cm −1 revealed the presence of gypsum (Ca 2 SO 4 ) as additional evaporitic mineral. One of the spectra gathered from the upper part of the core displayed two weak bands in the spectral range between 3600 and 3700 cm −1 . In detail, the peak at 3630 cm −1 matches the mentioned -OH vibration from muscovite, while the signal at 3695 cm −1 could be associated with additional clay minerals such as kaolinite, serpentine or chlorite 42 . In addition to those, a broad band at 3425 cm −1 was found associated with two phyllosilicate spectra. When compared to the Raman emission of organic functional groups described elsewhere 40 , the detected signal matches the characteristic position of the in-phase bending mode of aromatic amines (-NH2, Fig. 4c).</p><p>Raman spectra from the laboratory analyses of both drill cores, ADC1 and ADC2, showed a similar mineral composition. Indeed, quartz, anatase, plagioclase and K-feldspar were found to be the main mineral components of both UP and LP subsamples, while calcite and actinolite were exclusively detected in the upper part of the core. Besides the detection of mineral phases, the RLS ExoMars Simulator could detect Raman features from potential biomarkers, as displayed in Fig. 4a. A doublet at 2190 and 2250 cm −1 was clearly identified in both UP and LP samples that, according to the results presented in previous works, could correspond to the vibration mode of different functional groups containing nitrogen 40 . Similarly, the peak observed around 1445 cm −1 can be either attributed to the symmetric bending of -CH2 or the asymmetric bending of -CH3. Further potential organic peaks were detected on sample UP, as shown in Fig. 4a, with features appearing at 2800 and 2850 cm −1 that fall within the C-H stretching region (2800-3100 cm −1 ) 44 . Concerning sample LP, additional vibrational features from organic functional groups are shown in Fig. 4b,d, with peaks detected in the range between 1300 and 1700 cm −1 . More specifically, the three signals at 1340, 1380 and 1530 cm −1 can be assigned to the C-(NO2) functional group (both symmetric and asymmetric stretching), while the peak at 1644 cm −1 can be related to the stretching mode of C=N. As shown in Fig. 6, diffractograms from ADC1 subsamples displayed wider and less intense peaks. Considering that XRD analyses were run under the same measurement conditions, including the amount of powdered material, it can be deduced that ADC1 subsamples could contain some secondary phase of low crystallinity. By comparing the position of the detected peaks with XRD reference pattern, both subsamples are mainly composed of quartz with minor amounts of plagioclase and muscovite. In the case of LP sample, hematite signals were detected (33.15 and 35.68 2θ), while UP displayed minor amounts of amphibole.</p><p>The overall results gathered from the use of both spectroscopic and diffractometric systems are provided in Table 1. ADC1 and ADC2 drill cores are characterized by a complex mixture of organic and inorganic compounds. Summarized in Table 1, the mineralogical characterization obtained from the combined use of in-situ and laboratory Raman spectrometers is in good agreement with XRD data. Beside confirming the identification of the main mineral phases (quartz, feldspar and muscovite), the two RLS representative prototypes also detected additional minor compounds, whose concentration was often below the detection limit of XRD (anatase, calcite, amphibole and additional phyllosilicate, depending on the sample). Having in mind the forthcoming ExoMars mission, this result is extremely relevant as it demonstrates that the analytical strategy based on the multipoint Raman analysis of powdered samples could effectively help disclosing the composition of complex mineralogical mixtures. The two Raman spectrometers, operating under the same operational constraints of the RLS instrument, were able to detect phyllosilicate minerals, which are one of the main scientific targets defined for the ExoMars mission. Indeed, it is well known that phyllosilicates are capable of hosting microorganisms and accumulating biomarkers within their crystalline structure, thus potentially playing a key role in the preservation of life traces on Mars 45 . In fact, the large phyllosilicate deposits detected from orbit at Oxia Planum 46,47 were one of the main drivers in its selection as the landing site for the Rosalind Franklin rover.</p><p>The results obtained from the phyllosilicate-bearing samples agree with this thesis, as Raman spectra often presented features corresponding to different organics functional groups. Even though the Raman-based detection of organics in Atacama Desert samples was already achieved in previous works 34 , the great astrobiological relevance of the present research is based on the fact that (1) spectra were collected by Raman systems engineered to mimic the quality of RLS, and (2) drilling sites were remotely selected by the RCC team, who was operating the mission simulation from 11,000 km of distance having no more inputs than the data returned from the rover. The functional groups detected by Raman (including -C≡N, -NH2 and C-(NO2)) are compatible with the presence of nitrogen-fixing microorganisms in the drilled samples. Again, this result fits with previous works presented by Maza et al. 2019, who revealed the presence of six potential nitrogen fixers in the subsurface of the Atacama Desert 48 . Furthermore, it must be noted that LP samples returned the higher number of biomarkers spectra, suggesting that the microbial activity in the subsoil (below 15-20 cm of depth) is higher than in the surface. This gradient in microbial activity may be due to the fact that more favourable conditions for life proliferation can be found at higher depths (e.g., higher water content and lower exposure to UV radiation). Knowing that the analysis of subsoil samples is the core strategy for the ExoMars mission to detect traces of life on Mars, the Raman results here described are extremely promising as they confirm its efficacy.</p><p>Evaluation of RLS representative prototypes. By participating to the ExoFiT trial, the RLS team could evaluate advantages and disadvantages provided by the use of the RLS representative prototypes in ExoMarsrelated studies. Even though there are numerous studies evaluating the capabilities of the RLS ExoMars Simulator, this is the first work presenting Raman data gathered from the portable RAD1 system. For this reason, comparing the results of the two instruments could help evaluating the real scientific capabilities of the RAD1.</p><p>As shown in Table 1, the main mineralogical phases were correctly identified in RAD1 datasets, which results were in perfect agreement with spectra provided by the RLS ExoMars Simulator. However, when evaluating phases in minor proportions, some additional compounds could be detected by the laboratory setup. One of the main reasons is the fluorescence background, being more intense in spectra obtained with RAD1 at the LCC than it is in the laboratory ones. In the case of ADC2 subsamples, those containing a higher concentration of low crystalline phases (according to XRD), the fluorescence background covered almost completely the Raman vibrational features in the spectra. This difference can be justified by the use of different analytical approaches.</p><p>In the laboratory, spectra fluorescence was minimized by automatically performing laser-induced quenching on each spot of analysis (by using the same algorithm that implements RLS). However, this procedure was not feasible for in-situ analyses due to the mentioned time constraints and the stability problems of the spectrometer (triggered by adverse meteorological conditions).</p><p>It should be also noted that, despite the additional time required by florescence quenching, the number of spectra collected by the RLS ExoMars Simulator within the time constraints of the ExoFiT Trial was higher than those gathered by the RAD1 (manually operated). This result highlights that a more efficient characterization could be achieved through analysis automation. Learning from the Atacama trial experience, the RLS team is planning to couple the RAD1 system to a portable XYZ positioner as well as to implement its software with RLS algorithms for automatic multi-point analysis of samples. These improvements will allow to optimize data collection and to ensure a better simulation of the automatic operating mode of the RLS, thus increasing the scientific relevance of in-situ Raman studies of terrestrial analogue sites.</p><!><p>Using the data provided by the rover, the controlling team at the RCC the rover was capable of analysing the surrounding environments and identifying areas of scientific interest (PanCam and ISEM), investigating the textural features of the surface (CLUPI), determining the stratigraphy of the subsoil (WISDOM), extract drill cores (ExoMars drill emulator) and analysing their composition (RLS representative prototypes). Strictly focusing on Raman operations (the logistical and engineering challenges faced during the trial will be presented in a specific work), focusing on Raman operations (the logistical and engineering challenges faced during the trial will be presented in a specific work), the time frame dedicated to the spectroscopic analysis of drilled cores was found to be too narrow to achieve the number of spectra established for the nominal operation of RLS on Mars (between 20 and 39). As summarized in Table 1, the ability to detect minor or trace compounds of great scientific relevance (in this case of study, phyllosilicates and organics functional group) increases with the number of analysed spots per sample. In this sense, the RLS ExoMars Simulator missed the identification of the organic functional groups detected by RAD1 in sample ADC2-UP, this particular case evidences that more than 39 spectra per sample could be sometimes needed. Knowing the RLS will work in combination with MicrOmega, the additional information provided by the IR spectral images could be used to plan more targeted Raman analysis during real operations (for the ExoFiT trial the analysed spots were randomly selected), thus increasing the chances of detecting organics on Mars. However, if a scientifically interesting sample is collected from the Martian subsoil, the chances of detecting potential biomarkers could be increased by performing more than one cycle of spectroscopic analysis. Indeed, this procedure could help optimizing the use of the 32 single-use ovens equipped by MOMA to run GCMS analysis, thus enhancing the possibilities to fulfil the main objective of the mission.</p><!><p>During the Atacama ExoFiT test a complex series of operations, starting with the descent of the rover from the landing platform and ending with the extraction and analysis of drilled cores, were successfully carried out. Focusing on the analytical characterization of subsoil samples, the RLS representative prototypes demonstrated the key role that Raman spectroscopy could play in the fulfilment of the ExoMars mission objectives. By simulating the operational constraints of the RLS, the instruments used in this exercise disclosed the complex mineralogical composition of the samples, providing results qualitatively comparable to those obtained by a laboratory XRD system. In addition to the inorganic matrix, Raman spectrometers also detected several additional signals that could be assigned to biomarkers. In preparation of the upcoming ExoMars mission, this result confirms the capabilities of Raman spectroscopy, which was able to detect extremophilic microorganisms potentially colonizing the subsurface of Martian-like environments. Similar results on Mars would help in the selection of geological samples to be analysed by MOMA. In spite of the promising results, the comparison between RLS ExoMars Simulator and RAD1 data from sample ADC2-UP suggests that the nominal number of spots per sample the RLS will be nominally analyse on Mars (between 20 and 39) may not be sufficient to ensure the detection of trace compounds potentially present in the sample. This is why the ExoMars mission foresees an unprecedented cooperative approach (combined science), by which the instruments of the analytical laboratory will be able www.nature.com/scientificreports/ to analyse the same spot of the samples. More specifically, this capability will allow RLS to dedicate part of its operation to the analysis of sample spots previously identified by MicrOmega as regions of interest. Nevertheless, if during operations a sample of Martian subsoil reveals itself to be of high scientific interest, the possibility of running an additional cycle of combined MicrOmega-RLS analysis should be considered. Attending to the lessons learnt from the Atacama ExoFiT test, and recognizing the value of mission simulations in preparation for the ExoMars mission, the RLS team is planning to perform improvements (both hardware and software) of the portable RLS representative prototype, aiming to increase the scientific relevance of in-situ Raman studies of terrestrial analogue sites.</p><p>Received: 3 September 2020; Accepted: 30 December 2020</p>

Scientific Reports - Nature

7-Ketocholesterol induces P-glycoprotein through PI3K/mTOR signaling in hepatoma cells

7-Ketocholesterol (7-KC) is found at an elevated level in patients with cancer and chronic liver disease. The up-regulation of an efflux pump, P-glycoprotein (P-gp) leads to drug resistance. To elucidate the effect of 7-KC on P-gp, P-gp function and expression were investigated in hepatoma cell lines Huh-7 and HepG2 and in primary hepatocyte-derived HuS-E/2 cells. At a subtoxic concentration, 48-h exposure to 7-KC reduced the intracellular accumulation and cytotoxicity of P-gp substrate doxorubicin in hepatoma cells, but not in HuS-E/2 cells. In Huh-7 cells, 7-KC elevated efflux function through the activation of phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway. 7-KC activated the downstream protein synthesis initiation factor 4E-BP1 and induced P-gp expression post-transcriptionally. The stimulation of efflux was reversible and could not be prevented by N-acetyl cysteine. Total cellular ATP content remained the same, whereas the lactate production was increased and fluorescence lifetime of protein-bound NADH was shortened. These changes suggested a metabolic shift to glycolysis, but glycolytic inhibitors did not eliminate 7-KC-mediated P-gp induction. These results demonstrate that 7-KC induces P-gp through PI3K/mTOR signaling and decreased the cell-killing efficacy of doxorubicin in hepatoma cells.

7-ketocholesterol_induces_p-glycoprotein_through_pi3k/mtor_signaling_in_hepatoma_cells

1. Introduction<!>2.1. Chemicals, siRNA, and antibodies<!>2.2. Cell culture and treatments<!>2.3. Cell viability determination<!>2.4. Cellular accumulation of doxorubicin and efflux function of P-gp<!>2.5. Immunoblotting analyses<!>2.6. Isolation of lipid rafts and quantification of 7-KC and cholesterol<!>2.6. Real-time reverse transcription (RT)\xe2\x80\x93polymerase chain reaction (PCR)<!>2.7. Determination of superoxide radical, mitochondrial membrane potential, ATP content, and lactate production<!>2.8. Transfection of mTOR siRNA<!>2.9. Time-domain fluorescence lifetime imaging (FLIM)<!>2.10. Statistical analyses<!>3.1. Basal P-gp levels and effects of 7-KC on cell viability in Huh-7, HepG2, and HuS-E/2 cells<!>3.2. Effects of 7-KC on the accumulation of doxorubicin in Huh-7, HepG2, and HuS-E/2 cells<!>3.3. Effects of 7-KC on the cytotoxicity of doxorubicin in Huh-7 and HuS-E/2 cells<!>3.4. Effects of 7-KC on the efflux function of P-gp in Huh-7 and HepG2 cells<!>3.5. Differential effects of 7-KC, 7\xce\xb1-hydroxycholesterol, and cholesterol on the efflux function of P-gp in Huh-7 cells<!>3.6. Differential effects of 7-KC on the expression of P-gp in Huh-7 and HuS-E/2 cells<!>3.7. The main distribution of 7-KC in the non-lipid raft domains in Huh-7 cells<!>3.8. Tthe activation of PI3K/mTOR pathway by 7-KC in Huh-7 but not in HuS-E/2 cells<!>3.9. Effects of N-acetyl cysteine, oligomycin, and PI3K/mTOR inhibitors on 7-KC-mediated P-gp induction in Huh-7 and HepG2 cells<!>3.10. Alteration of NADH fluorescence lifetime and the stimulation of lactate production by 7-KC in Huh-7 cells<!>4. DISCUSSION

<p>The multidrug resistance (MDR)1 (ABCB1) gene-encoded P-glycoprotein (P-gp) pumps out a variety of xenobiotics and endogenous substances from inside cells to the extracellular region [1]. Its induction contributes to intrinsic resistance to chemotherapeutic agents before the drugs are taken and to acquired resistance after repeated cycles of chemotherapy. Hepatocellular carcinoma (HCC) usually has an exceptionally poor response to systemic treatment with chemotherapeutic agents [2]. Doxorubicin is commonly used as a chemotherapeutic agent in the treatment of hepatoma. The concentration of doxorubicin required for a 50% decrease of cell proliferation in P-gp (+) Hep3B and HepG2 cells was approximately 4-fold higher than that in P-gp(−) SK-HEP-1 cells [3], indicating that the level of functional P-gp represented one of the key factors affecting the cell-killing efficacy of doxorubicin. In a study of HCC patients, patients with a detectable level of P-gp protein in tumor sections had a lower disease-free interval and survival time [4]. However, P-gp expression levels did not exhibit a significant correlation with cell proliferation, mitotic counts, and the existence of immunodetected p53. In a study of liver samples of HCC patients, the fractions of patients carrying immunodetectable P-gp in groups that had (27 patients) and had not (16 patients) undergone previous chemotherapy were similar [5]. In addition, P-gp protein levels in tumor tissues tend to be higher than the levels in the surrounding normal tissues in HCC patients with or without cirrhosis [6,7]. Thus, we postulated that microenvironmental factor(s) in tumor tissues might up-regulate the expression of P-gp.</p><p>Recent evidence has emphasized the importance of the tumor microenvironment (e.g., alterations in the membrane lipid composition) in intrinsic resistance to chemotherapy [8]. Hypercholesterolemia appears to be a key paraneoplastic syndrome in HCC patients [9]. Oxysterols, including 7-ketocholesterol (7-KC), are generated by the oxidation of cholesterol either by autooxidation or by enzymes such as cytochrome P450s [10]. 7-KC is one of the main oxysterols found in healthy human plasma and is abundant in retina and atherosclerotic plaques. Exposure of cells to 7-KC elicited a variety of defense responses, including inflammation, apoptosis, and the stimulation of vascular endothelial growth factor [10]. The blood concentration of 7-KC was about 7–20 ng/ml (17 – 50 nM) [11,12] in healthy controls. In patients with chronic hepatitis, lung cancer, and rectal cancer, blood 7-KC concentrations were 2- to 4-fold higher than those in healthy controls [11,12]. The mean hepatic contents of 7-KC in different groups were in a wide range of 0.24 – 36.85 µg/g liver [11,13]. According to the density of liver (1.051g/ml) [14], the mean hepatic contents of 7-KC were 0.57 – 91.98 µM, which was higher than its blood concentration. However, the hepatic content or blood concentration of 7-KC in hepatoma patients has not been reported. In hepatoma-bearing rats with hyperlipidemia, the mRNA and protein levels of cholesterol efflux pumps ABCA1 and ABCG1 in tumor tissues were significantly higher than their respective levels in host or control livers [15]. In patients with familial combined hyperlipidemia, the cholesterol lowering agent atorvastatin efficiently decreased plasma 7-KC levels to those of normolipidemic controls [16]. In hypercholesterolemia patients, the ABCB1 mRNA level in peripheral blood mononuclear cells (PBMC) was significantly reduced after taking a daily dose of 10 mg atorvastatin for 4 weeks [17]. There was a significant correlation between the reduction of total cholesterol and the mRNA level of ABCB1 in PBMC. In hepatoma cell line HepG2, 24-h exposure to atorvastatin decreased the expression level and efflux function of P-gp [17]. However, this decrease was not affected by mevalonic acid lactone, suggesting that the intermediates produced during cholesterol synthesis might not be involved. However, the effects of cholesterol oxidation products on P-gp were not reported.</p><p>P-gp-mediated efflux requires an energy supply from ATP, which is primarily provided by mitochondrial oxidative phosphorylation and cytosolic glycolysis. Reduced nicotinamide adenine dinucleotide (NADH) is a key cofactor in these sources and it acts as a principal electron and proton donor in mitochondria [18]. Although it exists in both oxidized (NAD+) and a reduced (NADH) forms, only NADH is intrinsically fluorescent. This auto-fluorescence of NADH allows time-resolved fluorescence decay studies to be performed, thereby providing a noninvasive method to analyze energy metabolism in viable cells [18]. The fluorescence lifetime and the ratio of free (mainly localized in the cytoplasm) to protein-bound (mainly localized in the mitochondria) NADH are likely related to the NADH/NAD+ ratio [19]. Among oxysterols, 7-KC was first studied due to its marked pathological and toxicological effects [10] and elevated concentrations in patients with chronic hepatitis [11]. To examine the effect of 7-KC on P-gp, the efflux function and expression of P-gp were investigated in hepatoma cell lines Huh-7 and HepG2 and in the immortalized primary hepatocyte cell line, HuS-E/2 [20]. The signaling participated in the P-gp up-regulation and the changes in energetic machinery were illustrated.</p><!><p>Akt kinase inhibitor (AKI), bromopyruvate, cholesterol, doxorubicin, 17β-estradiol, 7-KC, N-acetyl cysteine, LY294002, 2-(N-morpholino)-ethanesulfonic acid (MES), 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), oligomycin, rapamycin, rhodamine 123 (Rh123), sodium oxamate, and verapamil were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol, NaCl, sucrose, and Triton X-100 were purchased from Merck KGaA (Darmstadt, Germany). Dihydroethidium and 5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1) were purchased from Invitrogen (Carlsbad, CA, USA). ON-TARGETplus™ (SMARTpool) mTOR and scramble siRNAs were purchased from Thermo Scientific-Dharmacon RNAi Technologies (Suwanee, GA, USA). Antibodies against phosphorylated and total Akt, the mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 kinase (S6K), and eukaryotic translation initiation factor (eIF) 4E-binding protein 1 (4E-BP1) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal antibody Mdr (H-241): sc-8313 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-labeled monoclonal anti-P-gp was purchased from Abcam (Cambridge, MA, USA). Rabbit polyclonal anti-caveolin-1 was purchased from BD Biosciences Pharmingen (Franklin Lakes, NJ, USA). Antibodies against α-tubulin and β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA).</p><!><p>HuS-E/2 cells (HPV18/E6E7-immortalized primary human hepatocytes) were generously provided by Dr. K. Shimotohno (Kyoto University, Japan) and were cultured following the method established by Aly et al. [20]. The doxorubicin-resistant breast cancer cell line MCF-7/ADR was cultured as described previously [21]. Huh-7 and HepG2 hepatoma cells were cultured using complete Dulbecco's modified Eagle's medium (DMEM), pH 7.2, containing 25 mM sodium bicarbonate, 1% non-essential amino acid, 1% glutamine, 10% fetal bovine serum (FBS, purchased from Biological Industries, Kibbutz Beit HaeMek, Israel), and penicillin/streptomycin. The serum-free medium contained 100 nM sodium selenite (Na2SeO3), 0.1% bovine serum albumin, and penicillin/streptomycin [22]. Cells were seeded and cultured for 24 h prior to drug treatment. 7-KC and cholesterol were dissolved in ethanol, and the final concentration of ethanol in medium was less than 0.1%. During cell treatment, 7-KC- and cholesterol-containing media were changed every 24 h.</p><!><p>Cell viability was monitored using MTT reduction [23], trypan blue exclusion, and lactate dehydrogenase (LDH) release analyses. In the trypan blue exclusion assay, cells were trypsinized and resuspended in a complete medium containing 0.04% trypan blue and the viable cells were enumerated using a hemocytometer. The activity of LDH released from cells to the medium was determined using a Cytotox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, WI, USA). The IC50 value of cell growth inhibition was calculated by curve fitting using Grafit software (Erithacus Software, Surrey, UK).</p><!><p>To determine the accumulation of doxorubicin, 80% confluent cells were exposed to 10 µM (Huh-7 and HuS-E/2) and 50 µM (HepG2) doxorubicin for 1 h. The mean fluorescence of doxorubicin retained in 2 × 104 cells was measured using a flow FACSCalibur cytometer (Ex: 480 nm; Em: 564–660 nm; BD Biosciences, San Jose, CA, USA). The Rh123 efflux function of P-gp was determined using a method modified from Pan et al. [23]. Primarily, cells were seeded on a 12-well plate (1 × 105/ well) for 24 h and then incubated with 5 µM Rh123 for 1 h. After three washes with ice-cold phosphate buffered saline (PBS) in the dark, cells were incubated with Rh123-free medium in the absence and presence of 100 µM verapamil (a P-gp inhibitor) for 3 h. The fluorescence of Rh123 retained in the cells was measured (Ex: 485 nm; Em: 538 nm) and the percentage of Rh123 exported from the cells (E) was determined as the decrease of cellular Rh123. The efflux function of P-gp was monitored in terms of the decrease of export of Rh123 in the presence of verapamil (Etotal − E+verapamil, where Etotal is the value in the absence of verapamil).</p><!><p>Cell lysate was prepared using 38 mM Tris·HCl buffer, pH 7.5, containing 0.15 M NaCl, 0.25% sodium deoxycholate, and 0.01% Triton X-100. Protein concentration was determined using Bradford reagent with bovine serum albumin as the standard (Bio-Rad, Hercules, CA, USA). Electrophoresis and the following electrotransfer of proteins (25 µg/well of cell lysate and 3.5 µg/well of sucrose gradient fractions were loaded) from the slab gel (7.5% or 10% polyacrylamide gel) to a nitrocellulose membrane were performed as described previously [23]. Immunoreacted proteins were visualized using a chemiluminescence kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Relative band intensity was analyzed by densitometry using the Image Master software (Pharmacia Biotech, Uppsala, Sweden) and normalized by the band density of internal controls (β-actin, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), or α-tubulin). To determine the expression level of cell surface P-gp, cells were collected, immunostained with FITC-labeled anti-P-gp, and analyzed using the flow cytometric determination. Fluorescent isotype control immunoglobulin G, FITC-IgG2a (Abcam, Cambrige, MA, USA) was used for immunostaining as the internal control. Mean fluorescence intensity was determined using FACS/Cell Quest software (BD Biosciences, San Jose, CA, USA).</p><!><p>Lipid rafts were isolated from Huh-7 cells using a method modified from the report of Royer et al. [24]. After 48-h exposure, 5×107 cells were washed with ice-cold PBS, treated with trypsin/EDTA (Biological Industries, Kibbutz Beit HaeMek, Israel), neutralized with medium, and centrifuged at 200×g for 5 min at room temperature. The cell pellet was washed with PBS and then treated with 1 ml of 25 mM MES buffer (pH6.5) containing 150 mM NaCl, 1% (w/v) Triton X-100, and a mixture of protease inhibitors (Roche Applied Science, Indianapolis, IN, USA). After 30 min at 4°C, the cells were homogenized on ice using a Teflon pestle-glass homogenizer for 10 strokes. Cell lysate was mixed with 1 ml MES and 2 ml 80% (w/v) sucrose in MES and layered on the bottom of centrifugation tubes. 4.5 ml of 30% and 3 ml of 5% sucrose in MES buffer were sequentially overlaid on the top of the mixture of cell lysate and sucrose. After 24-h centrifugation at 36,000 rpm in a SW41 rotor (Beckman Coulter, Inc., Fullerton, CA, USA), each fraction of 1.1 ml were collected from the top to the bottom of tubes, mixed, and stored at −20°C. An aliquot (1 ml) of each fraction was extracted with 2 volumes of dichloromethane and mixed. After centrifugation, 1 ml of the bottom layer was dried under N2 gas in the chemical hood. Samples were dissolved in a acetonitrile/methanol mixture and subjected to the liquid chromagraphy (LC)-mass spectrometry (MS, with atmospheric pressure chemical ionization (APCI)) analysis as described before [25]. Protein concentration of each fraction was determined as described above.</p><!><p>Cellular total RNA was prepared from cells using the TRIzol reagent following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). Total RNA (20 µg) was subjected to the RT reaction using RevertAid™ reverse transcriptase (ThermoFischer Scientific, Waltham, MA, USA). The cDNA products were further subjected to PCR amplification with SYBR Green using the LightCycler® 480 Real-Time PCR System (Roche Molecular Systems, Branchburg, NJ, USA). The primer sets used were as follows: MDR1, forward: CAGCTATTCGAAGAGTGGGC; reverse: CCTGACTCACCACACCAATG; and β-actin, forward: GAGCCACATCGCTCAGACAC; reverse: CATGTAGTTGAGGTCAATGAAGG. An initial enzyme activation step of 94°C for 4 min was followed by 40 cycles each of 55°C for 45 s and 72°C for 1 min. The threshold cycle (Ct) number was determined. The relative mRNA expression levels were normalized to the β-actin expression level, which allowed target cDNA calculation as 2−(Ct MDR1 − Ct β-actin).</p><!><p>To determine the superoxide radical, Huh-7 cells were incubated with 5 µM dihydroethidium in a complete medium for 1 h in the dark [26]. The fluorescence intensity of cells resuspended in PBS was measured using the flow cytometric determination. Mitochondrial membrane potential was monitored using a detection kit with the cationic JC-1 dye (Invitrogen, Eugene, OR, USA) following the manufacturer's instructions. The ratio of the fluorescence intensities of red (Ex: 550 nm; Em: 600 nm) to green (Ex: 485 nm; Em: 535 nm) fluorescence was determined. Total cellular ATP content was quantitatively determined using ATP bioluminescence assay kit HS II (Roche Diagnostics, Mannheim, Germany). Lactate concentration of the medium was determined using an L-lactate assay kit purchased from Eton Bioscience (San Diego, CA, USA). The amount of lactate was calculated by interpolation from the standard curve established following the manufacturer's instructions.</p><!><p>Huh-7 cells were transfected with mTOR siRNA (25 nM) and scramble siRNA (25 nM) using DharmaFECT transfection reagents (Thermo Scientific-Dharmacon RNAi Technologies, Suwanee, GA, USA) following the instruction manual and then cells were incubated in a CO2 incubator for 48 h. The knockdown of mTOR expression was examined by immunoblotting as described above. Transfected cells were exposed to 7-KC for 48 h and the Rh123 efflux function was determined as described above.</p><!><p>Time-domain FLIM was performed following the method of Ghukasyan et al. [27] to determine the fluorescence lifetime (τ) and the fractional contribution ratio of free to protein-bound forms of NADH (a1/a2). An oil immersion objective lens (40 ×, 1.3 N.A., Olympus), average power between 3 to 5 mW, and a bandpass filter of 450 ± 40 nm (Edmund Optics, Barrington, NJ, USA) were used. Images (256 × 256-pixel) of grouped cells were taken from 3 – 5 different locations per dish with minimum spacing of cells. The full width at half maximum of instrument response function (IRF) was measured to be about 200 ps. Fluorescence lifetime and fractional contribution (relative amplitude, a) were analyzed using the SymPhoTime software package (PicoQuant, Berlin, Germany) [19]. Curve fitting of the experimental data to a two-exponential decay model was performed using the equation F(t) = a1e(−t/τ1) + a2e(−t/τ2), where F(t) is the fluorescence intensity at time t. The a and τ values of the free and protein-bound NADH are indicated by 1 and 2, respectively. A time-optimized procedure was used to determine the quality of fitting with a minimum reduced χ2 value [27].</p><!><p>All data are given as the mean ± SD. The statistical significance of the differences between the control and treated groups was evaluated using Student's t test. The results from cells treated with increasing concentrations of 7-KC were analyzed using one-way analysis of variance followed by Dunnett's test (GraphPad Prism 3.02, GraphPad Software, La Jolla, CA, USA). A p value of less than 0.05 was considered statistically significant.</p><!><p>In Huh-7 and HepG2 cells, an immune-reacted protein band was detected with the same electrophoretic mobility of the band detected in a P-gp-overexpressed MCF-7/ADR cell line. In HuS-E/2 cells, the P-gp level was relatively low (Fig. 1A). MTT assay was performed to monitor the number of viable cells with functional mitochondria. In Huh-7 cells, cell viability was slightly elevated by 10 and 25 µM 7-KC, but viability declined when the concentration was greater than 37.5 µM (IC50 for growth inhibition: 46.4 ± 2.5 µM) (Fig. 1B). To ensure a concentration without cell growth inhibition (defined as "sub-toxic") of 7-KC in Huh-7 cells in later experiments, cell viability was further examined using trypan blue exclusion assays and LDH release to monitor the plasma membrane integrity. Cell viability was unchanged for 7-KC concentrations up to 10 µM (data not shown). The influence of 7-KC on HepG2 cells was similar to that on Huh-7 cells. The growth of HepG2 cells was stimulated at 25 µM and then started to decline at higher concentrations (IC50: 68.3 ± 2.2 µM), whereas there was no 7-KC-mediated growth stimulation in HuS-E/2 cells. Compared to Huh-7 and HepG2 cells, HuS-E/2 cells were more susceptible to 7-KC-induced cell death (IC50: 27.4 ± 2.5 µM). Consequently, cells were treated with 7-KC at sub-toxic concentrations (Huh-7, ≤ 10 µM; HepG2, ≤ 37.5µM; HuS, ≤ 10 µM) in the following P-gp studies. To examine the contribution of the higher P-gp levels found in hepatoma cells to their increased resistance to 7-KC-induced toxicity, the effect of verapamil (a P-gp inhibitor) on cell growth was studied in Huh-7 cells. Verapamil slightly enhanced the cytotoxicity of 7-KC at the concentrations higher than 37.5 µM (Fig. 1C). Because the toxicity enhancement only occurred when cells were exposed to a cytotoxic concentration of 7-KC, the enhancement by verapamil could not support the contribution of P-gp to the greater resistance to 7-KC toxicity in hepatoma cells.</p><!><p>Cellular clearance of doxorubicin was found to be approximately half by diffusion and half by P-gp-mediated efflux [28]. The P-gp substrate doxorubicin predominately accumulated in the nucleus of control and 7-KC-treated Huh-7 cells (fluorescence microscopy images not shown). After 1-h exposure to doxorubicin in the accumulation assay, cell morphology and MTT values were not affected in cells (data not shown). Pre-exposure to 7-KC for 48 h caused a concentration-dependent decrease of the doxorubicin accumulation in Huh-7 cells and this decrease was greater than that in HepG2 cells under the same exposure concentration (Fig. 2A). In contrast, doxorubicin accumulation in HuS-E/2 cells was not reduced by 7-KC at concentrations up to 7.5 µM. On increasing the concentration of 7-KC to 10 µM, cellular doxorubicin accumulation increased slightly in HuS-E/2 cells (Fig. 2A), which was the opposite of the responses in Huh-7 and HepG2 hepatoma cells. The following determinations of doxorubicin toxicity and the P-gp inductive mechanism in hepatoma cells were performed using Huh-7, not HepG2, because 7-KC had a stronger effect on Huh-7.</p><!><p>To examine the impact of 7-KC on the cell-killing efficacy of doxorubicin, a doxorubicin-treatment causing cytotoxicity was designed and the cytotoxicity was assessed by MTT assay and LDH release. Huh-7 cells were pre-exposed to 7.5 µM 7-KC for 48 h and then to doxorubicin for a further 24 h. Compared with doxorubicin treatment alone, results of MTT assay showed that pre-exposure to 7-KC significantly diminished the cytotoxicity of doxorubicin (Fig. 2B). Treatment of cells with verapamil alone did not affect cell viability, whereas the presence of verapamil enhanced the cytotoxicity of doxorubicin in 7-KC-pre-exposed cells, indicating that P-gp played a crucial role in the 7-KC-mediated increase of resistance to doxorubicin in hepatoma cells. Consistent with the results of MTT assay, 7-KC pre-exposure reduced the LDH release induced by doxorubicin in Huh-7 cells (Fig. 2C). In contrast, in HuS-E/2 cells, 7-KC pre-exposure (7.5 µM, 48 h) did not affect the cytotoxicity of doxorubicin (Fig. 2D). Neither did verapamil enhance the cytotoxicity of doxorubicin in HuS-E/2 cells. The failure of verapamil and 7-KC to affect doxorubicin cytotoxicity in HuS-E/2 cells is consistent with their low P-gp expression level (Fig. 1A) and the absence of changes in doxorubicin accumulation on treatment with 7.5 µM 7-KC (Fig. 2A).</p><!><p>To determine the efflux function of P-gp, Rh123 was used as a substrate. The basal export of Rh123 in Huh-7 and HepG2 cells were 21.6 ± 1.5% and 19.4 ± 5.9% (mean ± SD), respectively. However, the efflux function of HuS-E/2 cells was below the detection limit. To evaluate the competitive potential of 7-KC against Rh123, Huh-7 cells were co-exposed to 7-KC (7.5 µM) and Rh123 for 1h. The co-exposure could not interfere with the basal export of Rh123 (data not shown), suggesting that 7-KC did not compete with Rh123 for export. However, the P-gp efflux function was elevated by 43–68% after 24-h and 48-h pre-exposure to 7-KC at concentrations of 5–10 µM (Fig. 3A). Because the increase of efflux function after 48-h exposure to 7-KC was greater than that after 24-h exposure, P-gp induction was further studied after 48-h exposure. In HepG2, 48-h exposure to 10–37.5 µM 7-KC also stimulated Rh123 efflux by up to 67% (Fig. 3B). To examine the reversibility of this efflux stimulation, Huh-7 cells pre-exposed to 7.5 µM 7-KC (24 h (Fig. 3C) or 48 h (Fig. 3D)) were cultured in a complete medium without 7-KC for a further 24 h. After recovery in 7-KC-free complete medium, the increased efflux function returned to the basal level, indicating that P-gp induction by 7-KC is reversible.</p><!><p>Research has shown that serum has a high concentration of cholesterol (828 – 931 µM in FBS, data provided by Biological Industries, Kibbutz Beit HaeMek, Israel) and possibly exhibited trace amounts of oxysterols. Therefore, the effects of 7-KC and cholesterol on efflux function were determined in Huh-7 cells cultured in a serum-free medium as described in Materials Methods. It was found that 7-KC (7.5 µM), but not cholesterol (7.5 µM), elevated the efflux function of P-gp after 48-h culture in both a serum-containing medium (Fig. 4A) and a serum-free medium (Fig. 4B).7α-Hydroxycholesterol at 1–10 µM did not exhibit cytotoxicity (data not shown) and could not affect the Rh123 efflux function of P-gp in Huh-7 cells cultured in a serum-containing medium (Fig. 4A).</p><!><p>To illustrate the mechanism of P-gp induction by 7-KC, P-gp protein and mRNA levels were analyzed. Immunoblotting analyses of Huh-7 cell lysate showed that exposure to 5 and 7.5 µM 7-KC increased total P-gp protein by 52% and 53%, respectively (Fig. 5A). The flow cytometric detection of immunostained cell-surface P-gp showed that exposure to 5 and 7.5 µM 7-KC increased the surface P-gp protein level by 34% and 35%, respectively (Fig. 5B). However, 7-KC did not affect the MDR1 mRNA level (Fig. 5C). These results suggest that 7-KC up-regulates P-gp expression at a post-transcriptional step. In HuS-E/2 cells, the P-gp protein level was not induced after 48 h-exposure to 7.5 µM 7-KC (90 ± 18% of the control level) (blot not shown).</p><!><p>After centrifugation with a sucrose density gradient, 10 fractions were collected from the top (low-density) to the bottom (high-density). After 48-h exposure of Huh-7 cells to 7.5 µM 7-KC, 7-KC was found to distribute mainly into fractions 6 – 9 (Fig. 6A). The cholesterol enriched (Fig. 6A) and caveolin-1 (Fig. 6B) abundant fractions were found in fractions 3–5, indicating the fractions in which the lipid raft domains mainly located. The high density fractions 8–10 contained relatively higher concentrations of total protein than the low density fractions (Fig. 6B). This result revealed that this 7-KC exposure resulted in the main distribution of 7-KC in the non-lipid raft domains in Huh-7 cells.</p><!><p>In Huh-7 cells, the phosphorylation status of molecules participating in the PI3K/mTOR pathway was determined by immunoblotting (Fig. 7A). 7-KC at 5 and 7.5 µM stimulated the phosphorylation of Akt and the downstream kinase mTOR. Subsequently, the phosphorylation of two important downstream effectors of mTOR, p70S6K and 4E-BP1, were enhanced. In contrast, in HuS-E/2 cells, the phosphorylation status of mTOR was not enhanced by 48-h exposure to either 5 or 7.5 µM 7-KC (Fig. 7B).</p><!><p>To investigate the upstream signaling of P-gp induction, first, the effects of 7-KC on superoxide anion formation and mitochondrial membrane potential were determined. The 48-h exposure of Huh-7 cells to 7-KC increased cellular superoxide formation, whereas N-acetyl cysteine treatment decreased the constitutive production of superoxide anions (Fig. 8A). The increase of superoxide radicals by 7-KC was eliminated by 30-min pre-treatment with N-acetyl cysteine, a radical scavenger, followed by 48-h co-treatment with 7-KC. Determination of changes in mitochondrial membrane potential using the JC-1 dye showed that 7-KC increased the J-aggregates, suggesting disruption of the proton flux into the mitochondrial matrix (Fig. 8B). The ATP synthase inhibitor oligomycin increased the mitochondrial membrane potential. However, the potential remained high when cells were pre-treated (1 h) with oligomycin and then co-treated (48 h) with 7-KC and oligomycin.</p><p>Second, the effects of radical scavengers and signaling inhibitors on 7-KC-elevated efflux activity and protein level of P-gp were studied. Cells were pre-exposed to scavenger/inhibitor for 1 h before 48-h co-treatment with 7-KC. Although oligomycin elevated the mitochondrial membrane potential, as 7-KC did, oligomycin did not stimulate Rh123 efflux. N-acetyl cysteine and oligomycin did not suppress the Rh123 efflux induced by 7-KC. Single treatment with LY294002 (non-selective PI3K inhibitor) and AKI decreased the constitutive efflux function (Fig. 8C), whereas the mTOR inhibitor rapamycin could not affect it. On co-treatment with 7-KC, the LY294002, AKI, and rapamycin decreased the 7-KC-enhanced efflux activity to a basal level. In Huh-7 cells, rapamycin treatment decreased P-gp induction by 7-KC (Fig. 8D). In HepG2 cells, 7-KC-induced P-gp protein level was also decreased by rapamycin. In Huh-7 cells, the transfection of mTOR siRNA knocked down the expression of mTOR protein and suppressed the induction of Rh123 efflux by 7-KC (Fig. 8E). These results revealed that activation of the PI3K/mTOR pathway is essential for P-gp induction by 7-KC in Huh-7 and HepG2 cells.</p><!><p>The cellular total ATP level remained unchanged after exposure to 7-KC (Fig. 9A). However, time-resolved fluorescence analysis showed that 7-KC shortened the average lifetime (τ) of NADH, whereas cholesterol had no effect. Representative FLIM microscopy images and time-dependent amplitude changes are shown in Fig.9B and 9C, respectively. The ratio (a1/a2) of free to bound forms of NADH increased after 7-KC exposure, indicating that the decrease of average τ was mainly attributable to the increased fractional contribution of the short lifetime τ1 of free NADH or the decreased τ2 of the protein-bound NADH (Table 1). These changes suggested the perturbation of energetic sources by 7-KC. Consequently, the generation of glycolytic product lactate was investigated to examine the glycolytic shift. The exposure to 7-KC increased lactate production and this increase was reduced by rapamycin (Fig. 9D). Bromopyruvate and sodium oxamate decreased lactate production (Fig. 9D), but none of them reduced 7-KC-induced efflux activity (Fig. 9E). The contribution of glycolytic stimulation to P-gp induction is not obvious.</p><!><p>Increasing attention is being paid to the unique tumor microenvironment in cancer therapy. Our results showed that 7-KC (at a sub-toxic concentration of 5 and 7.5 µM, 48 h) but not cholesterol stimulated P-gp function in Huh-7 cells. 7-Hydroxycholesterol at a concentration up to 10 µM did not change the efflux function of P-gp. As a result of increased efflux by 7-KC, the accumulation and cell-killing efficacy of doxorubicin were reduced. In another experiment, Huh-7 cells were exposed to 1 µM 7-KC for 4 weeks with the subculture every 3–4 days. However, the Rh123 efflux function was not stimulated by 7-KC (data not shown). The lack of induction by this treatment could be attributed to factors such as inadequate exposure concentration and time-period, the influence of sub-culture process, and the differential effects of acute and chronic exposure. The concentration- and time-dependence of the effect of chronic 7-KC exposure and the P-gp modulation by the other oxysterols will be further investigated.</p><p>Compared with cholesterol, 7-KC has higher polarity and is less effective in forming ordered lipid domains and in phase boundary bilayer defects [29]. On the other hand, 7-KC has been reported to activate liver X receptor (LXR) [30], which has a dual function in the regulation of PI3K signaling [31,32]. The activation of PI3K signaling has been linked to lipid raft-associated receptors and kinases, such as epidermal growth factor receptor (EGFR) and Src kinase [33]. It has been reported that 7-KC became incorporated into the sphingolipid/cholesterol-enriched lipid raft domains of the plasma membrane when cells were exposed to 7-KC at a cytotoxic concentration of 50 µM [24]. Cell death was increased and the PI3K/Akt signaling was suppressed. However, our study of 7-KC at a sub-toxic concentration indicated that 7-KC mainly distributed in the non-lipid raft domains and the PI3K/Akt signaling was activated. 7-KC was able to form crystalline membrane domains different from cholesterol, which might be linked to the decreased cell death in aortic smooth muscle cells [34]. However, the association between 7-KC microcrystals and PI3K/mTOR activation has not been demonstrated. The mechanism by which 7-KC activated PI3K/mTOR signaling remains unclear.</p><p>Results of chemical inhibition of the signaling pathway, determinations of the phosphorylation cascade, and mTOR siRNA knockdown demonstrate that activation of PI3K/mTOR signaling participates in the 7-KC-mediated induction of P-gp, whereas the contribution of elevated ROS is small. Consistent with the down-regulation reported previously [35,36], our results also showed that constitutive Rh123 efflux activity was decreased by LY294002, AKI, and an inhibitor of extracellular signal-regulated kinase (ERK), PD98059 (data not shown). Contrary to the suppression by LY294002 and AKI, PD98059 did not suppress 7-KC-stimulated Rh123 efflux activity, suggesting that ERK might not be involved in this induction process (data not shown).</p><p>The present 7-KC treatment reveals a post-transcriptional induction via the stimulation of the phosphorylation of p70S6K and 4E-BP1, which execute the downstream signal of mTOR and promote the formation of active initiation complex of protein synthesis to accomplish translational regulation [37]. Atorvastatin treatment decreased the blood concentration of 7-KC in patients and the MDR1 mRNA level in HepG2 cells [16,17]. The decrease in 7-KC may not be the main contributor to atorvastatin-mediated down-regulation of P-gp, or atorvastatin may suppress MDR1 mRNA level via different signaling pathways. The post-translational regulation of P-gp has been reported to include phosphorylation through several protein kinases such as protein kinase C and Pim-1 [38]. However, the association between P-gp phosphorylation and efflux function remained inconsistent. To determine the effect of 7-KC on the phosphorylation status of P-gp, imunoblotting analysis of phosphorylated serine on the immunoprecipitated P-gp was performed. Our results showed that phospho-P-gp level that matches the increased total P-gp level was increased by 7-KC but not cholesterol exposure (data not shown). To further identify the contribution of phosphorylation to the functional induction of P-gp by 7-KC, effects of knockdown of protein kinases on the phosphorylation, ubiquitination, protein degradation process, and efflux function of P-gp need to be clarified.</p><p>7-KC reduced the cytotoxicity of doxorubicin in another hepatoma cell line HepG2, but not in HuS-E/2 cells. This irresponsiveness might be associated with the lack of 7-KC-mediated mTOR activation in HuS-E/2 cells. Normal and cancer cells exhibit distinct growth properties, including differences in the network/cross-talk among signaling pathways [39]. Our findings provide an evidence for the differential response of hepatoma and normal hepatocyte-derived cells to 7-KC. Huh-7 and HepG2 cells have several differences in their protein profiles, such as the mutation status of p53 [40]. Both p53-dependent and p53-independent induction of P-gp by xenobiotics have been reported in liver cancer cells [41,42]. The induction of P-gp by 7-KC occurred in both Huh-7 and HepG2 cells, suggesting that the p53 mutation in Huh-7 cells did not eliminate its responsiveness to P-gp induction. In our studies, the minimal concentration of 7-KC (5 µM) to show P-gp induction was higher than its blood concentration. However, cells were exposed to 7-KC for a short time-period of 48 h. A further study of chronic exposure to a pathological concentration of 7-KC in vivo is essential to evaluate the contribution of 7-KC to intrinsic chemoresistance in patients. Since 7-KC-mediated P-gp induction was found to be reversible after a 24-h recovery period without 7-KC, the reduction of 7-KC level may have beneficial effects in patients considered to receive chemotherapy.</p><p>Both 7-KC and oligomycin elevated the mitochondrial membrane potential, reflecting the reduced level of protons in the mitochondrial matrix. However, 7-KC but not oligomycin stimulated the efflux function of P-gp. Thus, the increased mitochondrial membrane potential could not directly contribute to P-gp induction. The reduced level of protons might result from mitochondrial hyperpolarization caused by ATP import from cytosolic glycolysis and from reverse ATP synthesis via ATP synthase. As a consequence, the total ATP level did not change and cell viability was unaffected. On the other hand, activation of the PI3K/mTOR pathway can also stimulate the aerobic glycolysis pathway and tumor growth [43]. A rapamycin-sensitive stimulation of lactate production by 7-KC was found in Huh-7 cells, supporting the mitochondrial hyperpolarization and glycolytic shift. NADH fluorescence is emitted mainly from mitochondria [19] and the free/bound ratio (a1/a2) of NADH was increased by oxidative phosphorylation inhibitor CoCl2 and by glycolytic inhibitor bromopyruvate [44], suggesting that a1/a2 might not be a selective marker of changes in the cellular energetic machinery. However, τ2 was increased when glycolysis was inhibited and decreased when oxidative phosphorylation was inhibited in cultured cells. Thus, a decrease in τ2 might be a marker of the glycolytic shift. Consistent with the 7-KC-mediated glycolytic stimulation and mitochondrial disruption, τ2 was decreased. Thus, energy generation might be shifted by 7-KC from mitochondrial oxidation to glycolysis, which is known as the Warburg effect [45]. Glycolytic intermediate pyruvate and glycolysis inhibitors (2-deoxy-D-glucose and iodoacetate) have been reported to increase and decrease the expression of P-gp, respectively [46]. However, in our study, neither sodium oxamate nor bromopyruvate suppressed 7-KC-induced efflux activity. P-gp induction by 7-KC may not be linked to enhanced glycolysis.</p><p>In summary, our findings demonstrate that 7-KC at a sub-toxic concentration stimulates the efflux function of P-gp, which engenders the reduced cell-killing effect of doxorubicin in Huh-7 cells. The up-regulation of P-gp by 7-KC occurred via the PI3K/mTOR pathway. 7-KC interfered with mitochondrial pH gradient and stimulated glycolysis. All of these events may conduct to the poor prognosis of cancer therapy. Further studies of the chronic effects of oxysterols at a pathological concentration in vivo are warranted.</p>

PubMed Author Manuscript

Finding the molecular scaffold of nuclear receptor inhibitors through high-throughput screening based on proteochemometric modelling

Nuclear receptors (NR) are a class of proteins that are responsible for sensing steroid and thyroid hormones and certain other molecules. In that case, NR have the ability to regulate the expression of specific genes and associated with various diseases, which make it essential drug targets. Approaches which can predict the inhibition ability of compounds for different NR target should be particularly helpful for drug development. In this study, proteochemometric modelling was introduced to analysis the bioactivity between chemical compounds and NR targets. Results illustrated the ability of our PCM model for high-throughput NR-inhibitor screening after evaluated on both internal (AUC > 0.870) and external (AUC > 0.746) validation set. Moreover, in-silico predicted bioactive compounds were clustered according to structure similarity and a series of representative molecular scaffolds can be derived for five major NR targets. Through scaffolds analysis, those essential bioactive scaffolds of different NR target can be detected and compared. Generally, the methods and molecular scaffolds proposed in this article can not only help the screening of potential therapeutic NR-inhibitors but also able to guide the future NR-related drug discovery.Electronic supplementary materialThe online version of this article (10.1186/s13321-018-0275-x) contains supplementary material, which is available to authorized users.

finding_the_molecular_scaffold_of_nuclear_receptor_inhibitors_through_high-throughput_screening_base

Background<!><!>Construction of proteochemometric modeling<!>Evaluation of proteochemometric modeling<!><!>Finding the molecular scaffolds for NR inhibitors<!>Discussion<!><!>Protein target descriptor<!>Inhibitor descriptor<!>Proteochemometric modeling<!>Model evaluation<!>Molecular scaffold searching<!>

<p>As a ligand dependent transcription factors, nuclear receptors (NR) can be activated by important molecules such as steroidal hormones, endogenous hormones, glucocorticoids and thyroid hormones [1, 2]. After activation, NR can regulate the expression of specific genes and then participate in several essential physiological processes such as development, homeostasis and metabolism of the organism [1, 2]. Since NR can affect the expression of enormous genes which associated with various diseases such as diabetes and hepatic adipose infiltration, it can be considered as an appropriate therapeutic target for new drug discovery. Till now, 48 nuclear receptors have been discovered in humans [3], 23 of them are certified as drug target by U.S. Food and Drug Administration (FDA). Meanwhile, over 13% FDA approved drugs were aimed at those nuclear receptors [4]. In that case, discover novel drugs as nuclear receptor inhibitors have acquired a particular significance for NR-related metabolic diseases treatment. In drug design, scaffold is the fixed part of a molecule which is the essential part for biological activity of molecule. Therefore, scaffold based strategies were widely used for drug discovery [5–7]. It can be noticed that finding a new scaffold often lead to the discovery of a new inhibitor classes which may have the potential to become future drugs [8–10]. In that case, finding novel bioactive scaffolds is an essential process in the area of drug design.</p><p>In order to discover the molecular scaffold of a class of molecules such as NR-inhibitors, massive structure of molecules with bioactivity need to be screened and clustered to finding the consensus structure domain. Traditionally, this screening evolving titration experiments is a time-consuming, expensive and labor-intensive process, which could be assisted by computer-aided drug design (CADD) [11]. In recent decades, different methods including virtual screening [12, 13], molecular docking [14, 15], de-novo drug design [16–18], pharmacophore modeling [19–21] and molecular dynamics [22, 23] were introduced to find bioactive molecules for further drug design. In the early 1960 s, quantitative structure activity relationship (QSAR) approach was established to discover the relationship between ligand and target [24]. In general, conventional QSAR based approaches consider structure information and bio-active value to efficiently predict the relationship between ligand and target. However, its prediction ability is limited to single target and enable to map multiple ligand-target relationship [25]. Also, the prediction ability of conventional QSARs were limited since only ligand information were used for model construction [25–27]. To avoid the shortages of QSAR, an approach relying on the description of both ligand and target to quantitatively analyze their relations was invented and termed as Proteochemometric (PCM) modeling in 2001 [28]. The main advantage of PCM modeling is to integrate information on both ligand and target to make the model applicable for multiple target screening, including GPCRs [29–31], proteases [32–34], kinases [35, 36], reverse transcriptase [37, 38]. However, according to author's knowledge, PCM for NR-inhibitor prediction was hardly reported.</p><p>In this article, two major steps including PCM modelling and scaffold finding were processed to guide the design of NR-inhibitors. Initially, based on a total number of 11 nuclear receptors and 9633 molecular compounds with EC50 values were derived from ONRLDB [39], a series of PCM modelling were generated to predict the inhibition ability for NR-inhibitors. After rigorous validation through both internal and external validation dataset, our PCM model was proved to have the potential ability for high-throughput NR-inhibitor screening. It should be noted that NR-targets validated in external dataset were not involved in our training set. That means for those NR proteins without enough bio-active data to establish a traditional QSAR models, our model may also have the ability to provide NR-inhibitor screening. Further, after molecular clustering based on our PCM model, novel bioactive scaffolds for NR-inhibitors can be discovered. The potential bioactive scaffolds for different NR targets were proposed for future drug discovery of NR-inhibitors.</p><!><p>10-fold cross-validation results of different machine learning methods</p><p>Results in Table 1 were calculated based on descriptor T1</p><p>aThis parameters can't be calculated in here (continuous predict values are needed to calculate AUC value)</p><p>Performance of PCM modeling. a Cross-validation performance of PCM model constructed by RF classifier based on four different protein descriptors. b AUC value of PCM modeling constructed by RF classifier under different cutoffs of bio-active data, this results were obtained by descriptor T1. *Precision score means the area under the precision-recall curve</p><!><p>Further, the contribution of chemical descriptor was also analyzed. After statistic analysis, it can be found that lipo-hydro partition coefficient (MolLogP in RDKit) contains the major contribution among all ligand descriptors, which means it might be the key element for molecular with potential inhibition abilities (Additional file 2: Fig. S1). It can also found that, for both active compound and inactive compound, the distribution of MolLogP follows Normal distribution with significant difference, which were calculate through T test (P value < 0.0001). Result showed that, lipo-hydro partition coefficient is important for the activity of NR inhibitor, active compounds normally contain MolLogP around 5.775, while the MolLogP of inactive compounds were around 5.380. Importance and P value of top 10 chemical structure descriptors can be found in Additional file 3: Table S2.</p><!><p>In this study, PCM modeling was systemically evaluated through both internal and external validations. By setting different cutoffs of bio-active data, results of different PCM models can be found in Fig. 1b, detailed information of model performance on all four protein descriptors can be found in Additional file 4: Table S3. Generally, all PCM models can gives outstanding performance in internal validation by achieving an AUC value above 0.870 on different cutoffs. For external validation, all PCM model can also achieves a satisfied performance with AUC value over 0.746. Above results indicate the excellent ability of our PCM model for NR-related inhibitors prediction. Also, with the increasing of cutoffs, the performance of PCM models increased synchronously. This probably caused by the fact that the unbalance between positive and negative data according to different cutoff. For example, when set EC50 ≤ 1 as positive data and EC50 > 1 as negative data, the ratio (positive/negative) of training set, testing set and external validation set were all close to 1 (Additional file 5: Table S4). After the cutoff rising to 10, those ratios were quickly increased to 12.14, 12.95 and 22.76 respectively (Additional file 5: Table S4). Several reports also pointed out that the 1 μM cutoff may be more reasonable because it contains less noise [40]. In that case, the cutoff of EC50 value was set as 1 for further analysis.</p><!><p>Scaffold clustering of NR-inhibitors, colors in back ground and in spot represents the experimental confirmed active compounds and model predicted active compounds respectively. Red means active compounds while green means inactive compounds, white color means scaffold contains both active and inactive compounds. a Scaffold clustering of NR1C1-inhibitors. b Examples of compounds contains scaffold S10. c Scaffold clustering of NR1C2-inhibitors. d Scaffold clustering of NR1C3-inhibitors. e Scaffold clustering of NR1H2-inhibitors. f Scaffold clustering of NR2B1-inhibitors</p><!><p>Generally, the prediction of our PCM model matched perfectly well with the experimental values. For three peroxisome proliferator-activated receptor (PPAR) protein targets, the top 10 clusters of each target including NR1C1 (Fig. 2a), NR1C2 (Fig. 2c) and NR1C3 (Fig. 2d) were detected and marked in each sub-graphs. For PPAR protein targets, both unique and overlapped scaffolds can be detected. For example, target NR1C1 contains 7 bioactive scaffolds (marked as S1 to S7), 2 inactive scaffolds (marked as S8 and S9) and 1 mixed scaffold (marked as S10) contains both active and inactive compounds. Among above, scaffold S1 and S6 were active in both NR1C1 and NR1C3 (Fig. 2d), while S8 and S9 were both inactive scaffold. On the other hand, different pattern can be found in target NR1C2 (Fig. 2c). In NR1C2, 7 new scaffold clusters marked as S11 to S18 were detected. Besides that, as a major inactive scaffold for NR1C1 and NR1C3, S8 was determined as active scaffold in NR1C2. Also, as an active scaffold in NR1C1 and mixed scaffold in NR1C3, scaffold S2 was defined as inactive scaffold for NR1C2. The results of two targets beside PPAR targets were quite different, totally new scaffolds were discovered and illustrated in Fig. 2e, f. All above illustrated that, even from the same protein family, the inhibitor scaffolds of different NR protein targets were still distinguishable.</p><p>Also, it should be noticed that, the bioactivity of different compounds rely on multiple factors such as side-chain composition, functional group, substituent and chirality. For instance, scaffold S10 N-benzylbenzamide contains different compounds including compound 1–3 (Fig. 2b). The molecular structure of three compounds is extremely similar except for the chirality. The stereogenic center of compound 1 (Benzenepropanoic acid, α-ethyl-4-methoxy-3-[[[[4-(trifluoromethyl)phenyl]methyl]amino]carbonyl]-, (αS)-) and compound 2 (Benzenepropanoic acid, α-ethyl-4-methoxy-3-[[[[4-(trifluoromethyl)phenyl]methyl]amino]carbonyl]-, (αR)-) are absolutely configured as S and R, respectively. Compound 3 was defined as mixture of stereoisomers which may combine with both S and R chirality.</p><!><p>Computer-aided drug design (CADD) can assist and shorten the process of new drug discovery. To achieve that, one essential issue is to per-estimate the activity of different compound against different target proteins. By introducing PCM model into CADD, relationship between multiple compounds and targets can be determined. Based on high-throughput screening of compounds, bioactive molecules can be clustered and essential molecular scaffolds can be detected to guide the future development of therapeutic drugs.</p><p>In order to process high-throughput screening of bioactive inhibitors for targets from NR families, 7267 bio-active data of 11 nuclear receptors were collected to establish an in silico model. Through both internal and external validation, our PCM models were proved to be sensitive for NR-inhibitor prediction which might be benefit from our descriptors. For target descriptors, generalized sequence similarity descriptors contain information from 30 background targets from NR families. Models based on those descriptors can achieve a better prediction performance on both internal and external validation set, which means those descriptors can be extended to multiple targets from NR families. For chemical descriptors, since lipo-hydro partition coefficient contains the major contribution for classification and parameter MolLogP is distinguishable for active and inactive compounds, this may provide a clue for future therapeutic NR-inhibitors discoveries.</p><p>Another essential issue for PCM model construction is to choose the suitable machine learning method. In this study, five different machine learning methods including both regression and classification approaches were tested to establish PCM modeling. Results showed that the performance of RF and DT classifier are significantly higher than other methods, which means above algorithms might be more applicable in the case of NR-inhibitors prediction.</p><p>After high-throughput screening of NR-inhibitors, bioactive molecules could be clustered according to structure similarity and molecular scaffold enriched in each clustered can be detected and might assist the process of drug design. In this article, the appropriate models selected after evaluations were used to molecular clustering for five major NR targets. Results showed that our PCM model can successfully predict those potential NR-inhibitors which agree well with the experimental EC50 values. For each NR target, our algorithms can able to predict those potential therapeutic inhibitors and discover the molecular scaffolds for future drug development. Currently, this method was established on NR proteins and it can be extended to other protein targets after the accumulating of experimental data.</p><!><p>Clustering tree of nuclear receptors. 7 different subtypes of NR were marked in different colors and 11 NR proteins used in this study were marked in red as well as its data distribution</p><!><p>Here, both sequence similarity descriptors and structure similarity descriptors were used to characterize those five nuclear receptors. Firstly, a 30 protein targets from NR families can be derived from Protein Data Bank (PDB) [42] as background. For 11 protein targets in our dataset, the sequence and structure similarity compared with those 30 background protein target structures can be calculated by pairwise alignment respectively. Sequence alignment was calculated by smith-waterman alignment [43], while structure alignment was calculated by using jFATCAT [44]. Therefore, two types of generalized target descriptor including sequence similarity descriptor (T1) and structure similarity descriptor (T2) can be obtained for each protein targets. For comparison, specific descriptors based on 5 protein target from our training set instead of 30 background protein target were also established, recorded as T3 (specific sequence similarity descriptor based on 5 protein target) and T4 (specific structure similarity descriptor based on 5 protein target). Two generalized target descriptors can be found in Additional file 10: Table S8-1, 2 and two specific target descriptors were also listed in Additional file 11: Table S9-1, 2.</p><!><p>Chemical structure descriptors were calculated by using RDKit (release version 2016). RDkit provides different chemical structure descriptors, which contains both chemical and physical properties such as Molecular Weight, Hydrogen Bond Donor Count, Hydrogen Bond Acceptor Count, Rotatable Bond Count and LogP etc. In addition, RDKit contains massive types of chemical descriptors derived from other tools and literatures, such as MOE-type descriptors for partial charges, MR contributions, LogP contributions, EState indices and surface area contributions integrated from molecular operating environment (MOE). In general, 187 descriptors were used to characterize the structure features of inhibitor (Additional file 12: Table S10).</p><!><p>In this study, 4 Proteochemometric models were created from training set based on different combinations of descriptors (T1-L, T2-L, T3-L, T4-L). All models were implemented in scikit-learn (Version 0.18.1) by using Random Forest (RF) with default parameters. For classification, different thresholds of EC50 were selected to distinguish positive and negative data. Here, three different thresholds (EC50 < 1 μm, EC50 < 5 μm and EC50 < 10 μm) were used for classification respectively.</p><!><p>For each combination of descriptors, 10-fold cross-validation was carried out for the model. The performance of four models was assessed by classification accuracy. Further, both internal and external validation data were tested from different aspects to evaluate the overall performance of our models, including the area under the ROC curve (AUC) value, accuracy, precision, recall and F-score, statistical parameters were defined in the following equations:1\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ ext{Accuracy}} = rac{TP + TN}{TP + FP + TN + FN}$$\end{document}Accuracy=TP+TNTP+FP+TN+FN2\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ ext{Precision}} = rac{ ext{TP}}{{{ ext{TP}} + { ext{FP}}}}$$\end{document}Precision=TPTP+FP3\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ ext{Recall}} = rac{TP}{TP + FN}$$\end{document}Recall=TPTP+FN4\documentclass[12pt]{minimal} sepackage{amsmath} sepackage{wasysym} sepackage{amsfonts} sepackage{amssymb} sepackage{amsbsy} sepackage{mathrsfs} sepackage{upgreek} \setlength{\oddsidemargin}{-69pt} egin{document}$${ ext{F-score}} = 2 \cdot rac{precision \cdot recall}{precision + recall}$$\end{document}F-score=2·precision·recallprecision+recallPositive samples are those with EC50 value below threshold. TP represents True positive, TN represents True negative, FP represents false positive and FN represent false negative.</p><!><p>For each protein target, the similarity of corresponding molecules were analyzed based on Rubberbanding Forcefield approach in DataWarrior [41] (release version 4.5.2). Initially, all molecules were translated into a series of descriptors to encode various aspects of chemical structures including both 2-D and 3-D structure information. After that, calculate the entire similarity matrix between all molecules and locate most similar neighbors to be considered for every molecules. Then, stepwise relocate all molecules to ensure similar molecules were located close to each other. Finally, molecules with structure similarity over 0.95 will be clustered together [41]. For each cluster, the major Bemis-Murcko scaffold [45] (covering over 80% of the molecules in this cluster) was defined as the representative scaffold. Note that for several clusters, no major scaffold can be detected, in that case, the maximum common substructures for each two scaffolds can be calculated through RDKit and the major substructure was defined as the representative scaffold. After that, the Bemis-Murcko scaffold for each cluster can be derived and analyzed.</p><!><p>Additional file 1: Table S1. 10-fold cross-validation results of different machine learning methods on four descriptors.</p><p>Additional file 2: Fig. S1. Distributions of MolLogP in both active compound and inactive compound.</p><p>Additional file 3: Table S2. Importance and P value of top 10 chemical structure descriptors.</p><p>Additional file 4: Table S3. Model performance of random forest classifier on four protein descriptors.</p><p>Additional file 5: Table S4. Data distribution of training set, testing set and external validation set.</p><p>Additional file 6: Table S5. Chemical name and smiles file of selected scaffold.</p><p>Additional file 7. Supplementary Data 1: information of 9633 compounds.</p><p>Additional file 8: Table S6. Data distribution of different NR targets.</p><p>Additional file 9: Table S7. Information of crystal structure used for descriptor generation.</p><p>Additional file 10: Table S8-1. Sequence similarity descriptors based on 30 NR proteins (T1). Table S8-2. Structure similarity descriptors based on 30 NR proteins (T2).</p><p>Additional file 11: Table S9-1. Sequence similarity descriptors based on 5 NR proteins (T3). Table S9-2. Structure similarity descriptors based on 5 NR proteins (T4).</p><p>Additional file 12: Table S10. Information of inhibitor descriptors.</p><p>Electronic supplementary material</p><p>The online version of this article (10.1186/s13321-018-0275-x) contains supplementary material, which is available to authorized users.</p><p>Tianyi Qiu and Dingfeng Wu contributed equally to this work</p>

PubMed Open Access

Induced effects of advanced oxidation processes

Hazardous organic wastes from industrial, military, and commercial activities represent one of the greatest challenges to human beings. Advanced oxidation processes (AOPs) are alternatives to the degradation of those organic wastes. However, the knowledge about the exact mechanisms of AOPs is still incomplete. Here we report a phenomenon in the AOPs: induced effects, which is a common property of combustion reaction. Through analysis EDTA oxidation processes by Fenton and UV-Fenton system, the results indicate that, just like combustion, AOPs are typical induction reactions. One most compelling example is that pre-feeding easily oxidizable organic matter can promote the oxidation of refractory organic compound when it was treated by AOPs. Connecting AOPs to combustion, it is possible to achieve some helpful enlightenment from combustion to analyze, predict and understand AOPs. In addition, we assume that maybe other oxidation reactions also have induced effects, such as corrosion, aging and passivation. Muchmore research is necessary to reveal the possibilities of induced effects in those fields. Results and discussionFenton's reagent (mixture of hydrogen peroxide and ferrous iron) is one of the most effective AOPs, which was developed in the 1890s by Henry John Horstman Fenton as an analytical reagent 13 . Fenton's reaction is based on the catalyzed decomposition of H 2 O 2 by ferrous iron to produce reactive ?OH 14 . It has been found effective in treating various industrial wastewater components including a wide variety of landfill leachate 15 , pesticides 16 and surfactants 17 , as well as many other substances. Oxidations of ethylenediaminetetraacetic acid (EDTA) by

induced_effects_of_advanced_oxidation_processes

H

<p>azardous organic wastes from industrial, military, and commercial activities represent one of the greatest challenges to human beings 1,2 . Advanced oxidation processes (AOPs) are alternatives to the degradation of those organic wastes [3][4][5] . In 1987, Glaze et al. 3 defined AOPs as ''near ambient temperature and pressure water treatment processes which involve the generation of hydroxyl radicals in sufficient quantity to effect water purification''. The hydroxyl radical (?OH) is a powerful, highly reactive chemical oxidant, which reacts very quickly with organic compounds. Recently, other studies have suggested that, besides ?OH, AOPs can also generate other oxidizing species, such as sulfate radicals 6,7 . It is generally believed that, depending upon the nature of the organic species, they are oxidized by radicals mainly through hydrogen abstraction or addition itself to double bonds of the contaminant 8 .</p><p>AOPs are important tools for environmental technology, so they have to be placed on more sound scientific and engineering basis. However, the knowledge about the exact mechanisms of AOPs is still incomplete 9,10 . The most difficult problem is how to chooce or design the most efficient AOPs system for the given pollutant. So, the reaction mechanisms, efficiency improvement, and their mathematical modeling will be the key subjects of the future research. Some investigators called AOPs as ''cold combustion'' 11,12 . Just because, similar with combustion, AOPs is a kind of oxidation process which could oxidize and mineralize the organic matter under mild conditions. But we tried to discover more similarities between them and achieve some enlightenment which is helpful to analyze, predict, understand and improve the oxidation efficiency of AOPs. Here we report a similar phenomenon exist in the AOPs: induced effects, which is a general property of combustion reaction. In ancient times, human ancestors drilled wood to make fire. As a Chinese saying goes, ''a single spark can start a prairie fire''. Both of them mean that combustion can be induced by a small fire. Our experimental results indicated that AOPs may also have induced effects. The following experimental findings directly or indirectly reflect the induced effects of AOPs from different angles.</p><p>Fenton's reagent were carried out in our experiments, and the results are shown in Fig. 1. The results indicated that the reaction rate is very slow in the early stage of the reaction, only about 22% COD of EDTA have been oxidized in the first 25 min. Meanwhile, the solution temperature also raised very slowly, from 32uC raised to 42uC in the first 25 min. However, after this early stage, a sudden violent oxidation reaction has occurred, and the temperature raised very quickly, reached 80uC in the 10 minutes. About 45% of EDTA oxidation have achieved in this 10 minutes. According to usually employed hydroxyl radical theory, it implies that a large number of ?OH produced at this stage. However, it is difficult to understand why a long waiting time is needed before producing those large number of ?OH.</p><p>We believe that this is a induction reaction which just like the burning process of a pile of wood from hardly ignition to strong burning, and finally burned out.</p><p>The oxidation efficiency of Fenton's reaction could be strongly accelerated by adding UV radiation 18,19 . Although 254 nm UV radiation could penetrate only a very short distance into the mixed solution of H 2 O 2 and EDTA (Fig. 2). The result shows that addition of UV radiation could cut the waiting time of occurring violent oxidation reaction from 25 to 15 minutes (Fig. 1a). This phenomenon can also be explained by induced effects. Firstly, UV radiation could quickly initiate the oxidation reaction in the range where UV radiation could penetrate. Then, already happened reactions induce the whole reaction. In fact, researchers have developed a variety of methods to induce advanced oxidation reaction in recent decades, such as optical 20 , electrical 21 , ultrasonic 22 and microwave 23 .</p><p>Inspired by the induced effects, we tried to feed easily oxidizable organic matter to promote the oxidation of refractory organic compound in the solution. The experiment results agree well with our notions. In the EDTA oxidation experiments using Fenton process, the method of pre-feeding potassium oxalate in the solution, similar with UV, could also cut the waiting time of occurring violent oxidation reaction. The reason for choosing the potassium oxalate as an example is that it will not add COD of the solution, and then not affect the calculation of COD removal efficiency. According to the result, the addition of 11 mM potassium oxalate could cut the waiting time from 25 to 20 minutes when 50 mM EDTA was treated by Fenton process (Fig. 1). This means that the pre-oxidation of potassium oxalate could induce the oxidation of EDTA. It will be easily understood if we connect it with a very common practice: oil usually used to quickly induce a log of wood combustion. Using easily biodegraded organic matter, such as sucrose and starch 24,25 , to increase the biodegradation is a common method in the biological process. But this strategy is rarely used in the physicochemical method. To be sure, using easily oxidizable organic matter to promote the efficiency of AOPs has important practical value for the degredation of refractory organic compounds.</p><p>tert-Butanol, which is a strong radical scavenger, was adopted as the indicator for the hydroxyl radical type reaction. As shown in Fig. 1, the addition of tert-butanol markedly reduced the AOPs efficiency, indicating that the ?OH was the main active species in the process. The results suggested that these procedures (UV, K 2 C 2 O 4 ) actually enhanced the hydroxyl radical production 26,27 .</p><p>In addition, it is well known that oxygen will not be consumed if there is no fuel in a combustion reaction. Similarly, when study the reaction of degradation organic compounds by UV/H 2 O 2 process, we discovered that H 2 O 2 will consumed very slowly if there is no organic compounds in the solution (Fig. 3). This is another phenomenon to reflect the similar of AOPs with combustion. Based on this experiment result, even though the exact mechanism involved are not known, it is understandable if we assume that organic compounds play an important role for ?OH production along with oxidant, catalyst, UV or other possible factors in the AOPs.</p><p>From the above experimental results and discussions, we hypothesize that AOPs may have induced effects, which is similar to combustion. Although the mechanism is unclear, the findings from those studies raise new thinking in the relationship of AOPs and combustion, especially about induced effects. For example, combustion reaction is induced by the heat, but what induced the reaction of AOPs? We hope others will take up the challenge to begin addressing this important issue. It not only helps us to understand the mechanism of AOPs in more depth but also help us to find new methods to improve its efficiency from the angle of induced effects. In addition, it will raise a bold ideas: maybe all oxidation reactions have induced effects, such as corrosion, aging and passivation. Muchmore research is necessary to reveal the possibilities of induced effects in those fields.</p>

Scientific Reports - Nature

Flavylium Salts: A Blooming Core for Bioinspired Ionic Liquid Crystals

AbstractThermotropic ionic liquid crystals based on the flavylium scaffold have been synthesized and studied for their structure‐properties relationship for the first time. The mesogens were probed by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X‐ray diffraction (XRD). Low numbers of alkoxy side chains resulted in smectic (SmA) and lamello‐columnar (LamCol) phases, whereas higher substituted flavylium salts showed Colro as well as ordered and disordered columnar (Colho, Colhd) mesophases. Mesophase width ranged from 13 K to 220 K, giving access to room temperature liquid crystals. The optical properties of the synthesized compounds were probed towards absorption and emission properties. Strong absorption with maxima between 444 and 507 nm was observed, and some chromophores were highly emissive with quantum yields up to 99 %. Ultimately, mesogenic and dye properties were examined by temperature‐dependent emissive experiments in the solid state.

flavylium_salts:_a_blooming_core_for_bioinspired_ionic_liquid_crystals

<!>Introduction<!><!>Introduction<!>Synthesis<!><!>Synthesis<!><!>Solid‐state properties<!><!>Liquid crystalline properties<!><!>Liquid crystalline properties<!>Flavylium salts with lamellar mesophases<!><!>Flavylium salts with lamellar mesophases<!><!>Flavylium salts with lamellar mesophases<!><!>Flavylium salts with lamellar mesophases<!><!>Flavylium salts with lamellar mesophases<!>Flavylium salts with columnar rectangular phases<!><!>Flavylium salts with columnar rectangular phases<!><!>Flavylium salts with columnar rectangular phases<!>Flavylium salts with rectangular and hexagonal phases<!><!>Flavylium salts with rectangular and hexagonal phases<!><!>Flavylium salts with rectangular and hexagonal phases<!>Flavylium salts with hexagonal phases<!><!>Flavylium salts with hexagonal phases<!>Flavylium salts with 3′‐substitution pattern<!>Dye properties<!><!>Dye properties<!><!>Dye properties<!><!>Conclusions<!>Conflict of interest<!>

<p>R. Forschner, J. Knelles, K. Bader, C. Müller, W. Frey, A. Köhn, Y. Molard, F. Giesselmann, S. Laschat, Chem. Eur. J. 2019, 25, 12966.</p><!><p>Ionic liquid crystals (ILCs) are an emerging class of soft matter materials which combine the best of two worlds, that is, the fluidity and adjustable polarity of ionic liquids with the anisotropic properties of liquid crystals.1, 2, 3, 4, 5, 6 The vast majority of ILCs consist of nitrogen‐containing cations, such as ammonium, pyridinium, imidazolium or guanidinium salts and analogues thereof, whereas cations with other heteroatoms (O, S, P, …) are less commonly employed.</p><p>Although oxonium ions usually only occur as short‐lived intermediates in reactions, their high reactive character can be tamed by embedding the positively charged oxygen in an aromatic system. Such pyrylium derivatives, for example, 2,4,6‐triphenylpyrylium salts, show strong fluorescence and anion–π interactions.7, 8 They have been successfully utilized for electron‐transfer reactions,9 (photo)organocatalysis,10, 11, 12 white‐light fluorophores13 and laser dyes.14</p><p>Liquid crystalline oxonium salts reported in the literature (Scheme 1) are solely based on di‐ or triphenyl pyrylium cations 1,15 1,4‐disubstituted benzenes functionalized with two pyrylium units 2 16 and condensed xanthylium derivative BNAX 3.17 We found it quite surprising that ILCs based on the probably most prominent organic oxonium salt, the flavylium cation A‐Fla‐B never has been reported to the best of our knowledge. Those salts are an important scaffold in natural and synthetic dyes. For example, anthocyanins, that is, hydroxylated and O‐glycosylated flavylium salts provide the largest family of water‐soluble plant dyes, which protect plants against photooxidation, serve as food colorants18 and attractant for insects.19 Key features of these natural dyes are their pH‐dependent colored species ranging from red to blue, their ability for complexation of metals and formation of different types of aggregates.20, 21 Synthetic flavylium derivatives have been successfully utilized for organic–inorganic hybrid pigments,22 or photosensitizer for dye‐sensitized solar cells.23, 24, 25, 26</p><!><p>Examples of liquid crystalline oxonium salts reported in the literature and the basic structure of the flavylium salts A‐Fla‐B.</p><!><p>Because of their nearly planar structure and the strong tendency for aggregation into dimers and higher aggregates,27 they are particularly attractive candidates for ILCs. Additionally, flavylium salts possess unique structural features, that is, the ionic moiety is located in the center of the mesogenic core rather than as peripheral headgroup and the molecular shape is unsymmetrical. We anticipated that the substitution pattern of the rigid A and flexible B ring should enable tailoring of both liquid crystalline self‐assembly as well as linear optical properties of flavylium salts. Here, we report the first flavylium ILCs showing a rich polymorphism and promising absorption and emission characteristics. The results are discussed below.</p><!><p>For the syntheses of the desired flavylium salts a protocol by Chassaing28 was applied were the A ring of the flavylium salt is derived from a phenol and the B and C rings are generated from an ethynyl ketone. As shown in Scheme 2, a series of hydroxy substituted arylaldehydes 5 a–e was converted into the corresponding alkoxy‐substituted arylaldehydes 6 a–e through Williamson etherification in 84–97 % yields, except for the 3,4,5‐trisdodecyloxyphenylcarbaldehyde 6 f, which was obtained in three steps from ethyl gallate 1 in 83 % overall yield.29 Aldehydes 6 a–f and benzaldehyde 6 g were then treated with ethynyl magnesium bromide to give the propargylic alcohols 8 a–g in almost quantitative yield after aqueous workup, followed by 2‐iodoxybenzoic acid (IBX) oxidation to the corresponding ethynyl ketones 9 a–g. Dakin oxidation with H2O2 and H2SO4 of aldehydes 6 a–f yielded in the phenol derivatives 7 a–f in 61–96 %.30</p><!><p>i) C12H25Br, K2CO3, DMF, 80 °C, 3 h; ii) 1. C12H25Br, K2CO3, NaI, CH3CN, 105 °C, 2 d; 2. LiAlH4, Et2O, RT, 1 h; 3. DDQ, 1,4‐dioxane, RT, 1 h; iii) H2O2, H2SO4, CHCl3, CH3OH, RT, 18 h; iv) ethynylmagnesium bromide, THF, RT, 3 h; v) IBX, EtOAc, 80 °C, 18 h.</p><!><p>Treatment of an equimolar solution of a phenol 7 a–f and an ethynyl ketone 9 a–g in EtOAc with an excess of trifluoromethanesulfonic acid resulted in the formation of the desired flavylium salt A‐Fla‐B (Scheme 3). The variation in the yields of the flavylium salt A‐Fla‐B was mostly due to the differences in solubility. Some derivatives, for example, V‐Fla‐1 and 2‐Fla‐2, show low solubility in EtOAc and precipitated directly from the reaction solution at room temperature. Others, like 3‐Fla‐1 and 3′‐Fal‐3′ display good solubility at room temperature and, therefore, were recrystallized at low temperatures. The crude products were purified by recrystallization from the reaction solution. With 7 a no formation of the product was observed, therefore the alkoxy substitution in meta‐position is crucial for the reaction. This is the reason why phenols 7 b and 7 c with an additional methoxy substituent were used to obtain the derivatives with one alkoxy sidechain attached to the A ring. The flavyliums salt 3‐Fla‐0 could not be isolated and 3′‐Fla‐0 could not be obtained in satisfactory purity. It should be emphasized that the solid flavylium salts were bench stable and solutions did not show any color change or loss of color upon storage over more than six months.</p><!><p>Molecular structures of the flavylium salts A‐Fla‐B prepared in this work.</p><!><p>Recrystallization of V‐Fla‐1 from EtOAc provided suitable crystals for single‐crystal analysis. The compound crystallizes with one ion pair in the asymmetric unit of the centrosymmetric space group P 1‾ . The flavylium cation is almost planar with a torsion angle of 5° between the chromenylium and the phenyl moiety. The oxygen atoms of the triflate anion works as acceptors for a couple of hydrogen bond interactions (Figure 1 a). Firstly, there are π(C−H) donors of the chromenylium and the phenyl moieties. The (H⋅⋅⋅O) interval of the relevant distances is 2.34– 2.39 Å. Secondly, a weaker interaction is evident with the methyl C−H function of the methoxy group. The (H⋅⋅⋅O) distance range is 2.54–2.70 Å. And finally, there is a weak interaction between a C−H donor of the alkyl chain and the O6 of the triflate anion with a (C29−H29⋅⋅⋅O6) distance of 2.48 Å. The cation built up a layer type stacking interaction with a pairwise 180° rotated orientation of the molecules forced by a slight π–π stacking interaction of the chromenylium cores (Figure 1 b). In detail, the pyrylium core interacts with the benzene part of the chromenylium and vice versa. The distance of the centroids is in both cases 3.66 Å. Additional each pair is also stabilized by a slight stronger π–π stacking generated only between the pyrylium cores with a distance of 3.56 Å. Remarkably, the phenyl groups of the flavylium moieties are not involved in π–π stacking interactions. This is most likely due to the strong ionic and dipolar interaction of the benzopyrylium moiety, surpassing the possible contribution of the π–π interaction from the phenyl group. This packing motive seems to be universal for flavylium cations and will be important for the following discussion of the liquid crystalline properties. In the bc view of the packing diagram there is a layer‐type orientation of the molecules along the c‐axis evident (Figure 1 c). The central part of the cation and the triflate anions form a polar layer which alternates with the nonpolar layer consisting of the aliphatic interdigitated chains.</p><!><p>Single‐crystal X‐ray structure representations of V‐Fla‐1 in the solid state31 (H=light blue, C=white, O=red, S=yellow, F=green, in b) and c) hydrogens are omitted for clarity). a) Hydrogen‐bond interactions of the triflate anion given by dashed lines. b) Stacking interaction of the flavylium cation. c) bc view along the a axis showing the interdigitated layer structure.</p><!><p>The thermotropic behavior of the synthesized flavylium salts A‐Fla‐B were examined by polarizing optical microscopy (POM), differential scanning calorimetry (DSC) as well as wide‐ and small‐angle X‐ray scattering (WAXS and SAXS). The results of the DSC experiments are summarized in Table S1 (Supporting Information) and an overview of the observed phases is presented in Figure 2.</p><!><p>Overview of the observed mesophase of the flavylium salts A‐Fla‐B (a detailed version of this diagram with the mesophase width can be found in Figure S1, Supporting Information).</p><!><p>Some compounds show decomposition of the material in the DSC. However, the TGA measurements of the series iV‐Fla‐B showed that the compounds are stable to 200 °C, except for iV‐Fla‐3, which decomposes at about 150 °C (Figure S7, Supporting Information). Flavylium salts with no side chain on the B ring, that is, V‐Fla‐0, iV‐Fla‐0, 2‐Fla‐0 and 3′‐Fla‐0 were non‐mesomorphic irrespective of the number and position of the side chain on the A ring and showed only Cr–Cr transitions. In addition, V‐Fla‐2 with one side chain at the A ring and two side chains at the B ring as well as V‐Fla‐3′ and iV‐Fla‐3′ with sterically crowded B ring were non‐mesomorphic. For clarity, the following discussion is organized according to the type of mesophase (lamellar, columnar rectangular and/or hexagonal) formed by the flavylium salt.</p><!><p>Flavylium salts with one or two side chains on the A ring and one side chain on the B ring resulted in lamellar mesophases. For example, vanillin‐derived flavylium salt V‐Fla‐1 with one dodecyloxy chain on both A and B ring displayed two liquid crystalline phases, that is, a melting transition at 129 °C followed by a mesomorphic transition at 201 °C and a clearing point at 214 °C upon first heating in the DSC (Figure S2 b, Supporting Information). Under the POM the lower‐temperature phase showed uncharacteristic textures, whereas Maltese cross textures and a strong tendency for homeotropic alignment were observed upon heating into the high‐temperature mesophase. Upon cooling from the isotropic liquid, the compound displayed bâtonnets textures (Figure 3 a), indicating a SmA mesophase. Upon further cooling into the low temperature phase fan shaped textures were visible (Figure 3 b) which can be observed in smectic and columnar mesophases, but also has been reported for lamello‐columnar LamCol mesophases.32</p><!><p>POM micrographs of a) V‐Fla‐1 at 205 °C (magnification 200×) and b) at 188 °C (magnification 100×). All pictures were taken between crossed polarizers upon cooling from the isotropic phase with a cooling rate of 5 K min−1.</p><!><p>X‐ray diffraction of V‐Fla‐1 showed upon cooling from the isotropic phase the typical diffraction pattern of an oriented SmA mesophase consisting of a sharp layer reflex (001) and the higher order reflex (002) in the small angle region (Figure S9 a, Supporting Information). The wide‐angle region displayed two broad halos at 4.66 and 3.64 Å resulting from the molten alkyl chains and presumably short aggregates of the mesogens, respectively (Figure 4 a). Both reflexes are oriented perpendicular to the layer reflexes. The experimental layer spacing d=29.7 Å from the SAXS measurement is significantly smaller than the molecular length obtained from single crystal structure analysis (L=41.7 Å). Usually, the layer spacing of a SmA phase is 5–10 % smaller than the molecular length, due to the axial disorder of the mesogens defined by the order parameter.33 In the case of V‐Fla‐1, the observed d/L ratio of 0.71 is unusual and cannot solely be explained by a low order parameter. However, the difference between flavylium ILCs and conventional mesogens with smectic phases is that their charge is located in the center of the molecule. The close proximity to the counter ion leads to an expansion of the effective cross section of the core. The resulting free space between the alkyl chains is then filled by interdigitation of the neighboring hydrophobic layers, forming an SmA1 phase. This packing model is further supported by analogy with the crystal structure of V‐Fla‐1, which reveals interdigitation of alkyl chains and a lattice parameter of c=26 Å similar to the experimentally determined layer spacing.</p><!><p>Diffractogram and the diffraction pattern of the oriented sample of V‐Fla‐1 in the a) SmA phase at 210 °C and b) LamCol phase at 170 °C after cooling from the isotropic state (cooling rate: 5 K min−1). c) 2D SAXS pattern of the LamCol mesophase and the χ‐scan of the diffraction peaks obtained by slow cooling (0.2 K min−1) from the SmA phase into the LamCol phase. d) Proposed packing of the molecules in the LamCol mesophase. e) Temperature dependent layer spacing of the (001) reflex (▪), the (002) reflex (•) and the (010) reflex (▴) in the SmA (hollow symbols) and the LamCol phase (filled symbols) of compound V‐Fla‐1. The measurement was performed via the second heating (rate: 2 K min−1).</p><!><p>Cooling of the sample into the lower‐temperature phase at 170 °C, resulted in a more pronounced wide‐angle reflex at 3.54 Å as a result of the growing intracolumnar stacks, which were already weakly present in the SmA phase (Figure 4 b). Additionally, a sharp reflex with a layer spacing of 10.04 Å is observed. The perpendicular orientation towards the layer reflex, as well as the similarity of the length to the b‐axis (10.27 Å) of the single crystal structure, led to the assumption that this reflex originates from the lateral distance of the short columnar stacks. The scan over χ of a slowly cooled sample revealed that this reflex is split into two reflexes with ±10° with respect to the center (Figure 4 c).</p><p>Therefore, we assume that the lower‐temperature phase is a lamello‐columnar phase LamCol, in which the layers are build up by short stacks of flavylium cations (Figure 4 d). Within these stacks the molecules are organized in an antiparallel manner as observed in the crystal structure, in order to enable π–π interactions and reduce charge repulsion. The splitting of the lateral intercolumnar distance can be explained by an alternating tilt of these mesogenic stacks with ±10° in respect to the layer normal from one layer to another, comparable to the anticlinic SmC phase. Due to the low number of observed reflexes and the sliding of the layers, further differentiation regarding the symmetry was not reliable. Further evidence for this hypothesis can be given by calculating the volume of the elemental cell using the length of the 001 and the 010 reflex, as well as the intracolumnar distance. With the volume in hand the number of molecules per elemental cell Z can be calculated according to Lehmann,29 which in the case of the lower‐temperature phase of V‐Fla‐1 resulted in Z=1.07 assuming a density of 1 g mL−1.</p><p>The occurrence of the LamCol phase can be rationalized by the strong tendency for the formation of vertical aggregates of the flavylium cation and a weak layer coupling.34 At high temperatures the stacking is unfavorable, therefore the calamitic shaped molecules form a SmA phase as expected. However, the presence of the wide‐angle reflex corresponding to the π–π reflex indicates, that aggregation already occurs in the SmA phase. Upon decrease of the temperature the molecular stacks are growing until the phase transition into the LamCol phase occurs.</p><p>Further information of the mesophase was obtained by the temperature‐dependent SAXS measurements (Figure 4 e). In the LamCol phase, the layer spacing decreased only very slightly with increasing temperature. At the LamCol–SmA phase transition the layer spacing dropped significantly by approximately 2 Å and upon further increase of temperature a negative thermal expansion of the (001) reflex was observed, which is typical for the SmA phase.</p><p>The DSC of the isovanillin‐derived flavylium salt iV‐Fla‐1 with one dodecyloxy side chain on both A and B ring showed only one mesophase between 47 and 131 °C (Figure S3 a, Supporting Information). Under the POM an uncharacteristic grainy texture was observed. The XRD pattern showed an intense (001) reflex and the higher order reflexes (002) and (004) (Figure S10 a,b, Supporting Information). In addition, one further reflex at 10.4 Å was observed as well as a diffuse halo centered around 4.84 Å and a π–π reflex at 3.63 Å. For this compound no oriented sample could be obtained, but due to the similarity of the diffraction pattern compared to V‐Fla‐1 we surmised that this phase is also a LamCol phase. However, it should be noted that the layer spacing of iV‐Fla‐1 shows a stronger dependency on the temperature than V‐Fla‐1 in the LamCol phase (Figure S10 c, Supporting Information).</p><p>Flavylium salt 2‐Fla‐1 with two dodecyloxy chains at the A ring and one chain at the B ring showed three transitions upon heating in the DSC. An endothermal melting transition at 46 and 100 °C followed by a first order transition at 126 °C with a small transition (less than 0.7 kJ mol−1) and a clearing transition at 145 °C was detected (Table S1, Figure S4 a, Supporting Information). Under the POM 2‐Fla‐1 showed filament‐like textures in the highest mesophase upon heating (Figure 5 a). Cooling from the isotropic liquid resulted in a pronounced homeotropic alignment, interrupted by occasionally occurring Maltesian crosses. Upon entering the lower‐temperature mesophase fan‐shaped textures were observed (Figure 5 b). In a polyimide coated cell, already the higher‐temperature phase showed fan‐shaped textures, characteristic for the SmA phase (Figure 5 c). The lower‐temperature phase showed broken fan‐shaped textures characteristic for the SmC phase (Figure 5 d). Additionally, the birefringence increases drastically. However, attempts to determine optical tilt angles in both polyamide and nylon test cells failed despite good alignment, because the lower‐temperature phase appears to be uniaxial, disproving a SmC phase. Cooling into the lowest temperature phase resulted in grainy and darkened textures.</p><!><p>Polarized optical micrographs of a) 2‐Fla‐1 at 135 °C (magnification 100x) upon heating, b) 2‐Fla‐1 at 115 °C (magnification 100x) upon cooling from the isotropic liquid, c) fan‐shaped textures at 135 °C and at d) 110 °C. 2‐Fla‐1 in a rubbed polyimide cell (homogeneous alignment, cell gap: 3 μm) upon cooling. 2‐Fla‐1 in a single side rubbed nylon cell, cell gap: 1.6 μm) at e) 135 °C and f) 110 °C.</p><!><p>To gain further insight into the observed phases, 2‐Fla‐1 was examined by temperature‐dependent XRD measurements. In the small‐angle region at 130 °C, a sharp reflex indexed as (001) of the SmA phase is observed (Figure 6 a, Figure S11, Supporting Information). The layer spacing of 31.1 Å is smaller than the molecular length due to interdigitation of the alkoxy side chains, as also seen in the SmA phase of V‐Fla‐1. The wide‐angle region displayed a diffuse halo at 4.75 Å oriented perpendicular to the layer reflex (Figure 6 b). Therefore, this phase has been identified as a partially interdigitated SmA phase.</p><!><p>a) SAXS and b) WAXS patterns of 2‐Fla‐1 at 130 °C, 120 °C and 90 °C (from top to bottom). b) Temperature dependent layer spacing of the 001 and 002 reflex on cooling from the isotropic state. Transition temperatures are given by the dashed lines. c) Temperature‐dependent layer spacing of the (001) reflex (▪) and the (002) reflex (•) in the LamCol phase (filled symbols), the SmA′ phase (gray symbols) and the SmA phase (hollow symbols).</p><!><p>The mesophase at 120 °C showed besides the (001) reflex the higher ordered reflex (002). The χ scan of the wide‐angle region of the oriented sample showed that the reflexes oriented perpendicular to the layer reflexes and no significant difference towards the SmA phase could be observed. However, the reflex corresponding the intramolecular distance is more prominent in this phase indicating a longer correlation length of the lateral intermolecular distance. The temperature dependent SAXS measurement revealed, that the layer spacing decreased continuously with increasing temperature from 102 to 145 °C (Figure 6 c). Considering the results from the POM and the X‐ray analysis, we assume that this phase is a SmA′ phase, with the difference that the layers are built up by flavylium dimers.</p><p>The lowest temperature mesophase at 90 °C showed the layer reflexes (001), (002), (003) and (004) along the meridian, as well as the intercolumnar reflex (010) oriented perpendicular to the layer reflexes. Thus, the overall appearance of the XRD pattern showed similarities to the LamCol phase of V‐Fla‐1. The layer spacing of 34.0 Å is larger as compared to V‐Fla‐1, showing that interdigitation is less pronounced in 2‐Fla‐1, due to the additional side chain. The intercolumnar distance of the columns is 10.6 Å and no splitting of this reflex could be detected. The WAXS showed a diffuse halo at 4.60 Å and the intracolumnar reflex at 3.50 Å along the equator.</p><!><p>In contrast to the non‐mesomorphic vanillin‐derived flavylium salt V‐Fla‐2 (melting point: 195 °C), the corresponding isovanillin‐derived flavylium salt iV‐Fla‐2 and iV‐Fla‐3 showed Colro mesophases between 52 and 199 °C and between 50 and 156 °C in the DSC, respectively (Figure S3 c, Supporting Information). Under the POM both compounds displayed spherulite‐like textures and line defects, characteristic for columnar mesophases (Figure 7).</p><!><p>Polarized optical micrographs of a) iV‐Fla‐2 at 196 °C (magnification 200×) and b) iV‐Fla‐3 at 120 °C (magnification 200×). All pictures were taken by cooling from the isotropic phase with a cooling rate of 5 K min−1.</p><!><p>The diffraction pattern of an oriented fiber of iV‐Fla‐2 consisted of sharp reflexes in the small‐angle region (Figure S12 a, Supporting Information). The reflexes could be assigned as (11), (02), (12), (22), (23) and (31) of a columnar rectangular ordered mesophase Colro with p2gg symmetry. The lattice parameters of the elemental cell are a=35.9 Å, b=50.2 Å and a Z value of 4, indicating that one disk is formed by two molecules (it must be noted that a p2mg symmetry, where one disc consists of one molecule, cannot be completely ruled out). The wide‐angle region of a fiber sample showed a diffuse halo of the molten alkyl chains centered around 4.71 Å. This reflex is split into two reflexes with an azimuthal angle of 39° with respect to the meridian (Figure 8 a). Furthermore, a relatively sharp reflex at 3.52 Å as well as an additional diffuse reflex at 3.44 Å was observed. The diffraction pattern of iV‐Fla‐3 (Figure S12 b, Supporting Information) showed fewer and less intense higher‐order reflexes compared to iV‐Fla‐2, but the same phase geometry of p2gg with slightly larger lattice parameters (a=36.3 Å, b=51.0 Å, Z=4) was observed. The wide‐angle region showed also three reflexes: a diffuse halo at 4.63 Å with a tilt of 39°, a relative sharp reflex at 4.06 Å with no tilt as well as an additional diffuse reflex at 3.39 Å with a tilt of 30° with respect to the meridian (Figure 8 b). Furthermore, a reflex is observed at 7.46 Å.</p><!><p>X‐ray diffractograms of a fiber sample of a) iV‐Fla‐2 at 110 °C and b) iV‐Fla‐3 at 120 °C with the corresponding 2D diffraction pattern with the azimuthal angles of the diffuse wide‐angle reflexes. The fitting of the wide‐angle area is given in grey with the sum (bold line) of the distinct Gauss peaks (dashed lines). White arrows indicate the direction of the fiber. c) Proposed packing model of the Colro phase and the stacking within the column (the representation of the discoid has been simplified, for a more detailed discussion see Figure 10 and the corresponding text).</p><!><p>The occurrence of a relative sharp reflex in the wide‐angle region in discotic phases is usually referred to the periodic arrangement of the aromatic cores, but for the flavylium salts with a Colro mesophase the situation seems to be different, considering that the intracolumnar distance of iV‐Fla‐3 would be too large for typical π–π stacking. To rationalize the wide angle reflexes I–III, and their different azimuthal angles in Figure 8, the mesogens can be divided into three parts: the alkoxy side chains I, the triflate anion II and the aromatic core III. From the diffraction pattern of the iV‐Fla‐2 fiber sample the tilt of the mesogens with respect to the column axis is mainly governed by the alkyl chains. The observed tilt angle of 39° can be obtained directly from the azimuthal angle. Presumably the almost spherical anions form the linear, non‐tilted backbone of the columns as depicted in Figure 8 c. The small difference in the distances of the anions (3.52 Å) and cations (3.44 Å) can be explained by the tilt between the aromatic cores of 12° calculated by using the formula α=cos−1 (dIII/ dII). This angle is quite small and, therefore, could not be determined directly form the diffraction pattern due to overlapping of both reflexes.</p><p>For iV‐Fla‐3, the tilt of the alkyl chains is similar to iV‐Fla‐2 but the tilt between the aromatic cores is much higher. Firstly, this becomes noticeable by the splitting of the corresponding reflex III with an azimuthal angle of 30° in the diffraction pattern and, secondly, by the increased anion–anion distance of 4.06 Å as a result of the aromatic tilt whereas the distance between the aromatic cores remains almost identical. By using the above mentioned formula, the calculated tilt of the flavylium cation is 33° and, therefore, in good agreement with the experimental value and supports the assumed packing of the molecules within the column. Further evidence can be found by comparing the width of the peaks in the wide‐angle area. The aromatic reflex in the less tilted iV‐Fla‐2 is sharper compared to the more tilted iV‐Fla‐3. A smaller tilt results in a longer correlation length and therefore in a sharper reflex. The results provide a useful tool to estimate the tilt of the columns in the Colr phases in ILCs for which no planar aligned sample can be obtained. Such detailed information about the intracolumnar stacking can be important, for example, for ionic conductivity.35 An comprehensive packing model for the columnar phases of the flavylium salts will be discussed below by using 2‐Fla‐3 as an example.</p><!><p>The vanillin‐derived flavylium salt V‐Fla‐3 as well as 2‐Fla‐2 and 2‐Fla‐3 showed a lower temperature Colro mesophase and a higher temperature Colh mesophase. Flavylium salt V‐Fla‐3 showed upon heating in the DSC a melting transition at 53 °C, a mesomorphic transition at 100 °C and clearing into the isotropic phase at 190 °C (Figure S2 d, Supporting Information). Under the POM uncharacteristic grainy textures were observed for the low‐temperature phase upon heating, whereas the high‐temperature phase showed line defects. Cooling from the isotropic phase into the high‐temperature mesophase resulted in dendritic growth (Figure 9 a). The hexagonal shape of the liquid crystalline germs indicates a columnar hexagonal mesophase in agreement with Bouligand.36 Upon cooling into the low‐temperature phase, the texture became grainy especially in previously homeotropic aligned areas (Figure 9 b).</p><!><p>Polarized optical micrographs of V‐Fla‐3 at a) 125 °C (inset: dendritic germ observed below the clearing point, picture taken with slightly uncrossed polarizers) and b) 75 °C between crossed polarizers. Textures of 2‐Fla‐3 at c) 185 °C and d) 75 °C. All pictures were taken by cooling from the isotropic phase with a cooling rate of 5 K min−1 and a magnification of 200×.</p><!><p>XRD experiments of V‐Fla‐3 (Figure S13) in the higher‐temperature mesophase at 140 °C revealed the reflexes (10), (11), (20) and (21) of the Colh mesophase (p6mm symmetry) with a lattice parameter of a=25.8 Å (Z=1). The WAXS region showed a diffuse halo at 4.51 Å and a reflex at 3.39 Å originating from the intracolumnar order. As compared to the Colr phase, the distances between the anions and those between the aromatic cores are identical and therefore are represented by a single reflex at 3.39 Å. Furthermore, a reflex at 6.71 Å was observed, which is twice the layer spacing of the intracolumnar distance. For the further discussion this reflex will be noted as π–π′.</p><p>In the lower temperature phase of V‐Fla‐3 at 75 °C the SAXS region showed the reflexes (01), (10), (12), (20), (03), (13), (22), (04), (30), (24) and (15) which could be assigned to a Colr phase with p2mm symmetry and lattice parameters of a=57.7 Å and b=41.9 Å with a Z value of 4, indicating that one discoid is formed by 4 flavylium salts. Presumably, the decrease of the alkoxy chain length at 7 position to a methoxy group in V‐Fla‐3 resulted in an different volume requirement as compared to iV‐Fla‐2, iV‐Fla‐3 and 2‐Fla‐3 carrying a dodecyloxy chain at this position. The WAXS region showed a similar behavior as discussed above for iV‐Fla‐3. The diffuse halo centered around 4.52 Å showed a splitting into two reflexes with an angle of 51° with respect to the meridian corresponding to the average distance and tilt of the alkyl chains. The sharp reflex at 3.96 Å showed no tilt, as it originates from the non‐tilted anions as seen above. The reflexes corresponding to the distances of the aromatic cores at 6.74 and 3.64 Å showed both a tilt of 23° respectively and are in agreement with the calculated tilt α=cos−1 (3.64 Å/3.96 Å)=23°.</p><p>The DSC of flavylium salt 2‐Fla‐2 with two dodecyloxy side chains at the A and B ring showed a transition at 119 and 131 °C into the mesophase as well as a mesomorphic transition at 165 °C with low enthalpy (−0.8 kJ mol−1) followed by clearing into the isotropic liquid phase at 180 °C (Figure S4 b, Supporting Information). Under the POM the compound displayed spherulite‐like textures and dendritic growth typical for columnar phases. The small angle diffractogram of compound 2‐Fla‐2 at 170 °C showed a single (10) reflex of a hexagonal mesophase with a lattice parameter of a=35.5 Å (Z=2, Figure S14, Supporting Information). The WAXS pattern showed a diffuse halo at 4.68 Å and an additional intracolumnar reflex at 3.49 Å. In contrast to the hexagonal phases of V‐Fla‐3, 2‐Fla‐3, 3‐Fla‐2 and 3‐Fla‐3 the π–π′ reflex could not be observed. As seen for the previous flavylium compounds iV‐Fla‐2 and iV‐Fla‐3, the lower temperature Colr phase shows p2gg symmetry with a lattice parameter of a=61.0 Å and b=34.5 Å, preserving the pseudohexagonal lattice of the higher temperature hexagonal phase as indicated by the ratio a/b=31/2.</p><p>For the compound 2‐Fla‐3 upon heating a glass transition at 59 °C, a mesophase‐to‐mesophase transition at 102 °C and a clearing into the isotropic liquid at 200 °C was observed. In analogy to V‐Fla‐3, the higher‐temperature mesophase showed typical columnar textures, which became grainy upon entrance into the lower‐temperature mesophase (Figure 9 c,d).</p><p>From the XRD result for 2‐Fla‐3 a slightly larger lattice parameter of a=27.4 Å in the higher‐temperature Colho phase and a halo at 4.52 Å as well as the intracolumnar reflexes at 3.44 Å and 6.75 Å were observed (Figure S15 a,b, Supporting Information). The small angle peaks of the lower‐temperature mesophase could be perfectly indexed as hexagonal phase with a=35.4 Å (Z=2), but since the wide‐angle region indicates a tilt similar to V‐Fla‐3, we assume a Colr phase.</p><p>Proper indexation could be achieved for the hk sets: 20/02 and 11/31 both with p2gg symmetry and a lattice parameter of a=61.3 Å and b=35.3 Å, as well as the rather unlikely case of 10/01 with p2mm symmetry and a lattice parameter of a=30.1 Å and b=17.7 Å. The wide‐angle region displayed a halo with a layer spacing of 4.52 Å and a tilt angle of 51°, resulting from the alkyl chains. The layer spacing of the anions is with a value of 3.96 Å larger than the π–π distance of the aromatic cores (3.55 Å). The tilt of these cores was determined to be of 23° by fitting of the χ scan (calculated: 25°).</p><p>To construct general packing models for columnar phases, usually nanophase segregation and π–π interactions are decisive. However, in the case of flavyliums salt the additional repulsive charge interaction located in the center of the aromatic core plays the major role. Therefore, most likely flavylium salts are stacked into columns in an antiparallel arrangement (Figure 10 a) similar to the crystal structure of V‐Fla‐1 (Figure 1). Further evidence for this model can be found in the observed π–π′ reflex, which corresponds to either the distance between the aromatic core with the same direction or the distance between the anions. This model can be applied to all compounds with a Colho phase with one molecule per disc (e.g., V‐Fla‐3, 2‐Fla‐3, 3‐Fla‐2, 3‐Fla‐3).</p><!><p>Proposed packing model of the flavylium salts in the a) Colho and the b) Colro mesophase viewed from the top (left) and in an side view (right).The flavylium cations are displayed as blue arrows (pointing towards the oxonium cation) and the triflate anions as red dots.</p><!><p>In the Colro phases, the XRD results revealed that two flavylium salts form one discoid. We assume that in addition to the intracolumnar interaction in the lower temperature Colro also intercolumnar anion–H bonds occur. In other words, the Colro phase can be considered as two antiparallel packed columns bond together, as depicted in Figure 10 b. Within one discoid the flavylium salts can be arranged either in a face‐to‐face manner or pointing into same direction. In both cases, the flexible B ring has to rotate out of plane to avoid steric repulsion. We assume that additional intercolumnar anion–H interactions stabilize the lower‐temperature Colro phase, whereas the higher‐temperature Colho phase features only intramolecular interactions. 2‐Fla‐2 can be as an intermediate case which forms a Colho but contains two molecules per disc.</p><!><p>Flavylium salt 3‐Fla‐2 possessing three dodecyloxy chains at the A ring and two at the B ring showed a single broad mesophase between −6 °C and 215 °C in the DSC (Figure S5 c, Supporting Information) and characteristic columnar textures under the POM. The XRD (Figure S16 a,b, Supporting Information) experiments revealed a Colho mesophase with a slightly larger a value of 28.6 Å (Z=1) as compared to the analogue 2‐Fla‐3 with an inverted substitution pattern. The wide‐angle region displayed the diffuse halo, as well as the π–π and the π–π′ reflex. Compound 3‐Fla‐3 showed also a Colho phase, ranging from 56 and 209 °C with similar textures and XRD result (for details see Table S2 and Figure S16 c,d, Supporting Information). No Colro phase was observed for the substitution pattern 3‐Fla‐B, presumably due to steric overcrowding in the above mentioned packing models shown in Figure 10. Additionally, the overall number of alkoxy side chains can be sufficient to stabilize the Colho phase over a wide temperature range.</p><p>In contrast to the normal thermotropic behavior of 3‐Fla‐2 and 3‐Fla‐3, the flavylium salt 3‐Fla‐1 lacked a stable mesophase. Upon heating from the solid phase, clearing into the isotropic phase was detected at 54 °C by DSC and under the POM (Figure S5 a, Supporting Information). Upon further heating no other peak could be observed. The cooling curve showed a broad endothermic peak at 20 °C and a sharp peak at 11 °C. These two transitions were enantiotropic and are also observed in the second and third heating. Surprisingly, under the POM (Figure S17, Supporting Information) the formation of columnar textures could be observed upon heating the substance to 78 °C. These textures were only observable in thin sample areas between glass or in a polyimide‐coated test cell of 4.6 μm thickness, whereas thick parts of the sample remain isotropic. At 143 °C these textures cleared into a second isotropic state. Upon cooling again typical columnar textures was observed clearing into a fluidic phase at about 78 °C, revealing the enantiotropic nature of this phase. This shows that under bulk conditions the compound 3‐Fla‐1 has no stable mesophase, but under planar anchoring liquid crystalline properties can be observed. Reentrant phases have already been reported for compounds in which two SmA phases were separated by a nematic phase, but the sequence Ire‐Col‐I is quite rare37, 38, 39, 40 and, to the best of our knowledge, an Ire phase has never been reported for thermotropic ionic liquid crystals.</p><p>XRD experiments showed a (10) and (11) reflex in the small angle region and a diffuse halo in the WAXS measurement (Figure 11 a). Considering that no π–π reflex is observed, the intracolumnar order is quite low in contrast to the previously described columnar mesophase. Fortunately, a partially oriented SAXS pattern of 3‐Fla‐1 could be obtained by slowly heating the sample into the columnar phase from the isotropic re‐entrant phase. The diffraction pattern consisted of a diffuse part and sharp reflexes orientated in a hexagonal manner (Figure 11 b).</p><!><p>a) WAXS diffractogram of 3‐Fla‐1 at 85 °C. The inset displays the SAXS pattern of a planar aligned domain obtained by slow heating into the Colh phase at 80 °C (the additional diffuse reflex is most likely due passing of the beam through another domain without mesophase). b) Proposed stacking of two flavylium cations V‐Fla‐3 in the liquid crystalline state.</p><!><p>We assume that the unusual phase sequence can be explained by a complex relationship of intermolecular forces, surface interactions, and space filling. The high‐temperature isotropic phase probably consists of monomeric flavylium salts, whereas the surface is covered by molecules due to polar anchoring. Cooling the sample causes more and more molecules to assemble on the surface aligned molecules by intramolecular interactions, leading to the formation of columns which ultimately results in the observed Colh phase. With decreasing temperature, the attractive π–π and ionic interactions increase. At some point these interactions become so strong that stable dimers of 3‐Fla‐1 are formed, which no longer display any liquid crystalline behavior, resulting in the phase transition into the lower‐temperature Ire phase.</p><p>This behavior can be explained by comparing 3‐Fla‐1 to V‐Fla‐3, with the inverted substitution pattern. In V‐Fla‐3, the higher number of side chains is attached to the flexible B ring. Torsion of this phenyl moiety causes the side chains in m‐position to rotate out of the aromatic plane and extend into the upper and lower mesogen (Figure 11 b). This allows the mesogen to effectively fill the void left by the mono‐substituted side. Additionally, the rotation of the crowded B ring disfavors the formation of a dimeric species by steric repulsion. In contrast, the higher substituted side in 3‐Fla‐1 is rigid, therefore space filling is only possible by strong coiling of the alkyl chains, leading to a rather spherical appearance, disfavoring liquid crystalline properties.</p><!><p>Considering that several of the above discussed flavylium salts showed quite high clearing points, we wanted to push the clearing temperature towards lower temperatures. A lot of research has been done with respect to reduce the clearing point by using thioether side chains,41 branched42 or swallow‐tailed43 side chains rather than linear alkoxy side chains. In contrast, our aim was to reduce the clearing point by varying the substitution pattern at the flavylium A and B rings. Therefore the 3′‐substitution pattern, derived from 2,3,4‐dodecylalcoxybenzaldehyde 5 e at the A and/or B ring was tested.</p><p>The clearing points of the 3′‐mesogens, that is, A‐Fla‐3′ and 3′‐Fla‐B could indeed be reduced compared to their A‐Fla‐3 and 3‐Fla‐B counterparts. This effect is more pronounced for derivatives with the 3′‐substituent attached on flexible B ring. For example, 3‐Fla‐3 shows a clearing point at 210 °C and strong decomposition in the DSC, 3′‐Fla‐3 enters the isotropic phase at 169 °C and 3‐Fla‐3′ already at 93 °C. 3′‐Fla‐3 showed only minor differences compared to 3‐Fla‐3′.</p><p>All compounds of this series A‐Fla‐3′ and 3′‐Fla‐B, except the non‐mesomorphic V‐Fla‐3′ and iV‐Fla‐3′ derivatives exhibited a columnar mesophase as indicated by the typical columnar textures observed under the POM (Figure S18, Supporting Information). In contrast to the A‐Fla‐3 and 3‐Fla‐B flavylium salts, the columnar phases shows almost no intracolumnar order. Similar lattice parameters were found for compound 2‐Fla‐3′ (a=27.2 Å), 3‐Fla‐3′ (a=27.7 Å) and 3′‐Fla‐3′ (a=29.4 Å) and the calculated Z‐value of 1 indicating one molecule per discoid (Figure S19, Supporting Information). The intercolumnar reflex of 3′‐Fla‐3′ is broad, almost comparable to the isotropic liquid. This behavior can be explained by either a very small correlation length of the intercolumnar reflex, or that the mesophase is only stable under planar anchoring conditions, similar to 3‐Fla‐1. This would also explain the missing clearing point in the DSCs. In contrast, the derivatives 3′‐Fla‐1 and 3′‐Fla‐2 showed larger a values with 29.7 Å and 31.4 Å respectively and a Z value of 2 indicating two molecules per disk. 3′‐Fla‐3 again shows a Z‐value of 1 and a lattice parameter of 27.7 Å. The packing model shown in Figure 10 cannot easily be transferred to these compounds, because the columnar phases are disordered and the alkoxy substituent in position 8 causes a steric hindrance.</p><!><p>To obtain insight into the optical properties of the flavylium salts A‐Fla‐B were examined by UV/Vis absorption and emission spectroscopy. Chloroform was chosen as the solvent, because all compounds showed good solubility in halogenated solvents. The results are summarized in Table S3 (Supporting Information). All compounds showed absorption maxima between 444 and 507 nm. The spectra of the vanillin‐derived series V‐Fla‐B are shown in Figure 12. The substitution pattern on the A ring had only a minor influence (<4 nm) on the absorption maximum, whereas some variations of the extinction coefficients were observed. However, the number and position of substituents at the B ring resulted in a red‐shift of the absorption maximum, which increased in the series V‐Fla‐0<V‐Fla‐1<V‐Fla‐3′<V‐Fla‐3<V‐Fla‐2.</p><!><p>a) Absorption spectra (bold line, represented by its extinction coefficient determined by linear regression of a concentration series ranging from 0.2×10−5  m to 7×10−5  m) and normalized emission spectra (dashed line) of vanillin‐derived flavylium salts V‐Fla‐B in CHCl3. Solutions of the corresponding flavylium salt under b) daylight and c) UV‐radiation (366 nm).</p><!><p>Fluorescence spectra were also measured in CHCl3 by exciting the molecules in correspondence of their absorption maxima. The flavylium salts showed emission maxima between 498–595 nm. The Stokes shift increased in the series V‐Fla‐1 (1467 cm−1)<V‐Fla‐2 (2416 cm−1)<V‐Fla‐0 (2721 cm−1) and V‐Fla‐3 (3298 cm−1). Interestingly, the V‐Fla‐3′ derivative showed a comparable small Stokes shift (1467 cm−1) as observed for V‐Fla‐1 but the fluorescence intensity was low. The number of alkoxy substituents on the flavylium salt had a strong impact on the absolute fluorescence quantum yields. Although V‐Fla‐0 with no substituent on the B ring was only weakly emissive (Φ F=4 %), the corresponding analogue V‐Fla‐1 with one substituent on the B ring displayed a very strong green emission with a quantum yield of 97 %. Similar high values have already been reported by Haucke for 7,4′‐dimethoxy substituted flavylium salts.44 Upon further increasing the number of side chains in the B ring, the quantum yield decreased considerably (V‐Fla‐2: 5 %). Flavylium salts with three alkoxy side chains were only weakly emissive (V‐Fla‐3:<1 %, V‐Fla‐3′:<1 %). With increasing the number of alkoxy side chains at the A ring the quantum yields also decreased. Although vanillin‐derived flavylium salt V‐Fla‐1 and isovanillin‐derived flavylium salt iV‐Fla‐1 showed almost quantitative fluorescence (97 and 99 %, respectively), already 2‐Fla‐1 showed a reduced value of 92 %. With three alkoxy side chains in 3‐Fla‐1 the quantum yields drastically dropped below 1 %, as seen for 3‐Fla‐1 and 3′‐Fla‐1. Possible fluorescence quenching resulting from H aggregates can be clearly ruled out due to the low concentration and the fact that no blueshift of absorption and emission within the series V‐Fla‐1, iV‐Fla‐1, 2‐Fla‐1, 3‐Fla‐1 and 3′‐Fla‐1 was observed.</p><p>The fluorescence quantum yields seem to strongly depend on the number of alkoxy chains at the B ring. The high emission of V‐Fla‐1, iV‐Fla‐1 and 2‐Fla‐1, which bear only one alkoxy chain at the B ring as compared to the only weakly fluorescent flavylium salts with either no substituent on the B ring (i.e. V‐Fla‐0, iV‐Fla‐0, 2‐Fla‐0) or two alkoxy chains on the B ring (i.e. V‐Fla‐2, iV‐Fla‐2, 2‐Fla‐2), might be rationalized by considering the canonical Lewis structures. As exemplified for flavylium salts V‐Fla‐0, V‐Fla‐1 and V‐Fla‐2 possible Lewis structures are shown in Figure S24 (Supporting Information). According to a previous TD‐DFT study by Woodford45 in the parent flavylium salt V‐Fla‐0 (M0) carrying an unsubstituted phenyl B ring two Lewis structures V‐Fla‐0 (M1) and V‐Fla‐0 (M2) with the positive charge located at C‐2 or C‐4 are favored over Lewis structure V‐Fla‐0 (M0) with the positive charge at O‐1. Thus, the phenyl ring possesses a high degree of rotational freedom resulting in decreased fluorescence quantum yields. In contrast, the presence of a para‐alkoxy group attached to the B ring in V‐Fla‐1 stabilizes the positive charge at C‐2 (or C‐4) through conjugation, resulting in Lewis structure V‐Fla‐1 (M3). This Lewis structure seems to be preferred due to the extended π system. Thus, conjugation of the B ring leads to planarization and rigidification resulting in strong fluorescence emission. Further evidence for the rigidification can be found in the asymmetric peak shape of V‐Fla‐1, as a result of a similar geometric of the ground and excited state.</p><p>When a second alkoxy group is present in the B ring as in V‐Fla‐2, the contribution of the Lewis structure V‐Fla‐2 (M3) to the overall electronic structure is diminished as compared to V‐Fla‐1 (M3) because the electron‐donating +M effect of the para‐alkoxy group is partially counterbalanced by the electron‐withdrawing −I effect of the meta‐alkoxy group. This increases the single bond character of the C2‐C1′ bond, resulting in an increased rotational mobility and thus decreased fluorescence quantum yield. This effect becomes even more pronounced in V‐Fla‐3, which shows a fluorescence quantum yield below 1 %. These hypotheses were further supported by preliminary DFT calculations (for details see Supporting Information) with simplified flavylium salts (alkoxy groups were replaced by methoxy), which indicated an elongation of the C2−C1′ bond by 6 pm for V‐Fla‐2 upon transition from the ground state to the excited state, whereas the C2−C1′ bond lengths remained almost constant for V‐Fla‐0 and V‐Fla‐1. Furthermore, the calculated oscillator strengths of V‐Fla‐1 was twice as large as compared to V‐Fla‐0, V‐Fla‐2. A previous computational study by Quina has shown that calculations on hydroxylated flavylium salts are challenging.46 Therefore, our results should be treated with care. However, the above proposed model serves as a useful rationale for the experimental results.</p><p>The lifetime of the excited state in solution was examined by TRSPC (time resolved single photon counting). The monosubstitution on the B ring, that is, V‐Fla‐1, iV‐Fla‐1, and 2‐Fla‐1, resulted in monoexponential emission decays with lifetimes around 3.3 ns (Table S3, Figure S20, Supporting Information). For the unsymmetrical substitution with two alkoxy side chains on the B ring, the emission decay profiles could be fitted with two components with shorter lifetimes, revealing that two emitting species are involved in the fluorescence process. A potential reason for the two decay times might be the formation of hemiketals and chalcones, which is known for flavylium salts.20 The two decay times may also be caused by the presence of rotamers with intact π system.</p><p>In the solid state only compounds V‐Fla‐1, iV‐Fla‐1, and 2‐Fla‐1 showed enough fluorescence intensity to obtain reliable data. The emission spectrum of V‐Fla‐1 was broad and ranged from about 550–850 nm at room temperature with the main contribution at 645 nm (Figure 13, Figure S21, Supporting Information). Additionally, the spectrum of V‐Fla‐1 was the only spectrum which showed a contribution at about 770 nm, close to the NIR regime. The compound iV‐Fla‐1 and 2‐Fla‐1 showed similar emission spectra but with different contributions of the single bands. In the solid state, the emission spectra were red shifted compared to the emission in solution (λ max=484 nm). Temperature‐dependent measurements were performed upon cooling the sample from the isotropic phase. With decreasing temperature, the emission intensity increased, and a red shift was detected. For the compound V‐Fla‐1 the emission intensity is lowest in the isotropic state and remains low in the SmA phase. Upon entering the LamCol the intensity increased with a slow slope which further increased in the solid state. For iV‐Fla‐1 a similar behavior has been observed, with the difference that the intensity increased abruptly upon entering the LamCol mesophase, in which the luminescence intensity remained almost constant over the whole phase. In the solid state, the intensity increased as expected. Compound 2‐Fla‐1 showed low intensity in the isotropic state and in the smectic phases. Upon entering the LamCol phase, the intensity increased in the same manner as iV‐Fla‐1 and iV‐Fla‐1 in the crystalline state. Hence, emission intensity is strongly dependent on the emitter supramolecular organization. Although the liquid phase and mesophases with pronounced fluidity, that is, the SmA and SmA′ phase, show only weak luminescence, the higher ordered phases, that is, LamCol and solid phase, show higher fluorescence.</p><!><p>a) Emission spectra of V‐Fla‐1 (black), iV‐Fla‐1 (red) and 2‐Fla‐1 (green) in the solid state at 50 °C under irradiation with UV light (350–380 nm, solution spectra of V‐Fla‐1 is given in dashed lines for comparison) and b) temperature‐dependent emission intensity at 665 nm upon cooling the sample from the isotropic liquid (cooling rate of 10 K min−1). For the orientation transition temperatures of the DSC are given as arrows.</p><!><p>When optical micrographs of V‐Fla‐1 were examined under UV light, different behaviors were observed depending on the temperature. In the isotropic phase, the intensity of the emitted light appears evenly distributed over the sample area (Figure 14), whereas, already in the SmA phase, this intensity was inhomogeneous. Upon decreasing the temperature, the contrast between bright and dark areas increased. Considering that fluorescence is anisotropic, the emission intensity depends on the orientation of the chromophore.47 For compounds iV‐Fla‐1 and 2‐Fla‐1, a similar behavior was observed (Figures S22 and 23, Supporting Information).</p><!><p>Optical micrographs of V‐Fla‐1 observed between crossed polarizers (left column) and under UV radiation (right column, without polarizer, exposure time: 8 seconds, the brightness of b) and d) has been increased by 40 % to ensure visibility) in the a–b) isotropic phase at 230 °C, c‐d) the SmA phase at 200 °C, e–f) the LamCol phase at 170 °C and g–h) the crystalline phase at 30 °C. The images were obtained by cooling of the isotropic liquid with a cooling rate of 10 K min−1.</p><!><p>Mesogens based on nitrogen cations are dominating the world of ionic liquid crystals and are therefore well explored. In this work, we have shown that the flavylium backbone provides a new functional mesogenic core for ILCs with interesting mesomorphic properties and outstanding emissive behavior.</p><p>The flavylium salts have been synthesized according to a modular principle by the condensation of a phenol derivative and an ethynyl ketone building block. Depending on the substitution pattern, various types of mesophases formed. The calamitic shaped molecules V‐Fla‐1, iV‐Fla‐1, and 2‐Fla‐1 formed lamellar phases (SmA, SmA′, LamCol), the higher substituted flavylium salts displayed discotic mesophases (Colho, Colhd, and Colro). The mesophase widths varied within ranges from 13 K (SmA phase of V‐Fla‐1) up to 220 K for the Colho phase of 3‐Fla‐2, which also displays liquid crystallinity at room temperature. We found that the observed mesophases are governed by the strong π–π and ionic interactions resulting in an antiparallel stacking of the flavylium cation within the columns. A general feature of these flavylium salts is the strong tendency for alignment, allowing the preparation of fiber samples, granting detailed insight into the mesophase by X‐ray diffraction. Special attention was given to the wide‐angle region, providing detailed information of the intracolumnar stacking, which is of interest for future applications of ILCs. In the rectangular phases, the triflate anion provided the linear backbone of the column, whereas the tilt of the aromatic core ranges from 12° iV‐Fla‐2 to 33° in iV‐Fla‐3. A comprehensive packing model for the discotic phase have been proposed, to enable the design of novel flavylium salts with tailored mesomorphic properties. Furthermore, we were able to report the first Ire phase in ionic liquid crystals under polar anchoring conditions for 3‐Fla‐1.</p><p>The flavylium salts show strong absorption in the visible area and the derivatives A‐Fla‐1 (A=V, iV, 2) show strong emission with almost quantitative absolute quantum yields as a unique feature of this substitution pattern. According to preliminary calculations for V‐Fla‐1, both the ground and excited state seem to remain rather rigid, explaining the spectral shape and the enhanced fluorescence. All other flavylium salts are either non‐rigid in the ground state (like V‐Fla‐0) or become nonrigid in the excited state (like V‐Fla‐2), which likely leads to additional non‐radiative pathways. These compounds also show weak emissive behavior in the solid state. The loss of luminescence intensity with increasing temperature could be reduced in the LamCol phase of iV‐Fla‐1 making it an interesting functional liquid crystal. Further work should demonstrate, how the mesophase behavior can be tailored by variation of the anion.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Solid Phase Synthesis and Spectroscopic Characterization of the Active and Inactive Forms of Bacteriophage S21 Pinholin Protein.

The mechanism for the lysis pathway of double-stranded DNA bacteriophages involves a small hole-forming class of membrane proteins, the holins. This study focuses on a poorly characterized class of holins, the pinholin, of which the S21 protein of phage \xcf\x8621 is the prototype. Here we report the first in vitro synthesis of the wildtype form of the S21 pinholin, S2168, and negative-dominant mutant form, S21IRS, both prepared using solid phase peptide synthesis and studied using biophysical techniques. Both forms of the pinholin were labeled with a nitroxide spin label and successfully incorporated into both bicelles and multilamellar vesicles which are membrane mimetic systems. Circular dichroism revealed the two forms were both >80% alpha helical, in agreement with the predictions based on the literature. The molar ellipticity ratio [\xce\xb8]222/ [\xce\xb8]208 for both forms of the pinholin was 1.4, suggesting a coiled-coil tertiary structure in the bilayer consistent with the proposed oligomerization step in models for the mechanism of hole formation. 31P solid-state NMR spectroscopic data on pinholin indicate a strong interaction of both forms of the pinholin with the membrane headgroups. The 31P NMR data has an axially symmetric line shape which is consistent with lamellar phase proteoliposomes lipid mimetics.

solid_phase_synthesis_and_spectroscopic_characterization_of_the_active_and_inactive_forms_of_bacteri

Introduction:<!>Solid Phase Peptide Synthesis<!>Protecting Group and Solid Phase Cleavage<!>Protein Purification and Spin Labeling<!>Peptide Incorporation into Lipid Mimetic Systems<!>Circular Dichroism<!>Continuous Wave Electron Paramagnetic Resonance Spectroscopy<!>31P Solid State Nuclear Magnetic Resonance Spectroscopy<!>Results and Discussion:<!>Optimization of the Solid Phase Peptide Synthesis<!>Optimization of Peptide Cleavage Conditions and HPLC Purification<!>Optimization of MALDI-TOF Sample Conditions and Matrix<!>Circular Dichroism of Active S2168 and Inactive S21IRS Pinholin<!>Continuous Wave EPR Measurements of Pinholin<!>31P Solid State \xe2\x80\x93 NMR Spectroscopy of Pinholin<!>Conclusion:

<p>The final step of the double-stranded DNA bacteriophage infection cycle is host lysis.[1-3] The mechanism for this lysis pathway involves three proteins, a small hole-forming inner membrane protein known as the holin, a muralytic enzyme known as the endolysin, and the spanin complex responsible for outer membrane disruption.[3, 4] The function of the holin protein is to permeabilize the inner phospholipid bilayer allowing the release of the endolysin to begin the degradation of the peptidoglycan.[5] This is accomplished by a harmless accumulation of the holin in the host cell membrane until the protein "triggers" at an allele-specific time. Triggering is the term used to denote when the holin reaches a critical concentration in the membrane and attains the functionality to permeabilize the membrane.[3] Due to the variation in mechanisms and sizes of lesions formed between different classes of holins the lesions have been termed "holes" to show distinction from channels and other such membrane permeabilization pathways.[2, 3]</p><p>Initially it was believed that all holins, like the λ S105 canonical holin, trigger to form micron-scale holes in the inner cell membrane.[3] These holes allowed for non-selective escape of fully folded and functional endolysin enzymes. However, more recently a second type of holin has been discovered.[2, 6] Instead of forming micron-scale non-selective holes in the cytoplasmic membrane, these holins form nanometer-scale holes that are only responsible for the depolarization of the membrane. [2, 6] Due to the small size of the holes, this new class of holin was named the pinholin. Unlike the large hole forming canonical holins the hole created by the pinholins is not large enough to allow for the nonspecific escape of functional endolysin from the cytoplasm. Instead these pinholins are paired with signal-anchor-release (SAR) endolysins.[2, 3] These proteins are named for a special N-terminal transmembrane helix that acts as an uncleaved signal sequence, resulting in a sec-mediated export. The SAR-endolysins accumulate in the periplasm as membrane-tethered, inactive enzymes. When the pinholins trigger and cause depolarization of the membrane, the SAR domain exits the bilayer, allowing the periplasmic catalytic domain to refold to its active form and begin degradation of the peptidoglycan.[6-8]</p><p>This study focuses on the optimization of the solid phase peptide synthesis and spectroscopic characterization of the pinholin membrane protein system. More specifically, the system under study is encoded by the S21 holin gene of the lambdoid bacteriophage φ21. The 71-codon S21 gene has a dual translational start motif.[9, 10] This results in the synthesis of two gene products, S2171 and S2168, resulting from translational initiations from codon Met1 and Met4, respectively. These two gene products are outlined in Figure 1. The S2168 is the functional or active form of the pinholin. This form of pinholin has two transmembrane domains (TMDs), the first of which (TMD1) externalizes from the membrane, while the second (TMD2) remains embedded in the bilayer and is essential for lytic functionality.[11-13] The second form, S2171, is known as the antiholin. The antiholin is responsible for the delayed triggering of the pinholin due to the addition of a positively charged lysine on the N-terminus (Figure 1) which drastically slows the externalization of TMD1. A basic schematic for the orientation of the pinholin in the membrane as well as hypothetical conformations of externalized TMD1 can be seen in Figure 2. The pinholin pathway begins with the accumulation of both forms of pinholin embedded in the bilayer, accumulating as dimers. Since the S2168/S2171 production ratio is ~2:1, the dimers are mostly S2168:S2168 homodimers and S2168:S2171 heterodimers.[14] Activation is thought to require externalization of TMD1 from the bilayer. Once both TMD1 segments are externalized, the pinholin dimer is licensed to continue in a pathway of oligomerization. When concentration of the activated pinholin dimers reach a critical concentration, the pinholin population triggers and causes massive and sudden depolarization of the inner cell membrane. [6, 15]</p><p>The structure, mechanism, and model pathway of the pinholin protein has been difficult to study primarily because pinholin is not only a hydrophobic membrane protein but also expresses lethal in function, thus prohibiting high-level biosynthesis. The pinholin system poses an interesting challenge as the length of the pinholin and antiholin proteins are near the limit of solid phase peptide synthesis. The resulting function of the pinholin pathway is well known, but the individual steps of the dimerization and oligomerization in the pathway are not well studied. The Young group has shown that the addition of the five amino acid sequence 'RYIRS' to the N-terminus of the S2168 pinholin, shown in Figure 1, prevents the externalization of TMD1 and ultimately the function of the pinholin pathway.[12] This is called the inactive IRS form of the pinholin. This presents an opportunity for recapitulating the holin pathway in vitro, using appropriate proportions of the wildtype pinholin (S2168) and the S21IRS, as a surrogate antipinholin to control the pathway. Utilizing this control, the structure and dynamics of the pinholin protein as it progresses through the lytic pathway can be spectroscopically studied with a variety of biophysical techniques.</p><p>This study represents the first time that solid phase peptide synthesis has been used to study any holin system in vitro. Circular dichroism has been used to determine alpha helical protein secondary structure as well as probe the initial oligomerization steps required in the proposed pinholin lytic pathway. The extant biophysical information on the oligomerization of pinholin was conducted on a truncated version of the protein where only TMD2 was present.[11, 14] However, this study uses the full length pinholin system. Electron paramagnetic resonance (EPR) spectroscopy was used to confirm pinholin incorporation into an in vitro multilamellar vesicle mimetic system. In conjunction with EPR spectroscopy, 31P solid-state NMR spectroscopy was used to discern the effect the different pinholin forms have on the membrane from the perspective of the phospholipids.</p><!><p>The solid phase peptide synthesis of pinholin peptides was conducted using a CEM Liberty Blue Peptide Synthesizer with Discovery Bio Microwave System. The synthesis used a NovaSyn TG amino resin, a composite of low cross-linked polystyrene with the PEG chains terminally functionalized with an amino group. All syntheses were run at a 0.1 mM scale with Dimethylformamide (DMF) as the base solvent. All Fmoc protected amino acid solutions were prepared at a 0.2 M concentration and coupled using a standard activator and activator base pair of DIC and oxyma, respectively. The coupling reactions were run at 90°C for 4 min while the Fmoc deprotection was run with 20% v/v piperidine in DMF at 93°C for 1 min.[16] As seen in Figure 1, the pinholin protein is naturally cys-less, requiring the introduction of only one site-specific cysteine into the primary sequence for site directed spin labeling EPR experiments.</p><!><p>The solid phase bound pinholin peptide was washed three times with dichloromethane and allowed to dry on a vacuum filter. The amino acid side chain protecting groups as well as the solid phase resin were cleaved from the peptide in a three-hour Trifluoroacetic acid (TFA) cleavage reaction.[17-19] The cleavage solution was then gravity filtered to remove the cleaved solid phase resin. TFA was evaporated from the reaction solution using inert nitrogen gas flow. Tert-butyl ether was added in excess to precipitate the pinholin peptide and separate from the still solubilized protecting groups.[16] The precipitated peptide was centrifuged down into a pellet by spinning for 15 min at 9000 rpm and the excess ether was decanted off. This procedure was repeated three times to ensure that all the protecting groups and scavengers were removed. Following the three ether washes the pinholin peptide was placed in a vacuum desiccator to dry for at least 8 hrs.</p><!><p>The crude pinholin peptide was purified by reverse phase high pressure liquid chromatography (RP-HPLC) using a C4 column running a two-solvent gradient. The first solvent was deionized water, the second was 90% HPLC grade acetonitrile. Both solvents were degassed and then acidified with 0.1% TFA by volume. The pinholin peptide was collected in fractions and the molecular weight of the peptide was confirmed using Matrix Assisted Laser Desorption Ionization – Time of Flight Spectrometry (MALDI-TOF). Collected fractions were dried using lyophilization to recover the pure pinholin.</p><p>The dry, pure peptide was then spin-labeled with S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate (MTSL), a nitroxide spin label at position Leu25. This position was chosen due to its location in TMD1, and therefore will test our ability to spin label the different conformations adopted by the active S2168 and inactive S21IRS pinholin. This was performed by dissolving the pinholin peptide and MTSL, at a 5x molar excess, in DMSO and letting it react while being stirred continuously for 24 hrs. The reaction was stopped by freezing the solution in liquid nitrogen and then dried using lyophilization.</p><p>The resulting crude spin-labeled pinholin was again purified using RP-HPLC and the same two-solvent system on a C4 semi-prep column to remove the excess MTSL. MTSL addition to the pinholin peptide was confirmed through MALDI-TOF mass, as shown in Figure 3. Collected pure peptide fractions were dried using lyophilization.</p><!><p>The pure peptide was incorporated into one of two different lipid mimetic environments, 1,2-Dimyristoyl - sn - Glycero - 3 - Phosphocholine (DMPC) / 1,2 - Diheptanoyl - sn - Glycero – 3 - Phosphocholine (DHPC) bicelles, or DMPC multilamellar vesicles (MLV). MLVs are a commonly used mimetic system and have been shown to be successful in mimicking a bilayer for membrane protein studies.[20, 21] MLVs were created by mixing the peptide dissolved in 2,2,2-Trifluoroethanol (TFE) with DMPC in chloroform at the desired protein concentration or protein to lipid ratio. Solvents were evaporated off using inert N2 gas and the remaining lipid/protein film was rehydrated using 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at a concentration of 20mM adjusted to a neutral pH of ~7.0. The protein/lipid solution was flash frozen in liquid nitrogen and then sonicated. This process was repeated 3 times to ensure MLV formation.</p><p>Bicelles were made by taking the dissolved peptide in TFE and adding it to a solution of DMPC and DHPC in chloroform at an optimized q value of 3.6.[22] Then the solvents were evaporated off using inert N2(g) and the same process as MLV formation was followed until sample became clear.</p><!><p>Circular Dichroism (CD) measurements on pinholin were performed on an Aviv Circular Dichroism Spectrometer Model 435 in a quartz cuvette with a 1.0 mm path length. Data was collected from 260 to 190 nm with 1 nm bandwidth at 25°C. CD data was collected on pinholin MLV samples prepared using the conditions outlined in the previous section.</p><p>CD spectral simulations and secondary structural content calculations were performed using DICHROWEB software found on http://dichroweb.cryst.bbk.ac.uk. [23] The CDSSTR algorithm was used for all simulations and compared back to reference data set SMP180 with a spectral width of 190-240nm.[24-28] Molar ellipticity ratios [θ]222/ [θ]208 were calculated to determine the presence of tertiary coiled-coil helices.[29, 30]</p><!><p>CW-EPR experiments were performed at the Ohio Advanced EPR Laboratory at Miami University. CW-EPR spectra were collected at X-band on a Bruker EMX EPR spectrometer using an ER041xG microwave bridge and ER4119-HS cavity coupled with a BVT 3000 nitrogen gas temperature controller. Each CW-EPR spectrum was acquired with 42 s field sweep with a central field of 3315 G and sweep width of 100 G, modulation frequency of 100 kHz, modulation amplitude of 1 G, and microwave power of 10 mW at room temperature.</p><!><p>The 31P solid-state nuclear magnetic resonance measurements were conducted at 25°C using a Bruker 500 MHz WB UltraShield NMR spectrometer with a 4mm triple resonance CP-MAS probe. 31P NMR spectra were recorded with 1H decoupling using a 4 μs π/2 pulse and a 4 s recycle delay, a spectral width of 300 ppm, and by averaging 4K scans. The free induction decay was processed using 200 Hz of line broadening. All figures were generated using the Igor software package.</p><!><p>This study reports the successful in vitro synthesis of both the active S2168 and inactive S21IRS forms of the pinholin system using solid phase peptide synthesis (SPPS). The full length active 68 and inactive IRS forms of the pinholin protein are composed of 68 and 73 amino acids respectively. The optimization of the sample preparation was confirmed through MALDI-TOF spectra that match the predicted molecular weights for each of the wild type pinholins, 7548 Da for the active S2168 and 8223 Da for the inactive S21IRS (Figures 1 and 2). A cysteine was substituted into the primary sequence for the leucine at position 25 for future nitroxide spin labeling. After synthesis, the solid phase resin and all amino acid protecting groups were removed in a three-hour reaction using a 30 mL cleavage solution of trifluoroacetic acid (TFA). The resulting peptide was precipitated from solution and washed 3 times using tert-butyl ether. Following the ether washes, the protein was purified using RP-HPLC and fractions were analyzed using MALDI-TOF to confirm the accuracy and overall purity of the synthesis.</p><!><p>Long hydrophobic peptides are difficult to synthesize, therefore the initial pinholin synthesis was of the first 20 amino acids (see Figure 1) with each of the following syntheses adding 10 more amino acids to the chain length. The peptide from each synthesis was analyzed using MALDI-TOF to determine which amino acids were not coupling completely. Coupling times and temperatures where adjusted to increase successful coupling as the chain length was extended to the full 73 amino acids of the inactive IRS pinholin. Since the pinholin system is naturally cys-less the incorporation of a cysteine at any position along the primary sequence allows for disulfide bond formation to the spin label at specific positions.</p><p>The MALDI-TOF results for the synthesis of both the active S2168 and inactive S21IRS pinholin wild type using the fourth cleavage condition in the following section can be seen in Figure 3. The target MW for the active S2168 and inactive S21IRS pinholin were 7546 and 8222 Da, respectively. The observed MW from Figure 3 for the active S2168 was found to be 7548 Da while the inactive S21IRS shows a peak at 8223 Da confirming a successful synthesis for both forms of the WT pinholin. Figure 3 also shows the MALDI-TOF data for the active S2168-L25C-MTSL and inactive S21IRS-L25C-MTSL forms of pinholin. The +185 Da shift in the m/z, 7722 for the active and 8391 for the inactive, confirms the successful spin labeling of both forms of the pinholin. In all cases there is a small peak appearing at one half the target MW corresponding to detection of a doubly charged pinholin ion.</p><!><p>The cleavage protocol was optimized by monitoring the cleavage reaction of various cleavage solutions over time. Four different cleavage conditions were tested based on type and number of certain amino acid side chain protecting groups present.[16] The cleavage conditions for four different solutions are as follows:</p><p>Cleavage condition 1[17] – TFA/EDT/thioanisole/water – 88/5/2/5</p><p>Cleavage condition 2[18] – TFA/phenol/water/thioanisole/EDT – 82.5/5/5/5/2.5</p><p>Cleavage condition 3[19] – TFA/thioanisole/EDT/anisole – 90/5/3/2</p><p>Cleavage condition 4[17] – TFA/triisoproylsilane/EDT/water – 94/1/2.5/2.5</p><p>Each of the different components of the cleavage reaction was measured out as a %v/v with a final cleavage solution volume of 30 mL. As each cleavage condition was tested a 5 mL aliquot of the reaction solution was removed at t = 30, 60, 90, 120, 150, and 180 minutes. Each aliquot was worked up following the procedure outlined in section 2.2. Finally, each time point was analyzed using MALDI-TOF to confirm the target protein molecular weight.</p><p>The MALDI-TOF results clearly identified cleavage condition 4 as the best cleavage condition for this system as it gave the sharpest target peak with the fewest impurity peaks. The MALDI peak at the target molecular weight does not begin to appear until an hour after the reaction begins. The MALDI results show no change in peak shape between the 2.5 and 3 hour aliquots indicating the completion of the cleavage reaction.</p><!><p>To optimize the MALDI-TOF analysis the pinholin peptide was dissolved in solutions of increasing amounts of acetonitrile in water. Each solution was spotted using one of three different matrices at a 1:1 ratio, α-Cyano-4-hydroxycinnamic acid (CHCA) matrix, 2,5-Dihydroxybenzoic acid (DHB) matrix, and Sinapic Acid (SA) matrix. The sample condition of 85% acetonitrile spotted with sinapic acid matrix gave the clearest resolution of MS peaks. Closer analysis of MALDI-TOF results revealed peaks at a higher m/z ratio indicating the presence of unremoved protecting groups during cleavage step. This process lead to the optimization of the cleavage reaction to remove these protecting groups.</p><!><p>CD spectroscopy is primarily used in the biochemical field to determine the global secondary structure of large macromolecules, such as peptides and proteins. This is accomplished through measuring the difference in absorption between left and right-handed circularly polarized light over a range of wavelengths.[31] The folding of the protein into a helical secondary structure was confirmed for both the active S2168 and inactive S21IRS pinholin using CD spectroscopy.</p><p>CD samples for both the active S2168 and inactive S21IRS forms of wild type pinholin were separately prepared in DMPC MLVs at a protein to lipid ratio of 1:500 with a final concentration of protein equal to 50 μM. Background scattering at lower wavelengths was minimized by subtracting the data collected from empty DMPC MLVs run at the same lipid concentration from the protein sample in DMPC MLVs. The resulting CD spectra of the active S2168 pinholin (blue) and the inactive S21IRS (red) are shown in Figure 4. Both forms of pinholin show a predominately helical structure with double minima at 208 nm and 222 nm and a large positive peak at 195 nm. The active S2168 pinholin showed a calculated helical content of 83%, while the inactive S21IRS pinholin secondary structure calculation determined 82% helical content. The percent normalized RMSD value calculated for both forms of the pinholin were less than 1.0%. [28] The helical content agrees well to the expected secondary structure of the protein based on previous studies in the literature.[11]</p><p>To give a more in-depth analysis of the CD spectra the molar ellipticity ratio of [θ]222/ [θ]208 was calculated for both forms of the pinholin. A molar ellipticity ratio exceeding a value of 1 is indicative of coiled-coil helical structures.[29, 30, 32] The molar ellipticity ratio [θ]222/ [θ]208 for both forms of the pinholin was found to be 1.4 and therefore, this coiled-coil structure was determined to be present for both the active S2168 and inactive S21IRS forms. This matches with the current predicted pinholin lysis pathway in which the pinholin begin to oligomerize, forming more coiled-coils, as more pinholin proteins accumulate in the membrane ultimately resulting in the lysis of the membrane.[11, 15] Further evidence of this will be shown in future experiments in which the oligomerization state of pinholin will be investigated as a function of concentration.</p><!><p>CW-EPR spectroscopy can be used to probe the dynamic and structural properties of both solution and membrane proteins. CW-EPR spectroscopy of these spin-labeled molecules can reveal information about the motion of the nitroxide side chain, solvent accessibility, and the polarity of the surrounding environment.[21, 33]</p><p>The successful spin labeling of the pinholin protein and incorporation into both bicelle and MLV lipid mimetic systems is shown in the CW-EPR spectra in Figure 5. This EPR data was also used to calculate spin labeling efficiency for each peptide which ranged from ~85-90% labeling efficiency. The EPR spectra for unbound nitroxide spin labels typically consist of three sharp peaks of relatively similar intensities, as seen in Figure 5A. Upon binding to a protein, such as pinholin, the MTSL will have a more restricted mobility due to the presence of the protein backbone. This decrease in the motion of the MTSL will broaden the lines in the EPR spectra and cause a decrease in their amplitude which is present in Figure 5B.[33, 34] The broadening of the EPR spectral linewidth is quantitatively determined by measuring the central line width. In Figure 5A the free MTSL spectra shows a central line width of 1.4 G, while the MTSL bound to the pinholin, 5B, has a central line width of 2.9 G. The line broadening of the EPR spectra from Figure 5A to 5B confirms the successful disulfide bond formation between the free MTSL and the Cys side chain of the pinholin due to a more restricted environment for the bound SL.</p><p>Incorporation of the pinholin into a lipid mimetic system should restrict the motion of the spin label even further through interactions with the lipid's hydrocarbon acyl chains. Since the broadening of the EPR spectra is proportional to the mobility of the spin label, the EPR spectra from pinholin incorporated into bicelles and MLVs show a greater degree of broadening than the labeled protein in solution (TFE) as seen when comparing Figure 5B to 5C, D. The central line widths of pinholin in bicelles versus pinholin in MLVs are 3.2 G and 3.3 G, respectively. The similarity of the central line width between the lipid incorporated samples is due to the similarity of the local environment around the spin label. Both bicelles and MLVs are good mimetics for lipid bilayers as opposed to a mimetic like micelles, which only mimic the hydrophobic environment of the membrane but cannot recreate the conditions of a bilayer. Therefore, the local environment and acyl chain packing around the spin label for both bicelles and MLVs will be similar in either mimetic.</p><!><p>Solid State NMR spectroscopy is a powerful biophysical technique which utilizes the presence of directionally dependent anisotropic interactions to probe the dynamics or kinetics of a system, specifically the membrane system for this study.[35] 31P SS NMR experiments were used to measure the chemical shift anisotropy (CSA) of the phosphorus head groups of DMPC lipids. [20] The degree in which the pinholin interacts with the 31P lipid head group will help to probe the differences between the roles of the active S2168 and inactive S21IRS forms of the pinholin in the lytic pathway from the perspective of the membrane.</p><p>31P SS-NMR samples were prepared using the wild type active S2168 and inactive S21IRS forms of the pinholin incorporated into DMPC MLVs at 1 mol%. More protein is required here to account for the lower sensitivity of NMR when compared to EPR spectroscopy. Empty DMPC MLVs, active S2168 pinholin in DMPC MLVs, and inactive S21IRS pinholin in DMPC MLVs at 25°C are shown in Figures 6A, B, and C, respectively. The shape of the static 31P SS-NMR spectra for the empty MLVs, active S2168, and inactive S21IRS are characteristic of lamellar phase lipid mimetics and show axial symmetry. The CSA width for each of the NMR spectra were found to be 48.0 ppm for empty DMPC MLVs, 44.7 ppm for active S2168 pinholin incorporated in DMPC MLVs, and 42.2 ppm for the inactive S21IRS pinholin incorporated in DMPC MLVs. The smaller CSA width for both the active S2168 and inactive S21IRS pinholin when compared to the empty MLVs confirms the interaction of the protein with the lipid mimetic system. The absence of an isotropic peak suggests the integrity of the MLV mimetic system is not compromised with the addition of the pinholin protein. The smaller CSA for the inactive S21IRS pinholin (42.2 ppm) indicates the inactive form is influencing the 31P DMPC lipid head groups more than the active S2168 form. These differences suggest that the active S2168 and inactive S21IRS pinholins behave differently once incorporated into the lipid bilayer. This difference will be further explored in future studies using both 31P T1 measurements and 2H lipid acyl chain experiments.</p><!><p>In this study we report the synthesis of both the active S2168 and inactive S21IRS forms of the pinholin protein using solid phase peptide synthesis. The measured CD data of both forms of the pinholin, matched the predicted alpha helical content. The CD molar ellipticity ratio also provided preliminary data of the helical packing required of the pinholin to attain functionality and will be explored to a greater extent in future works. CW-EPR spectroscopy was used to successfully show spin labeling of the pinholin as well as incorporation into both bicelles and MLV lipid mimetic systems. The success of this measurement opens the door for more in depth EPR structural and dynamic studies to be conducted in the future, such as pulsed EPR measurements.[36-40] 31P SS-NMR spectroscopy allowed for the study of the pinholin system from the lipid perspective and showed interactions of both forms of the pinholin with the lipid membrane, to varying degrees, through decreases in the CSA width when compared to the empty DMPC MLVs. These differences in the way the active S2168 and inactive S21IRS forms of the pinholin interact with the membrane suggest differences in the externalization of TMD1, and ultimately the role each form plays in the bacteriophage lytic pathway. Additionally, 31P T1 relaxation time measurements and 2H NMR experiments can be conducted to further probe the interaction of the pinholin with the acyl chain and lipid head groups to better understand how S2168 and S21IRS differ in their membrane interaction.</p>

PubMed Author Manuscript

Optimization of Replication, Transcription, and Translation in a Semi-Synthetic Organism

Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing non-canonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogs. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for information storage, as well as the most optimal unnatural ribonucleoside triphosphates for its retrieval. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.

optimization_of_replication,_transcription,_and_translation_in_a_semi-synthetic_organism

INTRODUCTION<!>Replication and templating of transcription.<!>Synthesis and first SAR analysis of ribonucleotide candidates.<!>Optimization of unnatural protein production.<!>Storage and retrieval of higher density unnatural information.<!>DISCUSSION<!>CONCLUSION

<p>In all natural organisms, information is encoded with the four-letter genetic alphabet, consisting of deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and deoxythymidine (dT), with the storage and retrieval of this information made possible by the formation of two base pairs, (d)A-dT/U and (d)G-(d)C (where "(d)" indicates that the nucleotides may be either deoxyribo- or ribonucleotides). Over 100 years ago, the newly defined field of synthetic biology set its central goal as the creation of new forms and functions,1 and perhaps the most general route to this goal is to increase the information that a cell can store and retrieve. Correspondingly, the last decade has seen significant effort and progress toward the identification of a fifth and sixth nucleotide that pair to form a third, unnatural base pair (UBP).2–4</p><p>Our efforts have focused on the development of UBPs formed between synthetic nucleotide analogs with predominantly hydrophobic nucleobases that pair via hydrophobic and packing forces, as opposed to the complementary hydrogen bonding used by natural nucleobases. Through a medicinal chemistry-like approach, based on the elucidation of structure-activity relationships (SARs) from the evaluation of over 150 nucleotide analogs,5 we discovered a family of UBPs that, when incorporated into DNA, are well replicated by DNA polymerases in vitro. Within this family, the UBPs formed between the synthetic nucleotides dNaM and either d5SICS or dTPT3 (dNaM-d5SICS and dNaM-dTPT3, respectively; Figure 1) have received the most attention and are amongst the most promising. Towards the goal of creating new forms and functions, we have used these UBPs as the basis of a semi-synthetic organism (SSO). The SSO, a strain of Escherichia coli constitutively expressing a nucleoside triphosphate transporter from Phaedactylum tricornutum (PtNTT2),6 imports the requisite unnatural nucleoside triphosphates from the media and uses them to replicate DNA containing the UBP, transcribe mRNA and tRNA containing the unnatural nucleotides, and then use the resulting cognate unnatural codons and anticodons to translate proteins containing non-canonical amino acids (ncAAs).7–12 In addition, the forces underlying the pairing of the unnatural nucleotides, as well as their physical properties have been explored by others.13–19</p><p>Although dNaM-d5SICS, and especially dNaM-dTPT3, are sufficiently well replicated in the SSO for the stable propagation of increased genetic information, they are still replicated less efficiently than a natural base pair, which has motivated continued chemical optimization. These efforts have identified several promising candidates, in particular dCNMO and dPTMO, which when paired with dTPT3, form UBPs that are better retained in the DNA of the SSO (Figure 1).10,20 In addition, we have also explored the ability of the SSO to retrieve the information encoded with the dPTMO-dTPT3 UBP via transcription and translation, and interestingly, found that its use results in more efficient expression of unnatural proteins than dNaM-dTPT3.10 This result clearly motivates elucidation of the SARs governing the templating of transcription. In addition, only the NaMTP and TPT3TP ribonucleoside triphosphates have been used to retrieve the increased information in the SSO. Given that these analogs were designed based on replication SARs, which may or may not be the same as those governing efficient transcription and translation, it remains unclear if they are optimal.</p><p>Here, we explore the ability of the unnatural information encoded by UBPs formed by dTPT3 and either dNaM or a dNaM analog, to be retrieved in the form of proteins containing ncAAs. We also report the first systematic evaluation of the efficiency with which this information is retrieved using thirteen analogs of NaMTP or TPT3TP, eleven of which are novel. The results identify an improved system of unnatural deoxy- and ribonucleotides that form the basis of an SSO that more efficiently stores and retrieves increased information, and for the first time, permits the efficient production of protein bearing multiple, proximal ncAAs.</p><!><p>While we have examined the in vivo replication of a wide variety of our UBPs, retention in a tRNA gene or in any actively transcribed gene has to date only been examined with dNaM, dPTMO, dMTMO, and dTPT3.9–10 To extend these studies, we first constructed plasmids with two dNaM-dTPT3 UBPs, such that the sequence AXC (here and throughout X refers to (d)NaM or a (d)NaM analog) was positioned to template codon 151 of sfGFP mRNA (sfGFP151(AXC)) and with the sequence GYT (here and throughout Y refers to (d)TPT3 or a (d)TPT3 analog) positioned to template the anticodon of the M. mazei Pyl tRNA (tRNAPyl(GYT)), which is selectively charged with the ncAA N6-(2-azidoethoxy)-carbonyl-l-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb PylRS).21–25 These plasmids were used to transform E. coli expressing the nucleoside triphosphate transporter PtNTT2 (strain YZ38) and harboring a plasmid encoding Mb PylRS. After transformation, colonies were selected and grown to an OD600 ~ 1.0 in liquid media supplemented with dTPT3TP (10 µM) and one of seven different dXTPs (Figure 2) added at varying concentrations (150 µM, 10 µM, or 5 µM). Cells were then diluted into fresh expression media containing the same unnatural deoxyribonucleotides as well as NaMTP (250 µM), TPT3TP (30 µM), and AzK (10 mM). After a brief incubation, T7 RNAP and tRNAPyl(GYT) expression was induced by the addition of isopropyl-β-d-thiogalactoside (IPTG, 1 mM). After an additional 1 h of incubation, the expression of sfGFP151(AXC) was initiated by the addition of anhydrotetracycline (aTc, 100 ng/mL).</p><p>After 2.5 h, plasmids were isolated and then the genes of interest were independently PCR amplified using d5SICSTP and dMMO2BIOTP, a biotinylated analog of dNaMTP.26 The resulting PCR product was analyzed via a gel mobility shift assay using streptavidin to quantify the UBP retained as a percent shift of the total amplified product (hereafter referred to as the streptavidin gel shift assay) (Figure 3A and 3B). At the highest dXTP concentration examined (150 µM), retention in the sfGFP151(AXC) gene was similar for each dXTP analog, varying from 99% for dNaMTP to 92% for dMTMOTP. Retention within the tRNAPyl(GYT) gene was slightly lower, ranging from 82% for dMTMOTP to 74% for dMMO2TP. With the addition of 10 µM of each dXTP, retention in the sfGFP151(AXC) gene ranged from 96% for d5FMTP to 73% for dNaMTP. With the tRNAPyl(GYT) gene, retentions were again generally slightly lower, ranging from 82% for d5FMTP and dPTMOTP to 71% for dNaMTP. At the lowest concentration (5 µM), retention ranged from 94% for d5FMTP to 64% for dMTMOTP in the sfGFP151(AXC) gene, and from 83% for d5FMTP to 71% for dMTMOTP in the tRNAPyl(GYT) gene. When the same concentrations were employed, the results with dNaMTP, dPTMOTP, or dMTMOTP are indistinguishable from those previously reported, and also as reported previously, cells grown with 5 µM dNaMTP did not survive.10 Generally, at the highest concentration, all dXTPs performed well with nearly quantitative retention of the UBP within the sfGFP gene. However, at lower concentrations it became increasingly evident that dCNMO-dTPT3, d5FM-dTPT3, and dPTMO-dTPT3 were replicated with significantly higher retention. Interestingly, retention in the tRNA gene was significantly less dependent on dXTP concentration.</p><p>To characterize the amount of protein produced, bulk culture fluorescence normalized to cell growth was measured 2.5 h after the induction of protein expression (Figure 3C). In the absence of AzK, fluorescence was generally low, with the exceptions of dPTMOTP and dMTMOTP at the lower concentrations, which appeared slightly higher. When AzK was added to the media, cells grown with each dXTP at either 150 or 10 µM generally showed significant and similar levels of fluorescence, with the exceptions of dNaMTP, which showed significantly less fluorescence at 10 µM than at 150 µM. At the lowest concentration (5 µM), the addition of AzK resulted in less fluorescence observed with dMTMOTP while it remained the same with dCNMOTP, d5FMTP, dClMOTP, dMMO2TP, or dPTMOTP. Again, as mentioned above, cells were unable to grow when provided with only 5 µM dNaMTP. Remarkably, the data indicate that while similar at high concentrations, at lower concentrations the use of each dXTP analog results in a greater AzK-dependent increase in fluorescence than does the use of dNaMTP. In particular, dCNMOTP, d5FMTP, and dClMOTP showed the greatest AzK-dependent increase in fluorescence, and were thus considered the most promising.</p><p>To directly assess the fidelity of unnatural protein production, cells were harvested 2.5 h after the induction of protein expression and the sfGFP produced was purified and subjected to a strain-promoted azide-alkyne cycloaddition reaction27 with dibenzocyclooctyne (DBCO) linked to a TAMRA dye by four PEG units. In addition to tagging the proteins containing the ncAA with a detectable fluorophore, conjugation produces a shift in electrophoretic migration, allowing quantification of protein containing AzK as a percentage of the total protein produced (i.e. fidelity of ncAA incorporation; Figure 3D).10 As with dNaM, use of each dXTP at the highest concentration (150 µM) resulted in virtually complete shifts of the purified protein, reflecting high fidelity incorporation of the ncAA. When grown in the presence of 10 µM of the dXTP, the fidelity of ncAA incorporation remained high for dCNMOTP, d5FMTP, dClMOTP, dMMO2TP, dPTMOTP, and dMTMOTP, but dropped precipitously for dNaMTP. Finally, at a concentration of 5 µM dXTP, the fidelity of ncAA incorporation remained similar to that observed at 10 µM for dCNMOTP, d5FMTP, dClMOTP, dMMO2TP, and dPTMOTP, but dropped for dMTMOTP (again due to viability, fidelity with dNaMTP could not be measured at this concentration).</p><p>The most promising members of the family that produced the greatest quantity of pure unnatural protein, especially at lower concentrations, are dCNMOTP and d5FMTP. However, relative to d5FMTP, the use dCNMOTP has been previously shown to result in higher UBP retention in more difficult to replicate sequences20. Thus, we conclude that the dCNMO-dTPT3 UBP is the most optimized for the storage and retrieval of increased information.</p><!><p>Retrieval of the information made available by the UBP has only previously been explored using NaMTP and TPT3TP. To begin to elucidate the SARs governing efficient transcription and translation in the SSO, we designed and synthesized nine novel NaMTP analogs (Figure 4A) and four novel TPT3TP analogs (Figure 4B). These analogs were designed to explore the role of nucleobase shape, aromatic surface area, and heteroatom derivatization. Generally, the synthesis of the XTP analogs proceeded via lithiation of the corresponding aryl halide, followed by coupling of the lithiated species to either the benzyl- or TBS-protected ribolactone. Reduction of the resulting hemi-acetal intermediate in the presence of boron trifluoride diethyl etherate and triethylsilane afforded the desired protected nucleoside in each case. Following deprotection, the resulting X nucleoside analogs were converted to triphosphates using standard Ludwig phosphorylation conditions (see Supporting Information).28 NaMTP and MMO2TP were synthesized as reported previously.29 The synthesis of the YTP analogs generally proceeded via intramolecular Curtius rearrangement of the corresponding acyl azide, followed by Lewis-acid mediated coupling to 1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribofuranose, resulting in pure β-anomer of the desired protected nucleoside. Following conversion of the pyridine to the corresponding thio-pyridone and subsequent benzoyl deprotection, the corresponding free nucleosides were converted to triphosphates using standard Ludwig phosphorylation conditions (see Supporting Information).28 5SICSTP was synthesized as reported previously.29</p><p>We initiated our SAR analysis with NaMTP and the nine XTP analogs. Based on performance in the dXTP screen described above, and to eliminate variable loss at the DNA level as a complicating factor, we encoded the unnatural information with the dCNMO-dTPT3 UBP. Additionally, we used our recently reported E. coli strain ML2,11 which expresses the nucleoside triphosphate transporter PtNTT2, but has also been genetically engineered for higher fidelity replication of the UBP by deletion of the gene encoding RecA (as is common in cloning strains) and overexpression of DNA Pol II. The same plasmid described above, harboring sfGFP151(AXC) and tRNAPyl(GYT), was used, and to focus the screen to a single unnatural ribonucleotide, the M. mazei Pyl tRNA was transcribed in the presence of 30 µM TPT3TP.</p><p>Cells were grown and induced to produce protein as described above, except that the various XTPs were provided at either high (250 µM) or low (25 µM) concentration in the expression media (Figure 5). Expressed sfGFP was purified 3 h after induction and ncAA content analyzed using the DBCO-mediated gel shift assay described above (Figure 5B). Remarkably, the use of each XTP at 250 µM resulted in sfGFP gel shifts of at least 63%. Along with the lack of observable shift in the absence of an XTP, this demonstrates that each XTP is imported by PtNTT2 and participates in transcription and translation at the ribosome with at least reasonable efficiency. While CNMOTP and 5F2OMeTP each performed well, resulting in gel shifts of 92% and 94%, respectively, NaMTP, MMO2TP, and 5FMTP performed the best, with protein gel shifts of 98%, 97%, and 98%, respectively. With similarly high levels of protein purity, we were able to compare the relative fluorescence produced using these three XTPs in the absence of any complications resulting from significant levels of natural sfGFP contaminant. Cells grown with 250 µM of either MMO2TP or 5FMTP produced 63% and 90% bulk fluorescence, respectively, compared to cells grown with NaMTP at the same concentration (Figure 5A).</p><p>At the lower concentration tested (25 µM), the use of NaMTP resulted in a lower fidelity of ncAA incorporation, with a protein shift that dropped to 86%. While 7 of the 9 NaMTP analogs also showed significant decreases in fidelity, the use of MMO2TP and 5FMTP each yielded protein shifts of 94%. Comparing the relative fluorescence of cells grown with these ribonucleotides (again possible due to the similar and high fidelity of unnatural protein produced), 5FMTP produces 34% more fluorescence than MMO2TP at this lower concentration.</p><p>We next performed similar experiments, except we supplied a constant amount of NaMTP (250 µM) and either a high (250 µM) or low (25 µM) concentration of a YTP analog. Consistent with previous reports9, the addition of TPT3TP at the higher concentration resulted in significantly reduced cell growth and little fluorescence relative to the control sample that did not receive a YTP (Figure 5C). In contrast, each of the other YTP analogs produced fluorescence above background, and protein shifts of at least 51% (Figure 5D). In particular, TAT1TP performed the best at this concentration, with its use resulting in at least 2.6-fold more fluorescence than any other YTP, while maintaining a protein shift of 96%. However, the addition of TAT1TP at this concentration did result in a modest level of reduced cell growth (Figure S1).</p><p>Consistent with previous reports,9–10 TPT3TP is somewhat less toxic when provided at lower concentrations, and correspondingly, when provided at 25 µM, cells produce significant quantities of pure protein. Under these conditions, TPT3TP is more optimal than SICSTP, FSICSTP, and 5SICSTP, producing 2-, 5-, and 6-fold greater fluorescence, respectively. Interestingly, the toxicity observed with TAT1TP at the higher concentrations was almost completely eliminated at lower concentrations (Figure S1), and its use resulted in even greater fluorescence than at the higher concentration. When provided at 25 µM, TAT1TP produces 41% more fluorescence than when provided at 250 µM, and interestingly, it produced 57% more fluorescence than when TPT3TP is provided at 25 µM. Most importantly, the use of TAT1TP at these concentrations resulted in the production of protein with 98% ncAA incorporation.</p><!><p>The screens described above identified dCNMO-dTPT3 as the most promising UBP for the storage of information, and TAT1TP and NaMTP or 5FMTP as the most promising ribonucleoside triphosphates for its retrieval. Thus, we next turned to exploring the concentrations of each ribonucleotide used to optimize the yield and fidelity of protein expression in the SSO. Upon transformation with the same plasmids used above, 10 µM dTPT3TP and 25 µM dCNMOTP were provided in the growth media, which was then also supplemented with TAT1TP at concentrations ranging from 100 µM to 12.5 µM, and either NaMTP or 5FMTP at concentrations ranging from 200 µM to 12.5 µM, all in series of 2-fold dilutions, and after the addition of 1 mM AzK, the cells were induced to express sfGFP.</p><p>Total sfGFP fluorescence observed in cells provided with TAT1TP and NaMTP was generally higher than that observed in cells provided with TAT1TP and 5FMTP (Figure 6A and 6B). In both cases, fluorescence was higher at lower concentrations of NaMTP or 5FMTP (due to increased production of contaminating natural sfGFP, see below). Additionally, cells generally produced higher fluorescence at higher concentrations of TAT1TP. However, due to a slight reduction in growth, cells provided with 100 µM TAT1TP produced less fluorescence than those provided with 50 µM TAT1TP.</p><p>Protein production was again quantified via the gel shift assay (Figure 6C and 6D). Generally, as the concentration of NaMTP decreased below 200 µM, incrementally lower fidelity of AzK incorporation into sfGFP was observed, while with use of 5FMTP this reduction in fidelity was only observed below a concentration of 50 µM. Clearly lower concentrations of 5FMTP can be used without compromising fidelity. Cells provided with a high concentration of 5FMTP (≥ 50 µM) produced high protein shifts at all concentrations of TAT1TP explored (100 µM, 50 µM, 25 µM, or 12.5 µM). However, when the concentration of 5FMTP was 25 µM or less, decreasing the concentration of TAT1TP resulted in a reduced protein shift. When NaMTP was provided at 200 µM, all concentrations of TAT1TP explored resulted in the production of protein with high fidelity ncAA incorporation, but with lower concentrations of NaMTP, decreasing the concentration of TAT1TP again resulted in a decreased protein shift.</p><p>In all, these studies revealed that the combined optimization of protein purity and yield is achieved with NaMTP and TAT1TP provided at concentrations of 200 µM and 50 µM, respectively, or with 5FMTP and TAT1TP both provided at a concentration of 50 µM. In terms of protein production alone, the use of NaMTP and TAT1TP is optimal, whereas the use of 5FMTP and TAT1TP results in slightly lower yields of pure ncAA-labeled protein, but requires significantly lower concentrations of the XTP.</p><!><p>With optimized unnatural nucleotides and conditions, we next sought to challenge the SSO by examining the storage and retrieval of information from a gene containing a higher density of UBPs. Towards this goal, we first validated the ability of the SSO to replicate DNA containing the sfGFP gene with the UBP positioned to encode codons 149 or 153, which are each separated from the codon described above (codon 151) by a single natural codon. Accordingly, expression plasmids were constructed, as described above, but in which the sequence AXC was positioned to encode either codon 149 or 153 (sfGFP149(AXC) or sfGFP153(AXC), respectively). Upon transformation of ML2, cells were grown in the presence of unnatural nucleoside triphosphates, corresponding to either our previously reported system (the deoxyribonucleotides dNaMTP and dTPT3TP and the ribonucleotides NaMTP and TPT3TP, denoted dNaM-dTPT3/NaMTP,TPT3TP),8–9 or the optimized system discovered in the current study (dCNMO-dTPT3/NaMTP,TAT1TP). UBP retention was then characterized using the streptavidin gel shift assay, as described above. High retention of the corresponding UBP in both sfGFP149(AXC) and sfGFP153(AXC) genes (≥95%) as well as in the tRNA gene (≥91%) was observed under both conditions (Table S1).</p><p>Total sfGFP fluorescence observed 3 h after induction revealed significant protein production from both constructs in the presence of AzK under both sets of conditions (Figure 7A). However, fluorescence from cells expressing the sfGFP149(AXC) construct provided with dCNMO-dTPT3/NaM,TAT1 was 58% higher than cells provided with dNaM-dTPT3/NaM,TPT3. In the case of the sfGFP153(AXC) gene, 43% more fluorescence was observed with dCNMO-dTPT3/NaMTP,TAT1TP than with dNaM-dTPT3/NaMTP,TPT3TP. Under both sets of conditions, approximately 2-fold more fluorescence was observed with sfGFP153(AXC) than with sfGFP149(AXC). Protein was purified and AzK incorporation was analyzed as described above (Figure 7B). With dNaM-dTPT3/NaMTP,TPT3TP, protein shifts of 86% and 94% were observed with sfGFP149(AXC) and sfGFP153(AXC), respectively. With dCNMO-dTPT3/NaMTP,TAT1TP, however, a 96% shift was observed with protein produced from either construct. These results clearly demonstrate that the two additional codon positions are both transcribed and translated efficiently, but again they are transcribed and translated better with the newly identified dCNMO-dTPT3/NaMTP,TAT1TP system.</p><p>We next constructed expression plasmids with an unnatural codon simultaneously encoded at two or all three of the positions examined (sfGFP149,151(AXC,AXC), sfGFP151,153(AXC,AXC), sfGFP149,153(AXC,AXC), and sfGFP149,151,153(AXC,AXC,AXC), respectively). ML2 cells were transformed, grown in the presence of either dNaM-dTPT3/NaMTP,TPT3TP or dCNMO-dTPT3/NaMTP,TAT1TP, and protein expression was induced as described above. While UBP retention in the tRNAPyl(GYT) gene remained high (≥88%) in all cases (Table S1), the biotin shift assay with the mRNA genes produced complex and uninterpretable band patterns, likely due to, at least in part, the formation of a mixture of complexes with single PCR products bound to multiple streptavidins. Thus, we proceeded to analyze the protein produced via conjugation to DBCO-TAMRA as described above (Figure 7B). Gratifyingly, relative to the shift observed with a single ncAA, a significantly further shifted band was observed for proteins expressed from the sfGFP149,151(AXC,AXC), sfGFP151,153(AXC,AXC), and sfGFP149,153(AXC,AXC) constructs, indicating the conjugation of two DBCO-TAMRA molecules to sfGFP bearing two AzK residues. When analyzing purified proteins expressed with dNaM-dTPT3/NaMTP,TPT3TP, quantification of these double shifted bands relative to total sfGFP revealed that 80%, 87%, and 83% of the protein, respectfully, had two AzK residues, and 20%, 13%, or 9% , respectfully, had a single AzK when using the sfGFP149,151(AXC,AXC), sfGFP151,153(AXC,AXC), or sfGFP149,153(AXC,AXC) constructs, respectively. With dCNMO-dTPT3/NaMTP,TAT1TP, 81%, 89%, and 93% of the protein had two ncAAs with sfGFP149,151(AXC,AXC), sfGFP151,153(AXC,AXC), and sfGFP149,153(AXC,AXC), respectively, while 19%, 11%, and 6% had a single ncAA. Cells transformed with the sfGFP149,151,153(AXC,AXC,AXC) construct expressed protein that produced an even further shifted band, clearly indicating the incorporation of three AzK residues. Quantification of each band relative to total sfGFP revealed that the use of dNaM-dTPT3/NaMTP,TPT3TP resulted in 39%, 24%, and 33% of the protein having three, two and one ncAAs, respectively, and with fluorescence and protein shifts that were highly variable (Figure 7A and 7B). In contrast, use of the dCNMO-dTPT3/NaMTP,TAT1TP system resulted in 90% of the produced protein having all three ncAAs, with the remainder having two.</p><p>To further verify the successful incorporation of all three ncAAs with the dCNMO-dTPT3/NaMTP,TAT1TP system, we analyzed the isolated sfGFP by quantitative intact protein mass spectrometry. Briefly, purified proteins were desalted using centrifugal filter devices (Amicon® Ultra-0.5 – Millipore), and analyzed by HRMS (ESI-TOF). The mass spectra acquired were subsequently deconvoluted using the Waters MaxEnt 1 software, which proved to be quantitative upon peak integration (Figure S2). In agreement with the gel shift assay, this analysis revealed that that 88% of the isolated protein contained the expected three AzK residues, while the remaining 12% contained two AzK residues and a single Ile or Leu residue (Figure 7C).</p><!><p>The previously reported SSOs stored information with the UBPs dNaM-dTPT3, dPTMO-dTPT3, or dMTMO-dTPT3, and retrieved that information using NaMTP and TPT3TP. To explore the optimization of the SSO, we examined retention of the UBP, transcription into sfGFP mRNA and tRNAPyl, and decoding at the ribosome, using a collection of previously and newly reported deoxy- and ribonucleotide triphosphate analogs. We first examined the ability to store information with seven different dX-dTPT3 UBPs. In each case, the strand context of the UBP was the same, with dTPT3 and dX positioned in the corresponding antisense (template) strands of the sfGFP and tRNAPyl genes, respectively. With high concentrations of each dXTP provided, each dX-dTPT3 UBP is retained at a high level in the mRNA gene, with variation between 92% for dMTMOTP and 96% to 99% for dCNMOTP, dPTMOTP, and dNaMTP. Retentions in the tRNA gene were somewhat reduced, varying between 74% to 82%. As the concentrations of dXTP decreased, retentions remained roughly constant in the tRNA gene, but decreased in a dXTP specific manner in the mRNA gene, decreasing to 64% for dMTMOTP, but remaining relatively high, at ~94%, for dCNMOTP and d5FMTP. The different concentration dependencies for retention in the tRNA and mRNA genes likely result from sequence context effects causing nucleotide insertion to be rate limiting in the mRNA and continued extension to be rate limiting in the tRNA gene. Exceptions were observed with dNaMTP, where at 10 μM retention decreased to 73%, and as reported previously10 the cells did not survive when dNaMTP was provided at 5 μM. In addition, with dCNMOTP and d5FMTP, retentions remained high in the mRNA (~93%) at even the lowest concentration examined. Loss of retention in the tRNA gene could result in less unnatural protein production, and perhaps more problematically, reduced fidelity of ncAA incorporation due to increased competition for decoding of the unnatural codon by "near-cognate" natural tRNAs. However, the fidelity of ncAA incorporation is clearly correlated with retention in the mRNA gene (Figure S3). Thus, the data demonstrates that each dX templates transcription of tRNA with sufficient efficiency and fidelity to not limit the fidelity of unnatural protein production. Based on these results, as well as those previously published20, d5FM-dTPT3, and especially dCNMO-dTPT3 are the most optimized UBPs, with their utility relative to dNaM-dTPT3 deriving principally from their higher retention and protein production at lower unnatural triphosphate concentrations.</p><p>We next examined the ability of ten different XTPs to mediate the retrieval of information stored by the dCNMO-dTPT3 UBP. Remarkably, all ten XTPs explored at high concentration are capable of mediating the production of proteins with at least moderate ncAA incorporation fidelity (98% to 63%). Generally, as XTP concentrations are decreased, the fidelity of ncAA incorporation decreased, indicating a reduction in the fidelity with which the unnatural mRNA is transcribed. This is consistent with the significant fluorescence observed when XTP was withheld.</p><p>The current study provides the first SARs for the transcription and translation of predominantly hydrophobic ribonucleotides. With the XTPs, when considering NaMTP, PTMOTP, and MTMOTP, it is clear that ring contraction and/or heteroatom derivatization is deleterious. However, with the monocyclic nucleobase XTPs, with the exception of ClMOTP, higher fidelity protein production is observed, relative to MTMOTP and PTMOTP, suggesting that the effects are more complicated than just aromatic surface area or heteroatom derivatization. It is likely that specific interactions between the unnatural nucleobases or with the polymerase are critical. Substitution at both the 4- and 5-positions of the monocyclic nucleobases has significant effects. Compared to 2OMeTP, a Cl or Br substituent at the 4-position (ClMOTP and BrMOTP) is modestly deleterious, while a methyl group at the 4-position (MMO2TP), reduces overall fluorescence, but with a significant increase in protein fidelity. A nitrile substituent at the 4-position (CNMOTP) results in the production of the greatest amount of unnatural protein, and also modestly increases the fidelity with which the ncAA is incorporated. Addition of a fluoro substituent at the 5-position (5F2OMeTP) also increases both protein production and fidelity, relative to 2OMeTP. The effects of substitution at the 4- and 5- positions appear at least approximately additive, as combining the 5-fluoro and 4-methyl substituents (5FMTP) allows for the high yield production of pure unnatural protein at lower concentrations, relative to the other unnatural triphosphates. However, while requiring higher concentrations, NaMTP provides the most optimal combination of yield and purity of the XTP analogs examined.</p><p>It is interesting to note that the SARs derived for the XTP analogs are distinctly different from those derived from the replication of dXTP analogs. For example, while dPTMOTP, dClMOTP, and especially dCNMO are more optimal than dNaMTP, ClMOTP and PTMOTP are modestly and significantly less optimal than NaMTP. Moreover, while dCNMO is the most optimal partner for dTPT3 discovered to date, the use of CNMOTP results in slightly reduced fidelity of ncAA incorporation, relative to NaMTP, although its use does result in the most unnatural protein production.</p><p>At the highest concentration, all five YTPs explored were effectively incorporated into the anticodon of tRNAPyl and capable of mediating the production of proteins with at least moderate ncAA incorporation fidelity (98% to 51%). However, the UBP retention data suggests that the fidelity of protein production is not sensitive to modest loss of unnatural tRNA, implying that for the YTPs that resulted in lower protein gel shifts, transcription of the tRNA was either very inefficient or low fidelity. TPT3TP, SICSTP, FSICSTP, and TAT1TP were all, at least slightly toxic at the highest concentration (Table S2), and bulk cell fluorescence increased with decreasing concentrations. Unlike with XTPs and transcription of the mRNA, fidelity of unnatural protein did not decrease, suggesting that the increased protein production was simply the result of increased cell growth. In contrast, 5SICSTP is not toxic (Table S2), and both unnatural protein production and fidelity decreased with decreasing concentrations, again presumably due to significantly compromised unnatural tRNA production.</p><p>When considering this YTP SAR, and using SICSTP as a reference, it is clear that a 7-fluoro substituent (FSICSTP) is quite deleterious, virtually ablating protein production, either due to effects on tRNA transcription or on translation, and only a small amount of protein is produced and with low fidelity. Addition of a methyl group at the 5-position (5SICSTP) reduces toxicity, but also appears to significantly reduce unnatural tRNA production. Ring contraction and heteroatom derivatization (TPT3TP) greatly improves both protein production and fidelity, but at high concentration it is the most toxic of the YTP analogs. Further heteroatom derivatization of the thiophene ring of TPT3TP, to produce the thioazole of TAT1TP, results in the production of even more pure unnatural protein than does TPT3TP, and importantly, with a substantial reduction in toxicity. Interestingly, unlike the case with the XTP SARs discussed above, the YTP SARs are relatively similar to those characterized with dYTP analogs.5 Given both the amount of protein produced and the fidelity of ncAA incorporation, TAT1TP is the most promising YTP identified to date.</p><p>Overall, the SARs identify the dCNMO-dTPT3/NaMTP,TAT1TP system as the most optimized for protein production, producing high amounts of protein with high fidelity incorporation of the ncAA. The use of dCNMO-dTPT3/5FMTP,TAT1TP produces protein with the same high fidelity, and while it produces slightly less protein, it requires the use of significantly less of the unnatural ribonucleotides. The utility of the dCNMO-dTPT3/NaMTP,TAT1TP system, relative to the previously reported dNaM-dTPT3/NaMTP,TPT3TP system, is particularly apparent with the encoding and retrieval of higher density unnatural information. As would be expected based on the results of the single labeled proteins, both systems produced protein with two ncAAs with high fidelity, but the dCNMO-dTPT3/NaMTP,TAT1TP system generally produced the desired protein in greater quantities. Moreover, when encoding three ncAAs, the dNaM-dTPT3/NaMTP, TPT3TP system produced triply labeled protein with significantly reduced and more variable fidelities and yields, while the fidelity and yield with the dCNMO-dTPT3/NaMTP,TAT1TP system remained reproducibly high. The contaminant, where an Ile or Leu replaced a single ncAA, is unlikely to result from unnatural tRNA production, as UBP retention in the tRNA gene was high and similar for the both systems, and even with small differences, the single ncAA-incorporation data suggest that they should not cause significant reductions in the fidelity of unnatural protein production. It is also unlikely to result from mRNA transcription, which should be identical for the two systems (in both cases the dTPT3 directs the incorporation of NaM into the mRNA). Thus, the origin of the Leu/Ile contaminant is likely to be loss of the UBP in the mRNA gene during replication (which, as mentioned above, we were unable to directly measure). This is also consistent with the most common mutation expected, (dX to dT), which would produce an Ile codon.</p><!><p>This study reports the first SARs underlying efficient transcription by T7 RNAP and translation at the ribosome in the E. coli based SSO. It is particularly interesting that the X and dX SAR appear unrelated, while the Y and dY SAR appear more similar; it is possible the replicative polymerase(s), likely Pol III and to a lesser extent Pol II,11 and T7 RNAP recognize distinct aspects of the (d)X nucleobases, but similar aspects of the (d)Y nucleobases. While the origins of this recognition will be pursued in future studies, the results have already identified a more optimal SSO, specifically an SSO that stores information with dCNMO-dTPT3, and retrieves the information it makes available with TAT1TP and NaMTP or 5FMTP. The optimization of the SSO is particularly apparent in its ability to produce protein with a higher density of ncAAs. The optimized SSO further attests to the ability of hydrophobic and packing forces to replace the complementary hydrogen bonds that underlie the storage and retrieval of natural information and represents significant progress towards the creation of a SSO with a fully unrestricted expansion of its genetic alphabet and code.</p>

PubMed Author Manuscript

Synthesis and high temperature thermoelectric properties of Yb0.25Co4Sb12-(Ag2Te)x(Sb2Te3)1−x nanocomposites

Nanocomposites are becoming a new paradigm in thermoelectric study: by incorporating nanophase(s) into a bulk matrix, a nanocomposite often exhibits unusual thermoelectric properties beyond its constituent phases. To date most nanophases are binary, while reports on ternary nanoinclusions are scarce. In this work, we conducted an exploratory study of introducing ternary (Ag2Te)x(Sb2Te3)1−x inclusions in the host matrix of Yb0.25Co4Sb12. Yb0.25Co4Sb12-4wt% (Ag2Te)x(Sb2Te3)1−x nanocomposites were prepared by a melting-milling-hot-pressing process. Microstructural analysis showed that poly-dispersed nanosized Ag-Sb-Te inclusions are distributed on the grain boundaries of Yb0.25Co4Sb12 coarse grains. Compared to the pristine nanoinclusion-free sample, the electrical conductivity, Seebeck coefficient, and thermal conductivity were optimized simultaneously upon nanocompositing, while the carrier mobility was largely remained. A maximum ZT of 1.3 was obtained in Yb0.25Co4Sb12-4wt% (Ag2Te)0.42(Sb2Te3)0.58 at 773 K, a ~ 40% increase compared to the pristine sample. The electron and phonon mean-free-path were estimated to help quantify the observed changes in the carrier mobility and lattice thermal conductivity.

synthesis_and_high_temperature_thermoelectric_properties_of_yb0.25co4sb12-(ag2te)x(sb2te3)1−x_nanoco

Introduction<!>Experimental procedures<!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Conclusions<!>Conflict of interest statement

<p>In the wake of severe environmental and energy crisis, thermoelectric (TE) materials, which can convert heat to electricity directly, have attracted much attention (Dresselhaus et al., 2007; Liu et al., 2012; Alam and Ramakrishna, 2013). The conversion efficiency of a TE material is determined by its dimensionless figure of merit ZT = α2T/ρκ, where α, ρ, κ, T are Seebeck coefficient, electrical resistivity, total thermal conductivity, and absolute temperature, respectively. The term α2/ρ is called power factor. The total thermal conductivity κ is composed of electronic component κe and lattice component κL.</p><p>A general challenge in single-phased bulk TE material is that all three TE properties, the electrical conductivity, the Seebeck coefficient, and the electronic thermal conductivity, are intimately but adversely inter-dependent, optimizing one often degrades others. In this regard, nanostructuring has provided a new route to partially decouple the TE properties, as evidenced in supperlattice (Venkatasubramanian et al., 2001; Harman et al., 2002; Zeng et al., 2009) and nanocomposites (Hsu et al., 2004; Zhao et al., 2005; Poudel et al., 2008; Fan et al., 2011; Lee et al., 2012; Liu et al., 2012; Alam and Ramakrishna, 2013), by enhancing the power factor and/or lowering the κL. Nanostructuring in bulk materials can be realized by directly incorporating nanoparticles in the host matrix (Fan et al., 2011; Lee et al., 2012), or creating nanoparticles in situ (Hsu et al., 2004), or reducing the host matrix particle size down to nanometer scale (Poudel et al., 2008). A number of approaches have been proposed to enhance the power factor, mostly via enhancing the Seebeck coefficient by (i) creating sharp features in the density-of-states, for instance, by introducing resonant states (Heremans et al., 2008; Ahn et al., 2010), and (ii) increasing the energy dependence in the relaxation times, for instance, by energy filtering effect at the interface (Heremans et al., 2005; Shakouri and Zebarjadi, 2009). Recently, a modulation-doping mechanism has been proposed to enhance the mobility therefore the electrical conductivity as compared to its uniform-doping counterpart (Zebarjadi et al., 2011; Yu et al., 2012). In the mean time, many ZT enhancements in nanocomposites are owing to the significant reduction of κL, via strong interface scattering of heat-carrying phonons.</p><p>CoSb3-based filled-skutterudites have been known as promising TE materials for medium-temperature power generation applications (Yang et al., 2007; Shi et al., 2011; Peng et al., 2012a; Rogl et al., 2014). Despite the tremendous progress in developing high performance filled-skutterudites, there is still room for further reduction of κL, especially in n-type filled-skutterudites, as compared to other state-of-the-art TE materials. The future of CoSb3-based filled-skutterudites is largely hinged upon further reduction of κL while remaining or even increasing the power factor.</p><p>To this end, introducing AgSbTe2 nanoinclusions in the host matrix of Yb0.2Co4Sb12 proved to be an effective approach. AgSbTe2 is by itself a promising TE material (Xu et al., 2010; Ma et al., 2013). Its extremely low κL (~0.6 W/m-K at room temperature; Rosi et al., 1961) has been attributed to strong anharmonicity (Morelli et al., 2008; Nielsen et al., 2013), cation force-constant disorder (Ye et al., 2008), and also prominent nanostructural features including naturally formed nanoscale modulations and nanodomains with Ag and Sb ordering (Ma et al., 2013; Carlton et al., 2014). In our previous study, we chose AgSbTe2 as the nanoinclusion in the host matrix of micro-grained Yb0.2Co4Sb12 skutterudite. We found that the addition of AgSbTe2 nanoinclusions simultaneously optimized {ρ, α, κ} and attained much improved ZT values when the nanoinclusion weight percentage was between 4 and 6 wt% (Fu et al., 2013; Peng et al., 2014). Importantly, there is a topological crossover for the AgSbTe2 inclusions from isolated nanoparticles to nano-coating between 6 and 8 wt%. Above this crossover, the composite with high AgSbTe2 content turned to behave like a conventional two-phase composite that can be described by an effective-medium model, and the ρ and α were degraded (Peng et al., 2014).</p><p>Building on these results, we intend to further optimize the TE performance of filled-skutterudite nanocomposite. Note that AgSbTe2 can be regarded as a solid solution between Ag2Te and Sb2Te3 from the metallurgical viewpoint, multi-phase nanocomposites, and complicated nanostructures can be formed by varying Ag2Te:Sb2Te3 ratios in AgSbTe2-based materials (Wang et al., 2008; Zhang et al., 2010a,b). Hence, in the present work we extend the study into a more complicated phase space by varying the Ag2Te:Sb2Te3 ratio but fixing the overall weight percentage of (Ag2Te)x(Sb2Te3)1−x at 4 wt%, in the hopes that the increased phase interfaces may enhance the α via energy filtering effect and reduce the κL via interface scattering.</p><!><p>Yb0.25Co4Sb12 and (Ag2Te)x(Sb2Te3)1−x (x = 0.36, 0.40, 0.42, 0.46) were separately synthesized. For Yb0.25Co4Sb12, stoichiometric amounts of Co powder (99.5%), Sb shot (99.99%), and Yb ingot (99.9%) were mixed and sealed in evacuated quartz tubes, which were slowly heated to 1323 K and held for 24 h, then cooled to 923 K and held for another 4 days, before furnace-cooled to room temperature. For (Ag2Te)x(Sb2Te3)1−x, appropriate amounts of Ag powder (99.9%), Sb shot (99.99%), and Te powder (99.99%) were mixed and sealed in evacuated quartz tubes, heated to 1273 K and held for 10 h, after that cooled to 923 K and then quenched in liquid-nitrogen. To obtain Yb0.25Co4Sb12-4 wt% (Ag2Te)x(Sb2Te3)1−x composites (which will be denoted by matrix-100x hereafter), the Yb0.25Co4Sb12 ingot was pulverized manually in an agate mortar, and the (Ag2Te)x(Sb2Te3)1−x ingot was milled in a planetary mill at 400 rmp for 5 h in vacuum. The Yb0.25Co4Sb12 coarse grains and (Ag2Te)x(Sb2Te3)1−x particles were then mixed in a planetary mill at 300 rpm for 40 min in vacuum, and finally hot pressed into circular pellets at 873 K and 100 MPa for 2 h in argon atmosphere.</p><p>Phase characterization was performed by powder X-ray diffraction (PANalytical X'pert PRO diffractometer with Cu Kα radiation). Scanning electron microscopy (FEI: Quanta 200, FEI: Sirion 200), and field–emission transmission electron microscopy (FEI: Tecnai G2 F30) equipped with energy-dispersive X-ray spectroscopy (EDS) were used to inspect the microstructure. For high temperature thermoelectric property measurements, the samples were cut by diamond saw into 8 × 8 × 2 mm3 square for the thermal conductivity measurement and 12 × 2 × 3 mm3 bar for the electrical resistivity and Seebeck coefficient measurements. All the TE property measurements were carried out from 300 to 773 K. The Seebeck coefficient-electrical resistivity measurements were performed simultaneously on an Ulvac-Riko ZEM-2 system. The thermal conductivity κ was calculated via the relation κ = DCd, where the thermal diffusivity D, specific heat C were measured on a laser-flash apparatus (Shinkuriko: TC-7000H) in vacuum. The density was obtained from the measured weight and dimensions. Room temperature Hall coefficient measurement was performed on a Hall effect measurement system (Ecopia: HMS 5500) via Van der Pauw method under an applied magnetic field of 0.55 T. The carrier concentration n and Hall mobility μH were estimated from the measured Hall coefficient RH and the electrical resistivity ρ via the relations, n = 1/eRH and μH = RH/ρ, where e is the electron charge.</p><!><p>The powder X-ray diffraction pattern of Yb0.25Co4Sb12 agrees well with a skutterudite structure, with the calculated lattice parameter to be 9.0438(2) Å. The enlargement of the lattice parameter compared to CoSb3 (JCPDS: 03-065-3144, 9.0347 Å) is consistent with the Yb-filling scenario. The powder X-ray diffraction patterns of (Ag2Te)x(Sb2Te3)1−x ingots are shown in the left panel of Figure 1. As known, there is some disagreement in the literature about the pseudo-binary Ag2Te-Sb2Te3 slice of the Ag-Sb-Te phase diagram (Maier, 1963; Marin et al., 1985; Matsushita et al., 2004), indicating the phase complexity of this Ag-Sb-Te system. According to the pseudo-binary phase diagram, proposed by Marin et al. (1985), single-phase rock-salt structured AgSbTe2 falls in the range of x = 0.42–0.45 in (Ag2Te)x(Sb2Te3)1−x; when x > 0.45 Ag2Te forms in addition to the rock salt phase; and when x < 0.42 Sb2Te3 forms in addition to the rock salt phase. What we observed in the present work is somewhat different: the sample x = 0.46 is composed of rock-salt structured AgSbTe2 (JCPDS: 00-015-0540), rhombohedral AgTe3 (JCPDS: 01-076-2328), and barely discernible Ag5Te3 (JCPDS: 01-086-1168). It should be noted that the characteristic peaks of AgTe3 are very close to those of AgSbTe2. With decreasing x, the peak intensity of AgSbTe2 decreases while the characteristic peaks of single elemental Te and Sb-Te compound emerge. In the work reported by Wang et al. on (Ag2Te)x(Sb2Te3)1−x (x = 0.44-0.50) synthesized via mechanical alloying—spark plasma sintering process (Wang et al., 2008), Ag5Te3 emerged instead of Ag2Te in addition to the primary rock salt phase in (Ag2Te)0.48(Sb2Te3)0.52 and (Ag2Te)0.46(Sb2Te3)0.54. These results indicate that the phase components of ternary (Ag2Te)x(Sb2Te3)1−x system intimately depend on the synthesis process. Also shown in the right panel of Figure 1 is the XRD pattern of hot-pressed matrix-42 composite, and the other composites show similar results. The pattern indicates only the host matrix structure and no second phases associated with (Ag2Te)x(Sb2Te3)1−x are detected, which is ascribed to the low content as well as the peak broadening effect of nano-phases.</p><!><p>(Left) The powder X-ray diffraction patterns of Ag2xSb2−2xTe3−2x ingots; (Right) The X-ray diffraction pattern of hot-pressed matrix-42 composite.</p><!><p>Mechanically polished and fractured surfaces were both prepared for microstructural analysis. Figure 2 shows the back scattered electron (BSE) SEM images and EDS result of the polished samples. The secondary phases show different contrast with the matrix, as verified by EDS result, and are distributed on the grain boundaries of Yb0.25Co4Sb12 host matrix. The calculated average grain size of the host matrix by line transection method is 3 μm, consistent with the result obtained on the laser particle size analyzer. Figure 3 presents some representative fractured surface SEM images. The micro-morphology of the composites is different from that of the matrix especially on the grain boundary. A high-magnification image in Figure 3C shows nanoparticles with size of about 200 nm distributed on the grain boundaries. Furthermore, TEM observations and EDS analysis were performed on the sample matrix-42. No Ag or Te were detected inside the coarse grain but were rich on the grain boundaries (Figure 4).</p><!><p>(A) BSE image of polished matrix-42 composite; (B) BSE image of polished matrix-40 composite; the red and blue points in (A,B) correspond to the host matrix and Ag-Sb-Te secondary phase respectively, as evidenced by EDS; (C) BSE image of polished and etched matrix-40 composite; (D) EDS results corresponding to the red and blue points in (C).</p><p>The fractured surface SEM images of the studied composites: (A) Yb0.25Co4Sb12 matrix; (B) matrix-40; (C) a high-magnification image of the marked range in (B).</p><p>(A) Bright-field TEM image of sample matrix-42 and (B) the EDS results corresponding to the red and blue points in (A).</p><!><p>Figures 5, 6 present the electrical resistivity and the Seebeck coefficient of the samples from 300 to 773 K, respectively. Consistent with the previous reports (Fu et al., 2013; Peng et al., 2014), the electrical resistivity of the composites was lowered while the absolute Seebeck coefficient was enhanced upon the addition of Ag-Sb-Te second phases, which is hard to achieve in conventional single-phased bulk material. The electrical resistivity of all materials increases with increasing temperature until it reaches a maximum, typical of heavily-doped semiconductor. Table 1 lists some room temperature physical properties of the samples studied. The carrier concentration, n, of the composites is slightly increased in relative to that of the pristine sample, presumably due to Te from the (Ag2Te)x(Sb2Te3)1−x doping the Sb-site of the host matrix through diffusion in the hot pressing process. Notably, the carrier mobility μH is retained despite an increased n. This observation is somewhat unexpected because the carrier mobility is usually degraded upon nanocompositing since the interfaces scatter charge carriers (Clinger et al., 2012; Lee et al., 2012). In conjunction with the results of microstructure analysis, one possible scenario is that the Ag-Te-Sb second phases on the grain boundaries have improved the inter-grain electrical conductivity. Similar phenomena have been reported in Ba0.3Co4Sb12/Ag (Zhou et al., 2012) and also in our previously reported Yb0.2Co4Sb12-AgSbTe2 nanocomposites (Peng et al., 2014).</p><!><p>High temperature electrical resistivity of the studied materials.</p><p>High temperature Seebeck coefficient of the studied materials.</p><p>Some room temperature physical properties of the studied materials.</p><!><p>Another interesting observation is the increased absolute Seebeck coefficient. For a series of samples that possess the same scattering mechanism and approximately the same effective mass, the absolute Seebeck coefficient is expected to be inversely correlated with the carrier concentration (Ganguly et al., 2011). However, as shown in Figure 6 the absolute Seebeck coefficient is increased compared with the pristine sample despite of an increased carrier concentration. Since, Ag-Sb-Te phases are distributed on the grain boundaries of the host matrix, energy filtering mechanism is expected to contribute to the enhanced Seebeck coefficient which preferentially scatters the low energy charge carriers (Xu et al., 2010). In addition, the increment is less than in the Yb0.2Co4Sb12-AgSbTe2 system (Peng et al., 2014). For example, the absolute Seebeck coefficient of matrix-40 is increased to 171 μV/K as compared to 162 μV/K of Yb0.25Co4Sb12 at 300 K, while that of Yb0.2Co4Sb12-4wt% (Ag2Te)0.5(Sb2Te3)0.5 is increased to 202 μV/K as compared to 152 μV/K of Yb0.2Co4Sb12 (Peng et al., 2014). This is ascribed to the different structure component of Ag-Sb-Te inclusion due to Ag/Sb ratio variation. Considering the structure component of Ag-Sb-Te inclusion is different from the pseudo-binary Ag2Te-Sb2Te3 phase diagram, further investigation of the synthesis process may be required to obtain the phase diagram consistent structure component. The lowered electrical resistivity in addition to the increased absolute Seebeck coefficient gives rise to an enhancement of the power factor (Figure 7).</p><!><p>High temperature power factor of the studied materials.</p><!><p>Figure 8 presents the κ and κL of the studied materials from 300 to 773 K. The κL is estimated by subtracting the κe from the κ using the Wiedemann–Franz relation κe = L0T/ρ, with the Lorentz constant given by L0 = 2 × 10−8 V2K−2, which is suitable for heavily doped semiconductors (Goldsmid, 1986). The κ and κL were decreased systematically upon the addition of Ag-Sb-Te inclusions, and the κL decrease was more significant than that of Yb0.2Co4Sb12-AgSbTe2 system (Peng et al., 2014). For example, κL of matrix-42 shows a 39% decrease compared with Yb0.25Co4Sb12 matrix at 300 K, while that of Yb0.2Co4Sb12-4wt% (Ag2Te)0.5(Sb2Te3)0.5 shows a 27% decrease compared with Yb0.2Co4Sb12 matrix (Peng et al., 2014). Since, the detailed interactions between heat-carrying phonons and interfaces can be complex and material dependent (Cahill et al., 2003; Medlin and Snyder, 2009), it appears that perhaps the most important material parameter underlying the lattice thermal conductivity reduction is the interfacial area per unit volume (interface density) (Dresselhaus et al., 2007). From the XRD results, it is known that the (Ag2Te)x(Sb2Te3)1−x series have complicated phase components, which will generally increase phase interface density and then decrease the κL. Similar phenomena had been observed in our previously investigated In0.2+xCo4Sb12+x composites, where the high interface density via the formation of InSb/Sb eutectic mixture on the grain boundaries considerably decreased the κL (Peng et al., 2012b).</p><!><p>High temperature thermal conductivity and lattice thermal conductivity of the studied materials, the lattice thermal conductivity of Yb0.2Co4Sb12 from literature (Fu et al., 2013) is present for comparison.</p><!><p>To help quantify the impact of Ag-Sb-Te nanoinclusions on the carrier mobility and the phonon scattering, the room temperature electron mean-free-path and phonon mean-free-path have been estimated. In degenerate semiconductors with a parabolic band and acoustic phonon scattering, the Seebeck coefficient (Jeffrey Snyder and Toberer, 2008), the carrier mobility and the electron mean-free-path can be expressed by: (1)α=8π2κB23eh2m∗T(π3n)2/3 (2)μ=eτcm∗ (3)12m∗vth¯2=32κBT (4)ln=vth¯ τc=3κBTm∗•m∗μe=3κBTm∗μ2e2</p><p>Where κB, h, m*, τc, vth, and ln are the Boltzmann constant, the Planck constant, effective mass, relaxation time, average thermal velocity, and mean-free-path, respectively. Per Equation (1), the effective mass was estimated to be in the range of 1.9 to 2.1m0. Incorporating Equations (2) and (3) to (4), the electron mean-free-path was estimated and listed in Table 1. Furthermore, the phonon mean-free-path can be estimated by the following relation: (5)κl=13Cvυslp</p><p>Where Cv, υs, lp are the constant volume heat capacity, sound velocity, and phonon mean-free-path, respectively. We choose υs = 3400m/s from literature (Sales et al., 1997). The calculated phonon mean-free-path is listed in Table 1. It can be seen that the phonon mean-free-path decreases substantially while the electron counterpart slightly increases. Though the theoretical analysis of thermal conductivity (electronic conductivity) is difficult for nanocomposite with varying-size inclusions, since the wave length and mean-free-path for phonons (or electrons) at certain energy are unknown for most materials system (Liu et al., 2012), it is necessary to note a fundamental difference between the electrical and heat transport. While those electrons near the Fermi level contribute to the electrical transport, phonon modes in the entire Brillouin zone generally contribute to the heat transport. As such, a multiple length scale microstructure is generally beneficial to effectively scatter heat-carrying phonons over a wider wavelength range and also over a wide temperature range, while imposing less influence on electron transport. Even, when the average wavelength of heat-carrying phonons is getting shorter at elevated temperatures, in which case the short-range defects are more effective in scattering phonons, the presence of long-range defects such as nanoinclusions and interfaces is indispensable to effectively suppress the lattice thermal conductivity. In this context, the microstructure characteristic of the multi-phase nanocomposites distributed on the grain boundaries of micro-grained matrix in the studied composites allows for a greater tunability of {ρ, α, κ} as a group.</p><p>Finally, Figure 9 presents the dimensionless figure of merit ZT of the studied materials from 300 to 773 K. The ZT values of the composites are enhanced in the entire temperature range, due to the simultaneous enhancement of the power factor and the reduction of the κL. A maximum ZT of 1.3 was obtained in matrix-42 at 773 K, a 42% increase compared with that of the matrix.</p><!><p>High temperature dimensionless figure of merit ZT of the studied materials.</p><!><p>Yb0.25Co4Sb12-4wt% (Ag2Te)x(Sb2Te3)1−x composites have been prepared via an ex situ melting-milling-hot-pressing process. The microstructure and high temperature thermoelectric properties have been investigated and correlated. Powder XRD analysis reveals the (Ag2Te)x(Sb2Te3)1−x series have complicated phase components, which closely depend on the conditions of synthesis process. In the composites, the Ag-Sb-Te inclusions are distributed on the grain boundaries of Yb0.25Co4Sb12 coarse grains. Compared to pristine sample, the electrical conductivity and absolute Seebeck coefficient of the composites are enhanced simultaneously. In the meantime the thermal conductivity and the lattice thermal conductivity are lowered substantially upon the addition of Ag-Sb-Te inclusions. Hence we have present a case study that multi-phase nanocomposites and complicated nanostructures formed by varying Ag2Te/Sb2Te3 ratios within (Ag2Te)x(Sb2Te3)1−x materials allows for a greater tunability of {ρ, α, κ} as a group. A maximum ZT of 1.3 was obtained in matrix-42 at 773 K, a 42% increase compared with the pristine sample. The present work reveals that the three thermoelectric parameters can be optimized simultaneously in CoSb3-based nanocomposites. Further investigation to clarify the underlying mechanisms is ongoing.</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

Facile Synthesis of Ag Nanocubes of 30 to 70 nm in Edge Length with CF3COOAg as a Precursor

This paper describes a new protocol for producing Ag nanocubes of 30 to 70 nm in edge length with the use of CF3COOAg as a precursor to elemental silver. By adding a trace amount of sodium hydrosulfide (NaHS) and hydrochloric acid (HCl) into the polyol synthesis, Ag nanocubes were obtained with good quality, high reproducibility, and on a scale up to 0.19 g per batch for the 70-nm Ag nanocubes. The Ag nanocubes were found to grow in size at a controllable pace over the course of synthesis. The linear relationship between the edge length of the Ag nanocubes and the position of localized surface plasmon resonance (LSPR) peak provides a simple method for finely tuning and controlling the size of the Ag nanocubes by monitoring the UV-vis spectra of the reaction at different times.

facile_synthesis_of_ag_nanocubes_of_30_to_70_nm_in_edge_length_with_cf3cooag_as_a_precursor

Introduction<!>Results and discussion<!>Conclusion<!>Sythesis of Ag nanocubes<!>Scale-up synthesis of Ag nanocubes<!>Instrumentation

<p>In recent years, there has been a continuous, strong effort in shape-controlled synthesis of silver nanostructures because of their remarkable properties and exciting applications in areas such as photonics, electronics, catalysis, sensing, and biomedicine.[1–8] Silver nanocubes stand out from various types of Ag nanostructures[9] (e.g., spheres, rods, bars, belts and wires) due to their sharp corners/edges, uniform size, and superior performance in a range of applications involving localized surface plasmon resonance (LSPR),[10–12] surface-enhanced Raman scattering (SERS),[13–17] biosensing.[18, 19] Silver nanocubes have also been used as a sacrificial template to generate gold, palladium, and platinum nanoboxes or nanocages,[20] which have started to show great potential in catalysis,[21] contrast enhancement in biomedical imaging,[22, 23] photothermal therapy[24] and drug delivery.[25] All of these applications require us to produce Ag nanocubes in high quality and relatively large quantity. In order to meet this challenge, we and other groups have developed a number of methods to produce Ag nanocubes, with notable examples including polyol reduction,[26–28] hydrothermal process,[29, 30] epitaxial growth on Au octahedronal seeds,[31] and ATP-mediated reduction in a polymer matrix.[32] However, most of these protocols have shortcomings in terms of reaction time, size control, yield, quantity, as well as reproducibility.</p><p>In the pursuit of a superior method, we recently started to switch to a new silver precursor, CF3COOAg, instead of the AgNO3 widely used in previous work. Our argument was that the nitrate group may decompose at an elevated temperature typical of a polyol synthesis to generate ionic and/or gaseous species, making the synthesis more difficult to understand and control. In comparison, the trifluoroacetate group is more stable. With this new precursor, we found that the presence of trace amounts of NaHS and HCl were necessary for the production of high-quality Ag nanocubes with sizes ranging from 30 to 70 nm. Compared with the previously reported methods for the preparation of Ag nanocubes,[26–32] this new protocol offers at least three significant advantages: i) the reaction rate is in an appropriate range, allowing us to control and fine-tune the size of Ag nanocubes by following the synthesis with UV-vis spectroscopy; ii) the synthesis becomes essentially not sensitive to the manufacturing source of ethylene glycol (EG), improving both the robustness and reproducibility of the polyol synthesis; and iii) it is relatively simple and straightforward to scale up the synthesis to produce Ag nanocubes in relatively large quantities and with high quality.</p><!><p>Figure 1 shows transmission electron microscopy (TEM) images of the Ag nanocubes obtained at different stages of a typical synthesis: 15, 30, 60, and 90 min, respectively, after the addition of CF3COOAg. During this period of time, the edge length of the nanocubes was increased from 30 to 42, 50, and 70 nm. The relatively slow growth rate allowed us to conveniently control and thus routinely tune the size of the Ag nanocubes by quickly taking UV-vis spectra from small aliquots sampled from the reaction solution. This new capability is a significant advantage over previously reported methods. For example, with respect to the NaHS-mediated polyol synthesis, the reaction was typically completed in 8–10 min and it only took about 2 min for the Ag nanocubes to grow from 25 to 45 nm.[26, 33] This short period of time makes it very difficult to follow the increase of size using a UV-vis spectroscopic method. As a result, we could only obtain Ag nanocubes of different sizes by monitoring the color changes associated with the nanocubes as they were growing,[26, 33] which was too vague to obtain Ag nanocubes having specific sizes. On the other extreme, the HCl-mediated polyol synthesis usually took 16 to 25 h to complete,[27] making it less practical to control the size of the Ag nanocubes by constantly monitoring the changes to UV-vis spectra.</p><p>In the present synthesis, the Ag nanocubes grew from 30 to 70 nm when the reaction time increased from 15 min to 90 min. As a result, we could easily take a small amount (several drops) of the reaction solution with a glass pipette, diluted with DI water in a cuvette, followed by recording of its UV-vis extinction spectrum. All of these could be completed in 1–2 min, during which the Ag nanocubes grew very little in size. Significantly, we also found that the synthesis was sufficiently robust so that frequent removal of the stopper from the flask had a minor impact on the path and yield of the reaction. Taken together, we could finely control the sizes of resultant Ag nanocubes according to their LSPR peak positions. To better understand the relationship between the sizes and the LSPR peak positions of Ag nanocubes, we analyzed the UV-vis spectra of Ag nanocubes with different edge lengths that were obtained from the syntheses described in Figure 1. As shown in Figure 2A, the major LSPR peaks of the Ag nanocubes displayed a continuous red-shift along with the size increase. When the edge length of the Ag nanocubes was increased from 30 to 42, 50, and 70 nm, the positions of the major LSPR peaks were located at 420, 436, 449, and 474 nm, respectively. The plot in Figure 2B suggests that there was a more or less linear relationship between the LSPR peak position and the edge length of the Ag nanocubes. The equation for describing the calibration curve was λmax = 1.3927 ℓ + 378.25 (R2 = 0.99), where λmax and ℓ are the peak position and edge length, respectively. In practice, this calibration curve should allow one to obtain Ag nanocubes of a specific edge length by monitoring the UV-vis spectra of the reaction solution.</p><p>To achieve a better understanding of the growth mechanism, aliquots were also taken from the early stages (<15 min) of a standard synthesis and then analyzed by TEM and high-resolution TEM. Immediately after the addition of CF3COOAg, the solution went into a whitish color, followed by the appearance of a slight yellow color in 1 min. As shown by the TEM image in Figure 3A, there were two sizes of nanoparticles co-existing in the product sampled at t = 1 min; the big nanoparticles had an average size of 57 nm while the small ones were around 13 nm in diameter. The high-resolution TEM image taken from a small particle gave a lattice fringe spacing of 2.0 Ǻ (inset of Fig. 3A), which is consistent with the {200} lattice spacing of face-centered cubic (fcc) Ag.[33] This information suggests that some of the small particles were single crystalline and made of Ag, which could serve as seeds and eventually grow into nanocubes under the influence of poly(vinyl pyrrolidone) (PVP). As a capping agent, PVP is able to selectively bind to the {100} facets of Ag nanocrystals, favoring the formation of nanocubes when the seeds are single crystalline.[9] In the present case, the molar ratio of PVP to CF3COOAg had to be higher than 2:1 in order to obtain Ag nanocubes in high yields. Energy-dispersive X-ray spectroscopy (EDX) analysis of the big nanoparticles gave an Ag-to-Cl atomic ratio of 50:45, indicating that these particles were made of AgCl. These results suggest that the formation of AgCl nanoparticles and the birth of Ag nanocrystal seeds both occurred at t < 1 min. After this point, the color of the reaction solution started darkening from slight yellow to deep orange. As shown in Figure 3, B–F, the number and size of AgCl nanoparticles decreased with the reaction time, accompanied by an increase for Ag nanocrystals in terms of both density and size. Eventually, Ag nanocubes started to appear in the solution around t = 11 min (Figure 3E).</p><p>In order to obtain Ag nanocubes in high yields and with good quality, we optimized the synthesis by adjusting the concentrations of both NaHS and HCl. As shown in Figure 4, each column showed the reactions performed under the same concentration of NaHS; from left to right, the concentration of NaHS was doubled from 0.125 to 0.25 and 0.50 μM. Each row showed the reactions performed under the same concentration of HCl; from top to bottom, the concentration of HCl was doubled from 0.105 to 0.21 and 0.42 mM. All the reactions were stopped at t = 30 min after the introduction of CF3COOAg. It is clear that the concentration of NaHS was a less important factor relative to the concentration of HCl as Ag nanocubes of good quality could be obtained in a range of NaHS concentrations (Fig. 4, A–C). From top to bottom, the size of Ag nanocubes became larger along with the increase of HCl concentration. For both concentrations of 0.105 and 0.21 mM, we could obtain Ag nanocubes of good quality (Fig. 4, A and D). When the concentration of HCl was increased to 0.42 mM, there were also some other types of particles in the products, including bipyramids, multiply twinned nanorods, irregular particles, in addition to the Ag nanocubes. Taken together, we found that the best result was obtained when the concentrations of NaHS and HCl were 0.25 μM and 0.21 mM, which represents the standard procedure used in this paper.</p><p>As shown in Figure 4, both NaHS and HCl had to be introduced into the synthesis when CF3COOAg was employed as a precursor to Ag nanocubes. In order to investigate the roles that NaHS or HCl played in this new protocol, we performed the synthesis with different combinations of reagents. The samples were collected at t = 1 and 30 min after the introduction of CF3COOAg, and then analyzed by TEM. As shown in Figure 5A, multiply twinned nanoparticles were obtained in the sample taken at t = 1 min of the synthesis when both NaHS and HCl were absent, and these particles then agglomerated into larger particles with irregular shapes at t = 30 min (Fig. 5B). Upon the addition of NaHS (Fig. 5C), there were 28% single-crystal nanoparticles with an average size of 14 nm co-existing with multiply twinned nanoparticles in the sample taken at t = 1 min. In this case, Ag2S clusters formed in the reaction solution could serve as primary nucleation sites to catalyze the reduction of CF3COOAg and thus formation of single-crystal Ag seeds.[26] The observation of single-crystal Ag seeds suggested that NaHS played the same role as in the previous work. After 30 min into the reaction (Fig. 5D), the small nanoparticles evolved into a mixture of agglomerated nanoparticles, nanorods, and a few single-crystal nanospheres and bipyramids. If HCl instead of NaHS was present (Fig. 5E), there were some cubic nanostructures with an average size of 57 nm observed in the sample. Our EDX analysis suggested that the chemical composition of these nanocubes was AgCl, which was consistent with the result reported in the HCl-mediated synthesis of Ag nanocubes.[27] The product sampled at t = 30 min consisted of irregular nanoparticles, nanorods, and a few nanocubes (Fig. 5F). However, if CF3COOAg solution was titrated into the reaction solution at a rate of 0.07 mL per min by using syringe pump, the product mainly consisted of Ag nanocubes, together with few nanorods and other irregular nanoparticles (Fig. S1). These results suggest that both NaHS and HCl are critical to the successful synthesis of Ag nanocubes.</p><p>Our previous studies suggested that a combination of an oxidatant and a ligand could result in a powerful oxidative etchant to selectively remove multiply twinned Ag seeds from a polyol synthesis.[34] The O2 from air and Cl− ions (from NaCl or HCl) has been validated as a typical example for generating single-crystal seeds and thus Ag nanocubes.[35] In order to verify this mechanism, we performed the synthesis under argon, with other conditions being the same as the standard procedure. As shown in Figure 5G, the product sampled at t = 1 min was similar to the sample taken from the standard synthesis (Fig. 3A). However, after 30 min into the reaction under argon, the majority of the product was Ag nanocubes, together with a small amount of nanorods and irregular nanoparticles (Fig. 5H). This observation suggests that oxidative etching only played a minor role in the formation of single-crystal seeds and Ag nanocubes in the presence of NaHS. In this case, the control over product morphology was mainly provided by Ag2S seeds formed at the very beginning of a synthesis. Of course, the presence of Cl− and O2 could help to remove the small number of twinned seeds that might also form parallel to the Ag2S-mediated nucleation process. As shown in Figure 3C, there were also some twinned seeds co-existing with single-crystal ones, but the final product only consisted of single-crystal nanocubes, indicating that the oxidative etching by O2/Cl− was also involved in the synthesis conducted in air.</p><p>The synthesis was further optimized by adjusting the reaction temperature. As shown in Figure 6 A and B, the synthesis was also performed at both 130 and 170 °C with other parameters being the same as the standard procedure. At these two temperatures, we obtained a mixture of Ag nanocubes and other types of nanoparticles, suggesting that the temperature of 150 °C used for the standard procedure seemed to be the optimal condition. To distinguish the roles of proton and chloride ions, HCl was replaced by NaCl for the standard synthesis. As shown in Figure 6C, there was no significant difference between the products obtained with either HCl (standard procedure) or NaCl. This observation indicates that protons were not a necessarily critical component in this synthesis. This is not surprising because protons are supposed to be generated during polyol reduction. To evaluate the capability of this new protocol for potential high-volume production of Ag nanocubes, we performed a scalp-up synthesis which used CF3COOAg at an amount of 20 times of the standard procedure, and we could still obtain Ag nanocubes of good quality in high yield (Fig. 6D).</p><p>For most applications, it is also important to find a simple and reliable way to store Ag nanocubes with good preservation of morphology for a relatively long period of time. As shown in Figure S2, freshly prepared Ag nanocubes could be stored on a solid support (in a dry state) for three months without losing their sharp corners and edges. In comparison, the Ag nanocubes became rounded at corners and edges when they were stored in de-ionized (DI) water for three months. We also found that the Ag nanocubes stored in the dry state could be easily re-dispersed in DI water via brief sonication.</p><!><p>In summary, Ag nanocubes with edge length from 30 to 70 nm were synthesized in large quantities by introducing CF3COOAg as a new silver precursor into the polyol synthesis. It was found that both NaHS and HCl were necessary to the production of Ag nanocubes with uniform shape, narrow size distribution, and high reproducibility. The results indicated that NaHS played an important role in the formation of single-crystal seeds, and Cl− ions acted as a ligand for oxidative etching to eliminate twinned particles. The relatively growth pace of Ag nanocubes over the course of synthesis and the linear relationship between the position of the LSPR peak and the edge length of the cubes make it easy to control and finely tune the size of every batch of Ag nanocubes by monitoring the UV-vis spectra. Another advantage of this method is that the reproducibility of the reaction and the quality of Ag nanocubes were not sensitive to the manufacturing source of EG, which was usually a major problem for the polyol synthesis of Ag nanocubes.</p><!><p>In a standard synthesis, 5 mL ethylene glycol (EG, J. T. Baker, lot no. G32B27) was added into a 100-mL round bottom flask (ACE Glass) and heated under magnetic stirring in an oil bath pre-set to 150 °C. 0.06 mL NaHS (3 mM in EG, Aldrich, 02326AH) was quickly injected into the heated solution after its temperature had reached 150 °C. Two minutes later, 0.5 mL of a 3 mM HCl solution was injected into the heated reaction solution, followed by the addition of 1.25 mL of poly(vinyl pyrrolidone) (PVP, 20 mg/mL in EG, MW≈55,000, Aldrich). The HCl solution was prepared by adding 2.5 μL HCl (38% by weight) into 10.30 mL EG. After another 2 min, 0.4 mL silver trifluoroacetate (CF3COOAg, 282 mM in EG, Aldrich, 04514TH) was added into the mixture. During the entire process, the flask was capped with a glass stopper except during the addition of reagents. After the addition of CF3COOAg, the transparent reaction solution took a whitish color and quickly became slightly yellow in 1 min, indicating the formation of the Ag seeds. The reaction was allowed to proceed for different periods of time and its color went through three stages of dark red, reddish grey, and brown as the edge length of the Ag nanocubes increased. The reaction solution was quenched by placing the reaction flask in an ice-water bath. All the samples were collected by centrifugation and then washed with acetone once to remove the remaining precursor and EG, and then DI water four times to remove excess PVP. We controlled the sizes of the Ag nanocubes monitoring their main LSPR peak positions using a UV-vis spectrometer from 15 to 90 min with an interval of 15 min. Briefly, a small amount (a few drops) of the reaction solution was taken out from the flask using a glass pipette and diluted with 1 mL DI water in a cuvette, followed by recording its extinction spectrum and compared with the calibration curve for wavelength versus edge length.</p><!><p>The scale-up synthesis of Ag nanocubes was carried out by following the procedure for the standard synthesis, except that the amounts of all reagents were increased by 20 times (or other different times). For example, 100 mL EG was added into a 250-mL round bottom flask (ACE Glass) and heated under magnetic stirring in an oil bath pre-set to 150 °C. After the temperature of the oil bath had reached 150 °C, 1.2 mL of the 3 mM NaHS solution and 2 min later, 10 mL of the 3 mM HCl was injected, followed by the addition of 25 mL of the PVP solution. After another 2 min, 8 mL of the CF3COOAg solution was introduced. The flask was capped with a glass stopper except during the addition of reagents. The procedures for size control and sample collection were the same as the standard synthesis.</p><!><p>The samples for transmission electron microscopy (TEM) were prepared by dropping 1.5 μL of the aqueous suspension onto a carbon-coated copper grid. The TEM images were captured using a microscope (FEI G2 Spirit Twin) operated at 120 kV. High-resolution TEM images were obtained using a JEOL 2100F operated at 200 kV. The energy-dispersive X-ray (EDX) spectra were captured using a field emission microscope (FEI, Nova NanoSEM 230) operated at accelerating voltages of 10–20 kV. The UV-vis spectra were taken using a Cary 50 spectrophotometer (Palo Alto, CA).</p>

PubMed Author Manuscript

Fear memory impairing effects of systemic treatment with the NMDA NR2B subunit antagonist, Ro 25-6981, in mice: attenuation with ageing

N-methyl-D-aspartate receptors (NMDARs) are mediators of synaptic plasticity and learning and are implicated in the pathophysiology of neuropsychiatric disease and age-related cognitive dysfunction. NMDARs are heteromers, but the relative contribution of specific subunits to NMDAR-mediated learning is not fully understood. We characterized pre-conditioning systemic treatment of the NR2B subunit-selective antagonist Ro 25-6981 for effects on multi-trial, one-trial and low-shock Pavlovian fear conditioning in C57BL/6J mice. Ro 25-6981 was also profiled for effects on novel open field exploration, elevated plus-maze anxiety-like behavior, startle reactivity, prepulse inhibition of startle, and nociception. Three-month (adult) and 12-month old C57BL/6Tac mice were compared for Ro 25-6981 effects on multi-trial fear conditioning, and corticolimbic NR2B protein levels. Ro 25-6981 moderately impaired fear learning in the multi-trial and one-trial (but not low-shock) conditioning paradigms, but did not affect exploratory or anxiety-related behaviors or sensory functions. Memory impairing effects of Ro 25-6981 were absent in 12-month old mice, although NR2B protein levels were not significantly altered. Present data provide further evidence of the memory impairing effects of selective blockade of NR2B-containing NMDARs, and show loss of these effects with ageing. This work could ultimately have implications for elucidating the pathophysiology of learning dysfunction in neuropsychiatric disorders and ageing.

fear_memory_impairing_effects_of_systemic_treatment_with_the_nmda_nr2b_subunit_antagonist,_ro_25-698

Introduction<!>Subjects<!>Pavlovian fear conditioning<!>Memory impairing effects of Ro 25-6981 (multi-trial fear conditioning)<!>Memory impairing effects of Ro 25-6981 (one-trial fear conditioning)<!>Memory impairing effects of Ro 25-6981 (multi-trial low-shock fear conditioning)<!>Novel open field<!>Elevated plus-maze<!>Acoustic startle and prepulse inhibition of startle<!>Hot plate nociception<!>Memory impairing effects of Ro 25-6981 in 3-month and 12-month old mice<!>Corticolimbic NR2B protein levels in 3-month and 12-month old mice<!>Drugs<!>Statistical analysis<!>Memory impairing effects of Ro 25-6981 (multi-trial fear conditioning)<!>Memory impairing effects of Ro 25-6981 (one-trial fear conditioning)<!>Memory impairing effects of Ro 25-6981 (multi-trial low-shock fear conditioning)<!>Memory impairing effects of Ro 25-6981 on fear memory in 3-month and 12-month old mice<!>Corticolimbic NR2B protein levels in 3-month and 12-month old mice<!>Novel open field<!>Elevated plus-maze<!>Acoustic startle and prepulse inhibition of startle<!>Hot plate nociception<!>Discussion<!>

<p>There is compelling evidence that glutamatergic neurotransmission at N-methyl-D-aspartate receptors (NMDARs) is a major molecular mechanism underlying multiple forms of learning and memory. Activation of NMDARs initiates a cascade of molecular events that underlie synaptic plasticity which are strongly implicated in learning and memory, and NMDAR blockade prevents the induction of some forms of long-term potentiation (LTP) and long-term depression (LTD) (Bliss and Collingridge, 1993; Malenka and Bear, 2004). Behaviorally, systemic or intracerebral administration of NMDAR antagonists impairs learning and memory performance on various tasks, including Pavlovian fear conditioning (reviewed in Bannerman et al., 2006; Morris et al., 1990; Nakazawa et al., 2004). Pavlovian fear conditioning is a behavioral paradigm in which rodents learn to associate an innocuous stimulus (e.g., auditory tone) with footshock. Fear learning is impaired by pre-training systemic or intra-amygdala administration of NMDAR antagonists (e.g., D,L-AP5, MK-801/dizocilpine) probably via disruption of plastic changes at thalamo-amygdala synapses (e.g., Fanselow and Kim, 1994; Gewirtz and Davis, 1997; Lee and Kim, 1998; Walker and Davis, 2000; Walker et al., 2005).</p><p>NMDARs are heteromers composed of an obligatory NR1 subunit and at least one or more NR2 (NR2A–NR2D) subunits (Laube et al., 1998; Rosenmund et al., 1998; Schorge and Colquhoun, 2003). An important but as yet unresolved issue is the relative contribution of NMDAR subtypes to NMDAR-mediation of learning and memory. NR2A and NR2B subunits are both highly expressed in forebrain regions implicated in fear conditioning, including the amygdala, but contribute distinct physiological and molecular properties to NMDARs (Cull-Candy et al., 2001; Liu et al., 2004; Loftis and Janowsky, 2003; Perez-Otano and Ehlers, 2005; Radley et al., 2007). Sensory experience and discrimination learning increases the ratio of NR2A/NR2B and the threshold for LTP-induction (Carmignoto and Vicini, 1992; Kirkwood et al., 1996; Quinlan et al., 2004). A similar profile is seen during ontogeny where NR2B expression decreases in favor of NR2A during late postnatal development causing shortening of excitatory postsynaptic potentials (EPSCs) and increasing the threshold for LTP-induction (Hestrin, 1992; Liu et al., 2004; Lopez de Armentia and Sah, 2003). Collectively, these findings support a working model in which the NR2B facilitates NMDAR-mediated synaptic plasticity and new learning, while NR2A may support memory stabilization by preventing excessive plasticity (Quinlan et al., 2004; Tang et al., 1999).</p><p>The respective roles of the NR2A and NR2B subunits at the behavioral level have yet to be fully elucidated, in part due to a paucity of subtype selective drugs, especially for NR2A (Bartlett et al., 2007; Kash and Winder, 2007; Neyton and Paoletti, 2006). Gene knockout of NR2A has been shown to produce deficits in hippocampal LTP and impair spatial working memory, instrumental discrimination learning and fear conditioning under certain conditions (Bannerman et al., 2008; Brigman et al., 2008; Kiyama et al., 1998; Sakimura et al., 1995; Sprengel et al., 1998). On the other hand, overexpressing NR2B in the mouse forebrain leads to superior fear conditioning and extinction (Tang et al., 1999). Furthermore, pre-training systemic or intra-amygdala administration of the NR2B-selective antagonists ifenprodil or CP101,606, or genetic disruption of tyrosine-phosphorylation of NR2B, impairs fear acquisition and extinction in rats (Bauer et al., 2002; Blair et al., 2005; Dalton et al., 2007; Nakazawa et al., 2006; Rodrigues et al., 2001; Sotres-Bayon et al., 2007; Walker and Davis, 2008). Recent data also show that pre-training siRNA knockdown or selective pharmacological blockade of NR2B with Ro 25-6981 in the anterior cingulate region of the prefrontal cortex impairs the acquisition of context (but not tone) fear memory in mice (Zhao et al., 2005). Finally, global knockdown of NR2B in mice (Takehara et al., 2004) or intra-hippocampal infusion of Ro 25-6981 in rats (Valenzuela-Harrington et al., 2007) impairs trace fear conditioning but not the delay form of tone (or context) fear conditioning in mice (Zhao et al., 2005). While these data support the role of NR2B to fear learning, they do not fully address the behavioral consequences of inactivating NR2B-containing NR2B receptors. For example, while the aforementioned work of Zhao and colleagues and Valenzuela-Harrington and co-workers demonstrates fear memory impairing effects of Ro 25-6981 injected directly into specific regions of cortex and hippocampus, the consequences of systemic treatment with the drug for this behavior is unclear. This issue is salient to the potential future clinical use of Ro 25-6981 or structurally similar compounds (Danysz and Parsons, 2002; Gogas, 2006).</p><p>The existing literature also does not adequately address the question of whether the functional contribution of NR2B is altered under certain 'pathological' conditions. In this context, glutamate and NMDAR dysfunction is associated in age-related cognitive decline that is seen on various assays for learning and memory in rodents (Magnusson, 1998; Rosenzweig and Barnes, 2003). Some studies have found that age-related learning deficits are concomitant with a loss of NMDARs, prominently NR2B (Magnusson, 2001; Ontl et al., 2004). Interestingly, there is also decreased NR2B expression in Alzheimer's disease (Maragos et al., 1987; Sze et al., 2001; Wang et al., 2000). Providing preliminary evidence that these changes may be of functional importance, mice in which NR2B is transgenically overexpressed are protected against deterioration of age-related learning on tasks including cued Pavlovian fear conditioning (Cao et al., 2007). However, whether the fear memory related effects of Ro 25-6981 vary as a function of ageing has to our knowledge not been studied.</p><p>The main objective of the present study was to characterize pre-training systemic treatment of the selective NR2B antagonist Ro 25-6981 for effects on strong (multi-trial) and weaker (one-trial, low-shock) forms of Pavlovian fear conditioning in mice. There is growing evidence that NMDAR blockade exerts effects on rodent locomotor activity, anxiety-related behaviors and sensorimotor gating (Boyce-Rustay and Holmes, 2006; Cryan and Dev, 2007; Geyer et al., 2001). Therefore, we also evaluated systemic Ro 25-6981 for effects on exploratory locomotion (novel open field), anxiety-related (elevated plus-maze) and sensory (acoustic startle, sensorimotor gating, hotplate nociception) behaviors. A final objective was to test whether fear memory impairing effects of Ro 25-6981 were altered in ageing mice.</p><!><p>With the exception of the ageing experiment, subjects were 2-4-month old male C57BL/6J mice obtained from The Jackson Laboratory (Bar Harbor, ME). Because it was not possible to obtain 12-month old mice from The Jackson Laboratory, C57BL/6 mice (C57BL/6Tac) were obtained from Taconic Farms (Germantown, NY). To provide appropriate controls for the age-comparison study, younger mice used in this experiment were 3-month old counterparts obtained from Taconic Farms in the same shipment as the 12-month old mice. Mice were pair-housed in a temperature and humidity controlled vivarium under a 12 h light/dark cycle (lights on 0600 h). All experimental procedures were approved by the National Institute on Alcohol Abuse and Alcoholism Animal Care and Use Committee and strictly followed the NIH guidelines 'Using Animals in Intramural Research.'</p><!><p>The memory impairing effects of Ro 25-6981 on Pavlovian fear conditioning were tested using 3 different paradigms (in separate groups of mice). One cohort was tested on a standard multi-trial delay conditioning paradigm (Kim and Fanselow, 1992; Yang et al., 2008) (for schematic, see Figure 1A). Previous studies have shown that pharmacologic and genetic inactivation of NMDARs or other glutamate receptors can preferentially impair fear learning under one-trial and low-shock forms of conditioning (Cravens et al., 2006; Feyder et al., 2008; Nakazawa et al., 2003; Walker and Davis, 2000). Therefore, additional experiments were conducted to test the memory impairing effects of Ro 25-6981 using a one-trial delay paradigm (for schematic, see Figure 1C) and multi-trial low-shock paradigm (for schematic, see Figure 1E).</p><!><p>The procedure was similar to that previously employed in our laboratory (Hefner and Holmes, 2006). Conditioning took place in a 35 × 25 × 22 cm chamber with transparent walls and a metal rod floor. To provide a distinctive olfactory environment, the chamber was cleaned between subjects with a 79.5% water/19.5% ethanol/1% vanilla extract solution. After a 180 sec acclimation period, the mouse received 3 pairings (60–90 sec interval) between a tone (30 sec, 80 dB white noise) and footshock (2 sec, 0.6 mA scrambled footshock), in which the shock was presented during the last 2 sec of the tone. The presentation of stimuli was controlled by the Med Associates Video Freeze system (Med Associates Inc., St. Albans, VT). Twenty-four hr after conditioning, tone-recall was tested in a novel context, in a different room from training. The novel context was a square chamber or cylinder with black/white-checkered walls and a solid-Plexiglas, opaque floor, cleaned between subjects with a 1% acetic acid/99% water solution. After a 180 sec acclimation period, the tone was presented for 180 sec. Freezing in all experiments was defined as absence of any visible movement except that required for respiration, and scored at 5 sec intervals by an observer blind to experimental group. The number of observations scored as freezing were converted to a percentage ([number of freezing observations/total number of observations] × 100) for analysis.</p><!><p>Mice were conditioned and tested as for multi-trial high-shock fear conditioning with the exception that there was only 1 tone-shock pairing as opposed to 3 pairings.</p><!><p>Mice were conditioned and tested using the same procedure as described for multi-trial high-shock conditioning with the exception that footshock intensity was 0.3 mA as opposed to 0.6 mA.</p><!><p>Mice were tested on a novel open field apparatus as previously described (Wiedholz et al., 2008). The mouse was placed in the perimeter of a white Plexiglas 40 × 40 × 35 cm square arena (50 lux) under 65 dB white noise to minimize external disturbances (Sound Screen, Marpac Corporation, Rocky Point, NC), and allowed to explore for 60 min. Total distance traveled in the whole arena and time spent in the center (20 × 20 cm) was measured by the Ethovision videotracking system (Noldus Information Technology Inc., Leesburg, VA).</p><!><p>One week after novel open field testing, mice were tested on the elevated plus-maze test for anxiety-like behavior with assignment of drug doses randomized. Testing was conducted as previously described (Handley and Mithani, 1984; Holmes et al., 2000). The apparatus consisting of 2 open arms (30 × 5 cm; 90 lux) and 2 closed arms (30 × 5 × 15 cm; 20 lux) extending from a 5 × 5 cm central area and elevated 20 cm from the ground (San Diego Instruments, San Diego, CA). The walls were made from black ABS plastic and the floor from white ABS plastic. A 0.5 cm raised lip around the perimeter of the open arms prevented mice from falling off the maze. Testing was conducted under 65 dB white noise to minimize external disturbances (Sound Screen, Marpac Corporation, Rocky Point, NC). The mouse was placed in the center facing an open arm and allowed to explore the apparatus for 5 min. Time spent in the open arms, and entries into the open and closed arms was measured by the Ethovision videotracking system (Noldus Information Technology Inc., Leesburg, VA).</p><!><p>Acoustic startle and prepulse inhibition of the startle response was measured as previously described (Millstein et al., 2006). Mice were placed in a clear Plexiglas cylinder in 1 of 4 SR-Lab System startle chambers (San Diego Instruments, San Diego, CA) for a 5 min acclimation period. A 65 dB broadband background noise was delivered during acclimation and throughout testing. During the test session, mice were presented with startle trials (40 msec, 120 dB broadband sound pulse) and prepulse+startle trials (20 msec noise prepulse sound followed, 100 msec later, by a 40 msec 120 dB broadband sound pulse). The prepulse+startle trials were preceded and followed by 5 pulse alone trials, which were not included in the analyses. Test trials consisted of 10 trials of 3 different intensities (3, 6, and 12 dB above background). Each trial type was presented 10 times with a variable interval of 12–30 sec between each presentation. Basal activity in the startle chambers was measured during no-stimulus trials. Startle amplitude was measured every 1 msec, over a 65 msec period beginning at the onset of the startle stimulus. The maximum startle amplitude over the sampling period was taken as the dependent variable. Whole-body startle responses were measured via vibrations transduced into analog signals by a piezoelectric unit attached to the platform on which the cylinders rested. Prepulse inhibition of startle was calculated as 100- [(startle response for prepulse+startle trials/startle response for startle-alone trials) ×100].</p><!><p>The hot plate test apparatus was a flat plate (Columbus Instruments, Columbus, OH) heated to 55°C on which the mouse was placed (Boyce-Rustay and Holmes, 2006). The latency to show a hind paw shake or lick was timed by an observer, with a maximum response latency of 30 sec.</p><!><p>Three-month and 12-month old mice were treated with Ro 25-6981 and tested using the multi-trial high-shock fear conditioning procedure described above (for schematic of the procedure, see Figure 2A). Twelve months was chosen as an age we hypothesized to be characterized by loss of NR2B function without the marked decrement in NMDAR function and learning that would occur in older mice (Magnusson et al., 2007). At completion of the tone-recall test, the vehicle-treated mice in this group were sacrificed by rapid cervical dislocation and decapitation. Brains were removed, flash-frozen in ice-cold isopentane and stored at −80°C for Western blot analysis of NMDAR levels as described below.</p><!><p>Micropunches (Zivic Laboratories Inc., Pittsburgh, PA) were taken from medial prefrontal cortex (2.0 mm diameter punch), dorsal hippocampus (2.0 mm diameter punch) and basolateral amygdala (1.0 mm diameter punch) and were dissected on ice. Tissue was homogenized by sonication in protease and phosphatase inhibitors (Sigma protease inhibitor cocktail and phosphatase inhibitor cocktails 1 and 2, 10 μM NaF, 1% Triton-X 100, 25 mM Tris, pH 6.8) by sonication and protein concentration determined by the BCA method. Samples were diluted with 4X sample buffer (Laemmli, 1970). β-mercaptoethanol was added to a final concentration of 5% (v/v) and samples were boiled for 10 min. Twenty-five (amygdala) or 30 (hippocampus and prefrontal cortex) μg of protein was subjected to discontinuous pH 7.5% SDS-polyacrylamide gel electrophoresis (pH 8.3) with a 4% stacking gel (pH 6.8) (Laemmli, 1970) using a triple-wide electrophoresis apparatus (CBS Scientific, La Jolla, CA). Proteins were transferred to a PVDF membrane overnight at 50 mA as described for nitrocellulose membranes (Towbin et al., 1979). The blot was washed in TTBS (150 mM NaCl, 25 mM Tris, 0.05% Tween-20, pH 7.3) and blocked for 1 hr in 5% non-fat powdered milk. The NR2B antibody (Chemicon/Millipore/Upstate, catalogue # AB1557P) was used at 1:1000. The blot was incubated overnight in primary antibody, washed 3 times in TTBS, and then incubated in HRP-conjugated goat anti-rabbit (Pierce, Rockford, IL) for 1 hr. Following 3 washes, immunoreactivity was detected using SuperSignal West Dura chemiluminescence detection reagent and collected using a Kodak Image Station 4000.</p><!><p>Ro 25-6981 (R-(R*,S*)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidine propranolol hydrochloride) (Tocris Cookson, Ellisville, MO) was prepared in a 0.9% saline vehicle and injected intraperitoneally in a volume of 10 mL/kg body weight. The doses and treatment-to-test interval for Ro 25-6981 were chosen on the basis of a previous study showing that i.p. injection of 10 mg/kg Ro 25-6981 30 min prior to conditioning impaired trace eyeblink conditioning in rats (Valenzuela-Harrington et al., 2007) and subcutaneous injection of 5 mg/kg Ro 25-6981 30 min prior to testing impaired spatial reversal learning in C57BL/6J mice (Duffy et al., 2008).</p><!><p>The effects of Ro 25-6981 treatment, age and brain region were analyzed by use of analysis of variance (ANOVA) and Fisher's LSD post hoc tests, using StatView (SAS Institute, Inc., Cary, NC). Mice that had tone recall freezing scores standard 2 deviations different from the grand mean were identified as statistical outliers and removed. Statistical significance was set at p<.05.</p><!><p>During conditioning, baseline freezing did not differ between treatment groups (0 mg/kg=1.1 ±1.1%, 1 mg/kg=0.0 ±0.0, 3 mg/kg=0.0 ±0. 0, 10 mg/kg=0.2 ±0.2). There was no significant effect of Ro 25-6981 on freezing to the final tone (0 mg/kg=54.5 ±7.4%, 1 mg/kg=50.6 ±6.9, 3 mg/kg=50.0 ±4.1, 10 mg/kg=42.5 ±6.6). During testing, baseline freezing did not differ between treatment groups (data not shown), while there was a significant effect of Ro 25-6981 treatment on freezing to tone (F3,63=3.13, p=0.03). Post hoc analysis showed that mice treated with 3 or 10 mg/kg Ro 25-6981 showed significantly less freezing than vehicle-treated controls (Figure 1B). These results demonstrate that pre-conditioning Ro 25-6981 treatment impaired fear recall in a multi-trial paradigm.</p><!><p>During conditioning, Ro 25-6981 treatment had no effect on baseline freezing (0 mg/kg=0.0 ±0.0%, 1 mg/kg=0.2 ±0.2, 3 mg/kg=0.0 ±0.0, 10 mg/kg=0.40 ±0.40). As expected, freezing to the single (unconditioned at the time of presentation) tone in this paradigm was minimal and not different between doses (0 mg/kg=2.9 ±2.9%, 1 mg/kg=1.5 ±1.5, 3 mg/kg=1.4 ±1.4, 10 mg/kg=4.3 ±3.1). During testing, baseline freezing did not differ between treatment groups (data not shown). Ro 25-6981 had a significant effect on freezing to tone (F3,51=2.83, p=.05). Post hoc analysis showed that 10 mg/kg Ro 25-6981 significantly reduced freezing relative to vehicle (Figure 1D). These data demonstrate that pre-conditioning Ro 25-6981 treatment also impaired fear recall in a one-trial paradigm.</p><!><p>During conditioning, Ro 25-6981 treatment had no affect on baseline freezing (0 mg/kg=0.0 ±0.0%, 1 mg/kg=0.4 ±0.4, 3 mg/kg=0.9 ±0.2, 10 mg/kg=0.4 ±0.2). There was a significant effect of Ro 25-6981 on freezing to the final tone (F3,58=3.94, p<.01), due to higher freezing in mice treated with 3 mg/kg relative to vehicle (p<.05) (vehicle=26.7 ±6.9%, 1 mg/kg=36.3 ±6.6, 3 mg/kg=45.3 ±5.3, 10 mg/kg=17.5 ±5.1). During testing, baseline freezing did not differ between treatment groups (data not shown). There was no significant main effect of Ro 25-6981 on freezing during tone recall when all doses were included in the analysis (F3,58=1.76, p=.16). However, planned post hoc comparisons indicated significantly lesser freezing at the 10 mg/kg dose relative to vehicle (Figure 1F). These results indicate that pre-conditioning Ro 25-6981 treatment impaired fear recall in a low-shock paradigm.</p><!><p>During conditioning, there was no effect of Ro 25-6981 or age on baseline freezing (3-month olds, 0 mg/kg=0.0 ±0.0%, 1 mg/kg=0.0 ±0.0, 3 mg/kg=0.0 ±0.0, 10 mg/kg=0.0 ±0.0, 12-month olds, 0 mg/kg=0.0 ±0.0%, 1 mg/kg=0.3 ±0.3, 3 mg/kg=0.0 ±0.0, 10 mg/kg=0.3 ±0.3). There was a significant Ro 25-6981 × age interaction for freezing to the final tone (F3,68=3.01, p=.04). Post hoc analysis showed that 10 mg/kg Ro 25-6981 significantly reduced freezing in 3-month, but not 12-month, old mice (p<.01) (3-month olds, 0 mg/kg=54.0 ±8.5%, 1 mg/kg=58.0 ±9.6, 3 mg/kg=57.5 ±7.0, 10 mg/kg=13.3 ±5.8, 12-month olds, 0 mg/kg=42.0 ±7.6, 1 mg/kg=52.0 ±8.5, 3 mg/kg=44.4 ±6.5, 10 mg/kg=42.0 ±8.7).</p><p>During testing, there was significant effect of age (F3,68=5.17, p=.03) but not Ro 25-6981 on baseline freezing. Post hoc analysis collapsed across dose showed that 12-month old mice generally froze more than 3-month old mice, although levels of freezing were actually very low in both groups (3-month old= 2.79 ±0.74%, 12-month old= 5.72 ±1.08%). There was a significant interaction between age and Ro 25-6981 treatment for freezing during tone recall (F3,68=3.01, p=.04). Post hoc tests showed that 10 mg/kg Ro 25-6981 reduced freezing relative to vehicle in 3-month, but not 12-month, old mice (Figure 2B). Freezing in vehicle-treated 3-month and 12-month old mice did not differ. These findings show that the memory impairing effects of preconditioning Ro 25-6981 treatment in a multi-trial paradigm were lost in 12-month old mice.</p><!><p>There was no significant main effect of region or age and no region × age interaction for NR2B levels (Table 1). Thus, loss of sensitivity to the memory impairing effects of Ro 25-6981 in 12-month old mice was not associated with significant loss of NR2B protein levels in various regions mediating this behavior.</p><!><p>Ro 25-6981 treatment had a significant effect on total distance traveled (F3,36=3.41, p<.03). However, post hoc analysis found no significant effect of any dose of Ro 25-6981 relative to vehicle (Figure 3A). Drug treatment significantly affected percent center time (F3,36=9.51, p<.01). Post hoc tests indicated significantly lesser center time in mice treated with 10 mg/kg Ro 25-6981 as compared to vehicle controls (Figure 3B). These results demonstrate that Ro 25-6981 treatment affected a measure of anxiety-like behavior but not exploratory locomotion in a novel open field.</p><!><p>Ro 25-6981 treatment did not significantly affect time spent in the open arm, center square or closed arms (Figure 3C), or the number of open, closed or total arm entries (Figure 3D). Thus, Ro 25-6981 treatment did not alter anxiety-like behavior in this assay.</p><!><p>Ro 25-6981 treatment had no effect on acoustic startle amplitude (Figure 4A) or baseline movement (data not shown). There was a significant effect of prepulse intensity (F2,60=265.82, p<.01) but no effect of Ro 25-6981 treatment and no drug × prepulse intensity interaction for percent prepulse inhibition (Figure 4B). These data show that Ro 25-6981 treatment did not affect startle or sensorimotor gating.</p><!><p>Ro 25-6981 treatment had no effect on latency to show a pain response in the hot plate test (Figure 4C), demonstrating that treatment did not affect this measure of nociception.</p><!><p>The NR2B subunit has emerged as a potential therapeutic target for a variety of neuropsychiatric and neurological conditions, including Alzheimer's and Huntingdon's disease, schizophrenia, and mood and anxiety disorders (Cryan and Dev, 2008; Danysz and Parsons, 2002; Gogas, 2006). The main findings of the present study were that systemic administration of the selective NR2B antagonist, Ro 25-6981, impaired the acquisition of fear memory in mice, and that this effect was modified by ageing.</p><p>The potential effects of systemically administered Ro 25-6981 on behaviors associated with NMDAR function, including locomotor exploration, anxiety-like behavior and sensorimotor gating, have not been well characterized in either rats or mice. At the dose range currently tested, Ro 25-6981 treatment had minimal effects on spontaneous locomotor exploration/activity (consonant with data in rats obtained by Kosowski and Liljequist, 2004) or anxiety-like behavior in the elevated plus-maze. Ro 25-6981 treatment also failed to alter responses on the hot plate assay, suggesting that anti-nociceptive actions were unlikely to account for drug effects on fear conditioning. In addition, we observed no effects of Ro 25-6981 treatment on acoustic startle reactivity or prepulse inhibition of the startle response, which is consistent with a previous study in rats which found that another NR2B selective antagonist, Ro 63-1908, failed to alter acoustic startle reactivity or sensorimotor gating at doses that impaired cognition (Higgins et al., 2003). The only significant effect currently observed was a decrease in center exploration in a novel open field at the highest dose tested, which may be indicative of an anxiety-like response to the drug (Cryan and Holmes, 2005) However, as no effect was seen in a second test for anxiety-related behavior, the elevated plus-maze, any effect on anxiety-like behavior does not appear to be robust. Indeed certain other NR2B-selective antagonists, including ifenprodil, have also failed to produce anxiety-related activity in mice (Dere et al., 2003). This provides an interesting contrast with the anxiolytic-like profile of subunit non-selective NMDAR antagonists (reviewed in Cryan and Dev, 2007) and gene knockout of the NR2A subunit (Boyce-Rustay and Holmes, 2006). Nonetheless, further studies will be needed to fully characterize the effects of Ro 25-6981 for potential anxiety-related effects, as well as sensorimotor gating and nociception, for example by using alternate assays or testing intracerebral region-specific injections. Notwithstanding, in the context of the present study, the absence of systemic Ro 25-6981 activity on these behaviors serves to exclude some of the potentially confounding effects on fear learning.</p><p>On a standard multi-trial delay cued fear conditioning paradigm, pre-training Ro 25-6981 treatment produced a significant deficit in tone recall measured twenty-four hours later. While a pre-training treatment experimental design does not dissociate drug effects on acquisition versus post-conditioning consolidation, the finding that Ro 25-6981 reduced freezing to the final tone presentation during conditioning is consistent with an impairment of fear acquisition rather than consolidation. Further supporting a selective effect of NR2B antagonism on fear acquisition, pre-training but not pre-recall intra-amygdala injection of the selective antagonist CP101,606 impaired subsequent fear recall in rats (Walker and Davis, 2008), while another study in rats found that systemic Ro 25-6981 injection prior to fear recall disrupted within-session extinction but also failed to affect fear recall (Dalton et al., 2007).</p><p>It was notable that Ro 25-6981's impairing effects on multi-trial conditioning were quite modest. It does not however appear that this is due to a failure to overcome a strong fear response produced by repeated tone × high-shock pairings in a multi-trial paradigm. This is because similarly modest effects were observed in the putatively less fear intensive low-shock multi-trial paradigm and 1-trial cued fear conditioning paradigms; both of which generally produced lower levels of fear than the multi-trial high-shock paradigm. Thus, the magnitude of the fear learning deficit produced by Ro 25-6981 is similar across strong and weak fear learning conditions, and thereby argues against a differential recruitment of NR2B as a function of conditioning strength. The alternative, and currently most parsimonious, interpretation is that selective blockade of NR2B-containing NMDARs does not disrupt fear learning to the same extent as more widespread blockade of NMDARs (at least when NR2B antagonists are delivered systemically). Another possibility is that because NR2B antagonists are more potent at diheteromeric than triheteromeric NR2B-containing NMDARs (Hatton and Paoletti, 2005; Kash and Winder, 2007), a proportion of NR2B-containing NMDARs could be relatively resistant to the memory impairing effects of Ro 25-6981.</p><p>The lateral amygdala is the most likely site of the fear impairing memory impairing effects of systemically administered Ro 25-6981. The lateral amygdala is the principle brain region mediating cued fear acquisition (Fanselow and Poulos, 2005; LeDoux, 2000; Maren and Quirk, 2004), and NR2B is expressed on the majority of thalamo-amygdala dendritic spines (Radley et al., 2007). Antagonism of NR2B-containing NMDARs via ifenprodil or genetic disruption of NR2B tyrosine phosphorylation disrupts synaptic plasticity in the basolateral amygdala (Bauer et al., 2002; Li et al., 1995; Nakazawa et al., 2006; Weisskopf and LeDoux, 1999). Furthermore, intra-amygdala injection of the NR2B antagonists ifenprodil or CP101,606 is sufficient to impair fear conditioning in rats while, in contrast, pre-training siRNA knockdown or selective pharmacological blockade of NR2B with Ro 25-6981 in the anterior cingulate impairs the acquisition of context (but not cued) fear memory and intra-hippocampal infusion of Ro 25-6981 impairs trace but not delay cued fear conditioning (Blair et al., 2005; Rodrigues et al., 2001; Sotres-Bayon et al., 2007; Valenzuela-Harrington et al., 2007; Walker and Davis, 2008; Zhao et al., 2005). However, given the modest fear memory impairing effects of Ro 25-6981 in the current study, it is noteworthy that the aforementioned memory impairing effects of pre-training intra-amygdala CP101,606 which, as with Ro 25-6981, is more 2B-selective than ifenprodil (Kash and Winder, 2007), were only evident within a narrow dose range (Walker and Davis, 2008).</p><p>Nonetheless, present data provide novel evidence that a NR2B-mediated component of fear learning may be compromised with ageing. The specific finding was that twelve month old C57BL/6Tac mice showed good basal fear, similar to that of three month old C57BL/6Tac counterparts but, unlike the younger mice, were resistant to the fear memory impairing effects of Ro 25-6981. We chose to examine the memory impairing effects of Ro 25-6981 at twelve months because rodents are not yet considered 'aged' and likely to exhibit global cognitive deficits (Barnes et al., 1997; Clayton et al., 2002; Magnusson et al., 2007). Rather twelve months perhaps more closely approximate to 'middle age' in humans when more subtle age-related changes begin to manifest. It should be made clear that for reasons of availability C57BL/6Tac mice were used for this ageing experiment rather than the C57BL/6J line used in the other experiments. However, vehicle-treated mice from the two lines showed very similar levels of fear (i.e., forty-eight percent freezing for C57BL/6Tac, forty-nine percent freezing for C57BL/6J). In addition, the three month old mice in this experiment were also C57BL/6Tac and showed a clear fear recall deficit at the highest dose of Ro 25-6981, replicating the effect of this dose in C57BL/6J tested across conditioning paradigms. Thus, the loss of the drug's efficacy in the twelve month old C57BL/6Tac mice appears to be a genuine effect of ageing rather than an idiosyncrasy of this line of C57BL/6 mice. Nonetheless, a final point to bear in mind is that our data do not demonstrate that ageing is only associated with a loss of the fear impairing effects of Ro 25-6981 and cannot not exclude the possibility that other positive (and as yet to determined) effects of Ro 25-6981 are not also diminished in older mice.</p><p>Magnusson and colleagues' work demonstrating that loss of NR2B at the protein expression level does not manifest until fifteen months in the frontal cortex of C57BL/6Nia mice and even later in hippocampus (Magnusson, 2000, 2001; Magnusson et al., 2002; Magnusson et al., 2007; Ontl et al., 2004). Consistent with their data, we found no evidence of reduced NR2B protein levels in the amygdala, dorsal hippocampus or medial prefrontal cortex of the twelve month old C57BL/6Tac mice. This suggests that the loss of sensitivity to Ro 25-6981 reflects functional alterations preceding the loss of protein itself, perhaps involving improper synaptic targeting or coupling to downstream signaling mechanisms (Malenka and Bear, 2004). Another, and not necessarily exclusive, possibility is that the ratio of NR2B to NR2A, at the protein and/or functional level, changes with ageing having the effect of reducing the importance of NR2B relative to NR2A for fear memory formation. These mechanisms await elucidation but could provide insight into how NMDAR-mediated learning processes are dynamically regulated with ageing.</p><p>In summary, the present study provides further evidence that selective blockade of NR2B-containing NMDARs is sufficient to impair the acquisition of conditioned fear behavior. This effect was demonstrated using various conditioning procedures, but was overall quite modest. The same dose range of Ro 25-6981 had minimal effects on locomotor exploration, anxiety-like behavior, nociception, or sensorimotor gating. Ro 25-6981's learning impairing effects were also absent in twelve month old mice. These data further support a role for NR2B-containing NMDARs in fear learning and suggest that this role may diminish with ageing. Further studies along these lines could ultimately have implications for understanding the contribution of NMDAR to the pathophysiology and treatment not only of fear-related neuropsychiatric conditions such as post-traumatic stress disorder, but also other disease states in which NMDARs are implicated including age-related cognitive dysfunction and Alzheimer's disease.</p><!><p>Fear memory impairing effects of Ro 25-6981 on multi-trial, one-trial and low-lock shock fear conditioning. (A) Schematic of the multi-trial high shock protocol. (B) Pre-training treatment with 10 mg/kg Ro 25-6981 reduced fear recall relative to vehicle (0) (n=16–18/dose). (C) Schematic of the one-trial high-shock conditioning protocol. (D) Pre-training treatment with 10 mg/kg Ro 25-6981 reduced fear recall relative to vehicle (0) (n=13–14/dose). (E) Schematic of multi-trial low-shock protocol. (F) Pre-training treatment with 10 mg/kg Ro 25-6981 reduced fear recall relative to vehicle (0) (n=15–16/dose). **p<.01, *p<.05 vs. vehicle (0). Data in Figures 1–4 are Means ±SEM.</p><p>Fear memory impairing effects of Ro 25-6981 on multi-trial high-shock fear conditioning as a function of ageing (A) Schematic of the multi-trial high-shock conditioning protocol. (B) Pre-training treatment with 10 mg/kg Ro 25-6981 significantly reduced fear recall relative to vehicle (0) in 3-month but not 12-month old mice (n=8–10/dose/age). **p<.01 vs. 0 (vehicle).</p><p>Effects of Ro 25-6981 on exploratory locomotion and anxiety-like behavior. In the novel open field, Ro 25-6981 did not alter total distance traveled (A), while the highest dose decreased percent center time (B) (n=10/dose). In the elevated plus-maze, Ro 25-6981 did not alter time spent in the open arms, center square or closed arms (C), or open, closed or total arm entries (D) (n=11/dose). **p<.01 vs. vehicle (0).</p><p>Effects of Ro 25-6981 on sensory functions. Ro 25-6981 did not alter the startle response (A), prepulse inhibition of startle (B) (n=10–12/dose) or hotplate nociception (C) (n=7–10/dose). PP=prepulse intensity level above 65 dB background noise.</p><p>NR2B protein levels in amygdala, hippocampus and medial prefrontal cortex of 3-month old and 12-month old mice. Data are mean ±SEM net intensity of immunoblots. n=6–9/region/age group.</p>

PubMed Author Manuscript

Algae biodiesel - a feasibility report

BackgroundAlgae biofuels have been studied numerous times including the Aquatic Species program in 1978 in the U.S., smaller laboratory research projects and private programs.ResultsUsing Molina Grima 2003 and Department of Energy figures, captial costs and operating costs of the closed systems and open systems were estimated. Cost per gallon of conservative estimates yielded $1,292.05 and $114.94 for closed and open ponds respectively. Contingency scenarios were generated in which cost per gallon of closed system biofuels would reach $17.54 under the generous conditions of 60% yield, 50% reduction in the capital costs and 50% hexane recovery. Price per gallon of open system produced fuel could reach $1.94 under generous assumptions of 30% yield and $0.2/kg CO2.ConclusionsCurrent subsidies could allow biodiesel to be produced economically under the generous conditions specified by the model.

algae_biodiesel_-_a_feasibility_report

Background<!><!>Background<!><!>Background<!><!>Background<!>Current research review<!>Algae research in the public sector<!><!>Laboratory studies on algae<!>Collection and classification<!>Biochemistry and physiology<!>Molecular biology and genetic engineering<!><!>Molecular biology and genetic engineering<!>Outdoor studies on algae<!>Algae pond studies<!>Resource assessment<!>System analysis<!>Algae research in the private sector<!>Process overview<!><!>Process overview<!>Species of algae<!><!>Open ponds<!><!>Water resources<!>Carbonation<!>Mixing systems<!>Other factors<!>Photobioreactors<!>Harvesting biomass<!>Generating biodiesel<!><!>Generating biodiesel<!>Current and future research directions<!>Economic feasibility assessment<!><!>Economic feasibility assessment<!><!>Economic feasibility assessment<!><!>Economic feasibility assessment<!><!>Economic feasibility assessment<!><!>Current policy<!>Renewable fuel standard<!>Biodiesel tax credit<!>Small Agri-Biodiesel Producer Credit<!>Biorefinery assistance<!>Bioenergy program for advanced biofuels<!>Import duty for fuel ethanol<!>Conclusions<!><!>Conclusions<!><!>Competing interests<!>Authors' contributions<!>Acknowledgements

<p>Due to concerns about high or unpredictable energy prices, the uncertain continued availability of fossil fuels, and the desire to derive energy from sources not under the control of hostile nations, the United States has long supported the production of biofuels through various incentive programs. Beginning with the passage of the Energy Tax Act in 1978, which provided a 100% gasoline tax exemption for alcohol fuel blends [1], the United States' policy has been greatly in favor of incentivizing the expansion of the use of biofuels. There are several reasons that biofuels are even more viable now than at any time in the past several decades. First, oil prices are significantly higher now than they were in the past and are not likely to fall to those low levels again. Biofuels are always seen as a more attractive option whenever fuel prices rise. Therefore, research into biofuels could be more cost-effective now, in an age of higher gas prices.</p><p>Second, though clean energy and environmentalism were concerns in the nineties, they are much more prominent on the nation's policy agenda in the present. Fears regarding global warming and related potential environmental catastrophes have made the government much more open to considering expensive policy options with positive environmental externalities. Since environmental concerns are being weighted with much more importance today, biofuels are much more attractive now, especially when created from a feedstock that avoids the environmental detriments of large-scale farming.</p><p>Third, energy independence is more important to the U.S. government today than it was back in the nineties. Now, with the wars in Iraq and Afghanistan, a loss of progress in the Arab-Israeli conflict, and increased fears of terrorism as a result of the September 11th attacks, any energy policy that can make the United States self-sufficient, i.e. not having to rely on such an unstable region for fuel, will be much more popular. Since biofuel is entirely a domestic product, it fits these criteria quite well.</p><p>Finally, the current recession may be an important impetus to investment in projects like production facilities for new types of biofuel. Much has been made of the importance of "shovel-ready" projects such as public works improvements for combating the recession. Indeed, the American Recovery and Reinvestment Act of 2009 earmarks over $61 billion for energy generation, efficiency improvements, and general research, including $800 million for projects specifically related to biomass [2]. It is clear that the government is currently interested in programs like the development of biofuel production capabilities as a way to stimulate domestic investment as well as to improve fuel generation and efficiency.</p><p>Despite these benefits, however, the time is not necessarily right for just any type of biofuel. There are many types of biofuels currently being researched and produced. Ethanol, biodiesel, and other oil-based fuels exist that can be either used directly in vehicles or that can be used after engine modifications or in blends with petro-fuels. We are choosing to look at biodiesel for several reasons. First, biodiesel can be used directly in diesel engines, whereas ethanol must be mixed with regular gasoline in order to work in gasoline engines (except those specially modified for ethanol only). Second, biodiesel takes less energy to make than petrodiesel does, making its net energy produced higher, even though the outputs of petro- and biodiesel are similar. Biodiesel also has lower emission rates of certain pollutants, such as SOX, CO, and particulate matter. Biodiesel eliminates tailpipe emissions of SOX completely [3]. Most importantly, biodiesel is renewable, and we can control its production levels and methods in a variety of ways to ensure the desired outcomes. As petroleum gets harder to extract from the earth and therefore more costly, biodiesels will remain as cost-efficient as the processes required to produce them, and these processes can change with new technology.</p><p>While diesel and gasoline engines are quite similar, the differences are important. Diesel fuel will self-ignite when pressurized in the cylinder, whereas gasoline needs a spark from a spark plug to combust. Diesel fuel also has more carbon atoms per molecule than gasoline, thus the energy density of diesel is greater than that of gasoline (Table 1). Diesel engines are relatively more efficient than gasoline engines as well, though they are required to work at higher temperatures, so some energy is lost to heat. In order to make a regular diesel engine run on biodiesel, no conversion is necessary. This goes for all blends, from B2 (2% biodiesel, 98% conventional diesel) all the way to B100 (100% biodiesel). One small concern is that in cold weather (temperatures below 30 degrees F) biodiesel viscosity will increase, blocking fuel lines. This problem can be solved by mixing in additives, such as a higher percentage of petrodiesel, or by installing heaters for the fuel lines.</p><!><p>Average density and heating values of biodiesel and diesel fuels</p><!><p>Biodiesel is more environmentally friendly than petrodiesel in many respects. One thing that biodiesel improves over petrodiesel is lubrication ability. In petrodiesel, environmental regulations require reduced sulfur content, but this sulfur was needed to increase lubrication. Biodiesel, however, does not need sulfur for lubrication and therefore is better for the environment in that regard [4]. Biodiesel vehicles also have significantly lower emissions when compared to standard diesel vehicles (Table 2).</p><!><p>Engine emission results, in % difference from no. 2 diesel [4]</p><!><p>Biodiesel not only burns more cleanly, but it may also have the advantage of being cleaner in its production process, depending on how one produces it. Recycled vegetable oil, for example, is an extremely clean feedstock for biodiesel because it has already been produced and used for other things. Relative to the production processes for these oils, the conversion costs tend to be slim or even negligible by comparison. Other feedstocks, however, are not as clean to make, and some may even counter-productively use more resources and release more carbon than petroleum-based fuel.</p><p>With all this in mind, we have chosen to consider not the broad category of biodiesels but rather the much more specific subcategory of algae fuel. There are many possible biofuel feedstocks, and there are several that are currently getting much more funding and attention than algae, but we chose algae because it seems to be the best hope for producing a fuel that might one day be cost competitive with petroleum fuels. The major feedstocks currently being used to produce oil for biodiesel are corn, soybean, rapeseed, yellow grease, and oil palm. Algae has the capability of yielding many times as much oil as the other feedstocks per unit of growing area, and that corn and soybeans are especially inefficient in this respect (Figure 1).</p><!><p>Oil Yields of Feedstocks for Biofuel. Numbers sourced from [20].</p><!><p>Algae biomass has the potential to grow yields far higher than any other feedstock currently being used. It has the possibility of a much higher energy yield per unit, so it can be much more efficient. However, little funding is being put into algae research currently. The main feedstocks being grown for biodiesel now are seeds, such as corn, soybean, rapeseed, and peanut. Yellow grease, which is used animal and vegetable fats (restaurant cooking oil and other fats) is also a very popular feedstock for biodiesel. As with other recycled oils, yellow grease biodiesel production is low cost, but it will not scale up to larger production, and thus production levels cannot be maintained as demand increases since it is recycled from other places where demand is not increasing.</p><p>The problem with the virgin oil feedstocks (not recycled) that are currently being put forward as good stocks for biodiesel production is that they are all farming-intensive. Plants such as corn and soy must be fertilized, irrigated, and maintained, and all those processes use up valuable resources, create soil erosion problems, and emit greenhouse gasses, as well as polluting in other ways (nitrogen runoff into water sources, for example). Some scientists claim that growing corn to make biofuels is more carbon-intensive than simply using petro-fuels instead [5], though this is widely disputed and depends on how one accounts for the costs of farming and fertilizer production. Another problem is that using food crops for biofuel increases the price of the food crop, which can lead to higher world food prices as we saw in 2007 [6]. While corn has gotten most of the bad press for being a very energy- and water-intensive crop to crow, all these farmed feedstocks have similar problems regarding energy and cost inputs and outputs.</p><p>Algae, on the other hand, can be easy to grow, and it does not require additional fertilizers or pesticides like many farmed crops do. It simply requires CO2 and sunlight to grow. It can be grown in grey water or wastewater, and in fact nitrogen-rich waste ponds are some of the better places to grow algae. It also can be grown on marginal land, so it does not take away from land used in farming for food. Algae gets its energy from the sun, so as with farmed crops, the energy output from algae biofuels does not require the direct input of other forms of chemical energy. Furthermore, the carbon released through biodiesel combustion was initially fixed from CO2 gas through photosynthesis. Thus, algae biodiesel is carbon neutral. In addition, algae have a much higher oil yield than any other feedstock currently being researched. Algae also have potential added benefits that have not yet been researched fully. One of these is the possibility of selling carbon credits or buying emitted CO2 from factories, further reducing overall greenhouse gas emissions. Another possible benefit could be selling the leftover nutrient-rich biomass from the algae to animal-raising farms as feed for livestock, as well as burning the leftover biomass for electricity to power the facility itself or to sell back to the grid.</p><p>Many scientists have recognized the problems with all these feedstocks for biofuel and have looked to genetically-engineered bacteria as the solution. Such bacteria, E. coli for example, can multiply much faster than any plant or algae, and they can be facilely engineered to produce precisely what the scientist wants. This solution seems like the ideal one for biofuel production. However, many have pointed out that bacteria merely reassemble current chemical energy sources at an energy cost, whereas algae harness solar energy and CO2 that would have otherwise been unused.</p><p>In the body of our paper, we will provide an overview of algae-related research up to the present time and then explain the scientific processes of producing biodiesel from algae. We will then conduct an economic feasibility analysis, taking into account public policy measures that could change the costs of production. Our conclusions will follow, showing the possible scenarios in which algae biodiesel could become cost-competitive as well as the scenarios in which it is not.</p><!><p>Research on algae fuel has been limited in the past, although the fluctuation in oil prices has ignited renewed interest in algal biodiesel for its high oil yield. Most research into the efficiency of algal-oil production continues to be done in the private sector, while government emphasis on algae research varies from country to country. For this paper, we will focus on one of the largest public funded program dedicated to algae research in the United States.</p><!><p>In the United States, the earliest government funded research on algae began in 1978 under the Carter administration. It was known as the Aquatic Species Program (ASP) and was funded by the Department of Energy (DOE) under the Office of Fuels Development. The ASP was just one component of the larger Biofuel Program under the DOE that aims to develop alternative sources of energy domestically in the United States, and its report was completed in 1998 [7]. Prior to 1980, the ASP started out focusing on using algae to produce hydrogen, but the DOE gradually shifted its emphasis on technologies that could have a large-scale impact on national energy consumption after 1980 and therefore prompted the ASP to focus on algae's ability to produce biodiesel. The ASP can be divided into two components of research – laboratory studies and outdoor studies. While the laboratory studies are involved with investigating algae's composition and oil yield, outdoor studies are concerned about testing large-scale systems and analyzing the cost-efficiency. Both components of research were carried on concurrently during the eighteen years of the program and build on each other's findings in the process. A summary of the research timeline of the ASP is located in Table 3.</p><!><p>Timeline for the aquatic species program</p><!><p>Within the laboratory studies, research is generally broken down into three types of activities: 1) collection, screening and classification of algae, 2) biochemical and physiological studies on lipid production, and 3) molecular biology and genetic engineering. The logical order of the three activities is very important to laboratory studies. Scientists are required to first gather a substantial amount of information on algae through collection and classification. Then, once adequate information is gathered, research can focus on oil production through understanding the biochemistry and physiology of algae. A natural next step is therefore to use such knowledge to genetically manipulate the metabolism of algae to improve its oil production.</p><!><p>Due to the large diversity of algae population, researchers were first interested in finding the algae that produced the most oil, has the fastest growth rate, and can grow under severe conditions such as extreme heat, pH, or salinity. Therefore, a large-scale operation took place from 1980 to 1987 dedicated to the gathering and screening of algae species. Collection first began in western Colorado, New Mexico, and Utah because it was believed that these harsh habitats will produce algae strains that can adapt to extreme environmental conditions. Subcontractors of the program were paid to collect algae strains from southeast regions such as Florida, Mississippi, and Alabama. Universities also joined the early collection efforts and collected large quantities of algae strains from various regions of the continental U.S. as well as Hawaii. By 1987, the collection consists of over 3,000 species of algae. The classification process began as soon as new strains enter the laboratories around the country and the resulting database was unprecedented in scale and serves a strong foundation for future research into algae.</p><!><p>It quickly became apparent that no one single species was going to meet all the qualifications the ASP envisioned. Therefore, the research switched gear and concentrated on studying the biochemistry and physiology of oil production in the hope of learning how to improve the performance of existing organisms. Several major discoveries occurred in 1985, 1986, and 1988, when scientists discovered the so-called "lipid trigger" that lead to an increase of oil excretion in algae. "Lipid triggers" are chemical elements in algae nutrient that when removed, "starve" the algae such that a rapid buildup of oil droplets within the cells occurs. The first of these discoveries identified nitrogen as the trigger, with studies confirming observations that nitrogen depletion could lead to an increase level of oil present in many species of algae. The downside of the lipid trigger is its inhibitive effect on cell growth and therefore slows down the overall production rate. In 1986, the National Renewable Energy Laboratory (NREL) made another discovery while studying silicon depletion (Si-depletion) in diatoms (Cyclotella cyptica). They found that Si-depleted cells direct carbon more toward lipid production and less toward carbohydrate production. Hence, NREL researchers began to look for key enzymes in the lipid synthesis pathway to identify the critical factor for controlling oil production in algae.</p><!><p>By 1988, researchers have successfully identified the enzyme Acetyl CoA Carboxylase (ACCase), which has shown positive correlation with lipid buildup during Si-depletion. These findings quickly prompted scientists to successfully clone the ACCase gene and to develop tools for expressing foreign genes in diatoms. In the 1990s, the ASP program accelerated rapidly and focused heavily on the genetic engineering front. At around the same time, another line of research that focused on the carbon metabolic pathway also yield a substantial discovery (Figure 2). Instead of focusing on the lipid synthesis, scientists identified key enzymes involved in the synthesis of carbohydrate and ways to disable them, thus diverting carbon to flow down the lipid synthesis pathway. However, the benefits of these findings have yet outweighed the loss from inhibitive growth rate due to depleted cells. Molecular research still needs to balance the efficiency of lipid production with algae growth, because those are two essential criteria for a viable algae farm environment.</p><!><p>"Carbon's Metabolic Pathways" by NREL [3]. Carbon goes through numerous metabolic pathways to synthesize compounds needed by the cells. Here's a representation of two possible pathways. Diatoms store carbon in lipid form or carbohydrate form. The result of the NREL experiments suggests that it might be possible to alter which path the cells use for storage (e.g. more toward lipid synthesis, less to carbohydrate synthesis).</p><!><p>At the current rate, laboratory studies will be a long-term effort, even after demonstration of potential for improving lipid production in algae and successful genetic reproduction. Many other factors are still required for algal mass culture, and some of them cannot be demonstrated in laboratories. Factors such as competitiveness, predation resistance, and harvestability are only feasible in outdoor testing. A strictly laboratory-based R&D program may lose touch with the realities of the eventual applications, thus, outdoor research must be carried out in parallel with laboratories studies</p><!><p>Within the outdoor studies, research is also broken down into three categories: 1) wastewater treatment, 2) pond studies 3) system analysis and resource assessment.</p><p>Similar to the laboratory studies, the order of the above categories help describe the trajectory of algae research in the outdoor environment. Wastewater treatment was in development well before 1980 due to its essential role in urban planning. Since many early practices rely on expensive and sometimes environment-unfriendly chemicals or in using considerable amounts of energy, algae were used to provide a cheap and efficient alternative to those old practices. While waste streams serve as desired breeding grounds for algae populations, another gain from algae-based wastewater treatment is the end product – algae biomass, which can be used as a biofuel feedstock. Most past experiments evaluate a combined wastewater treatment/fuel production system based on microalgae. When the Aquatic Species Program took on this track, the emphasis had moved from algae based wastewater treatment to dedicated algae farming.</p><!><p>Research into algae for mass-production has mostly focused on microalgae, the reasons being that microalgae have a more simplistic structure, a fast growth rate, and high oil content. From 1980 to 1987, the ASP funded two parallel efforts to develop large scale mass culture systems of microalgae. One was called "High Rate Pond" (HRP) design, developed at UC Berkley. The other was called "Algae Raceway Production System", developed by the University of Hawaii. The HRP design was ultimately selected by the ASP for the scale-up procedure and the "Outdoor Test Facility" (OTF) was constructed at an abandoned water treatment plant in Roswell, New Mexico. Between 1988 and 1990, the 1,000 meter pound achieved over 90% utilization of CO2. Best results were obtained using native algae species, which naturally had the fastest growth rate in their native climate. The OTF also demonstrated production of increased quantity of algae oil using both nitrogen and silicon depletion strategies from lab studies. The overall productivity was much lower than initially expected due to cold temperature days at the test site. The facility was closed down in 1990 and serve as a proof-of-concept for large scale open pound operation. Other outdoor projects were also funded over the course of the program including a subcontracted project with the Solar Energy Research Institute in Fairfield, California, which will be discussed more extensively in a subsequent section, and a three-year project in Israel. In the late 1980s, the Georgia Institute of Technology successfully developed the Algal Pond Model, a computer modeling tool for predicting performance of outdoor pond systems.</p><!><p>Resource Assessment aims to address the issue of resource availability and utilization: Where can such technology achieve the maximum potential? Various resource analyses indicated significant potential of land, water, and CO2 resources in the southwestern United States, which provided the most suitable location for large-scale algae farming. For example, one study conducted by NREL concluded that there is a potential for production of several quads (1015 Btu) of biodiesel fuels in the southwestern U.S. alone. However, this does not take into consideration of the spread of resources in this vast region. It will be difficult to find many locations where all the resources for microalgae cultivation mentioned above are readily available. Furthermore, most coal-fired power plants in the United States are located in the north, or in otherwise unfavorable climates, so only a small fraction of power plant CO2 resources would be available to microalgae systems. Therefore, the resource potential estimated by some of the studies must be significantly discounted.</p><!><p>Engineering design and cost analyses, together known as systems analysis, aim to address important questions relating to cost-efficiency of microalgae system: how much impact can algae technology have on current state of energy consumption? This is required both by the mission of DOE as well as the inherent need to justify budgetary decisions. The study analyses generally supported the view that microalgae biomass production could be performed at sufficiently low cost as to plausibly become a renewable energy source. The system analyses studies conducted under the ASP are much more accurate compared to earlier studies in the 1970s. Two systems – opened pond and closed photobioreactor – are the heart of the ASP program. One the one hand, the closed algae system provides better control over environmental conditions and biological contaminants, and higher productivities and harvesting rates. But the cost is extremely high and unfeasible at the current rate. On the other hand, large open pond systems are much more affordable, but at the same time due to hydraulic and CO2 supply limitation, productivity rate is still relatively low.</p><p>However, the most important issue involved in these engineering design and cost analyses are not the cost estimates, but the biological assumptions on which such designs are based. There has been a dramatic increase in projected productivities (from 50mt/ha/y in 1977 to 300mt/ha/y in 1993). This large increase is partially due to significant advances in scientific measurement, but also driven by clear necessity. Therefore, the main problem facing R&D involves less with engineering design, but more on dealing with microalgae cultivation, species control, and overall lipid harvest productivity. Future research will focus on these biological issues in the quest for low-cost production processes.</p><p>The total cost of the Aquatic Species Program is $25.05 million over a twenty-year period, compared to the total spending under the Biofuel Program ($458 million over the same period). In 1995, the DOE eliminated funding for algae research within the Biofuel Program. Under pressure to reduce budgets, the Department chose a strategy of more narrowly focusing its limited resources in one or two key areas, the largest of these is the development of bioethnaol.</p><!><p>At present, most companies in the private sector are early stage start-ups that involved heavily in R&D rather than commercialization, many of them younger than five years old. To this day, none has launched a successful full-scale commercialization of biodiesel from algae. Most of the challenges facing these private companies are finance related, since a substantial amount of resources is required to set up the algae farming operation and venture capital is relatively scarce in this particular segment in comparison to other green initiatives. At the same time, many private companies' R&D results produced innovative concepts and approach to biodiesel production. Unfortunately, we were not able to access most of the private research conducted within these companies. Nonetheless, we can look a few promising firms and their unique approach to algae commercialization, which is publicly available.</p><p>A few private firms have attracted media attention with their recent success in raising funding. Companies such as Massachusetts's Greenfuel Technologies Corporation and California based Solazyme all utilized special closed systems. Greenfuel Tech builds algae bioreactor systems, which not only directly feeds recycled CO2 to the algae but also carefully control the algae's intake of sunlight and nutrients. Solazyme, on the other hand specializes in using synthetic biology and genetic engineering to tweak algal strains for better biofuel yields. The company grows its algae in fermentation tanks without sunlight by feeding it sugar; both firms have already struck deals with biodiesel firms for the next stage of commercial expansion. Another California based firm called LiveFuels looks to continue the Aquatic Species Program's research in using open-pond algae systems. The firm is trying to develop green crude to be integrated directly into the nation's existing refinery infrastructure. Similarly, Solix Biofuels, a Colorado based company is also working on a biocrude, but using a closed-tank bioreactor set-up. The company has said that construction will begin shortly on its first, large-scale bioreactor at the nearby Belgian Brewery, where CO2 waste produced during the beer-making process will be used to feed the algae. Companies such as Seattle based Inventure Chemical and Israel based Seambiotic have teamed up to construct pilot commercial plants to produce algae for specific commercial applications. The combined effort will utilize high-yield oil-rich algae strains that Seambiotic has developed and grown in its open pound system coupled with Inventure's patent-pending conversion processes to produce ethanol, biodiesel and other value-added chemicals. A new start-up Aquaflow Binomics from New Zealand is focusing on harvesting wild algae that can be grown in wastewater and local city waste streams, which doesn't require extra land or feedstocks. The company has been working with Boeing on algae-to-bio-based jet fuel since last year.</p><!><p>Since the large DOE funded Aquatic Species Program was halted in the 1996, however, much of the publicly funded research into algae biodiesels have taken place on a laboratory scale. The methods most commonly used in the literature, therefore, are associated with this small scale environment, and do not necessarily provide viable options for algae production when scaled up. Private ventures have no doubt furthered the research in this area, but since we do not have access to these documents, the following report on the current process by which algae is farmed and processed into biodiesel is extrapolated based upon these publicly available documents.</p><p>It is perhaps worthy of note that algae have been grown and harvested for a variety of reasons ranging from the production of algae as feed for zooplankton to the production of medically significant compounds or β-carotene. Although these processes often require different specifications for algal growth than those required for viable algal biodiesel production, it is also important to note that such large-scale algal farming efforts of the past can be used as roadmaps for the growing process, which may be of use for this particular endeavour.</p><p>It should also be noted that although several firms such as the New Zealand company, Aquaflow, have proposed to harvest wild algae, thereby bypassing the farming step of the process, most companies have proposed a process that involves the integration of growing, harvesting, and conversion of biomass into biodiesel. Water, nutrients, organic solvents, and carbon may be recycled through this process. Thus, there may be tangible advantages achieved through vertical integration of the algae biodiesel production process, which may reduce the amount of waste produced by the production process and reduce the costs from inputs. An overview of such a system is shown in Figure 3.</p><!><p>Summary of the integrated process of algal biodiesel production [10]. Water, inorganic nutrients, light, and CO2 are provided to the algal cultures in the algal 20 biomass production stage. Biomass is then separated from the water and nutrients in the biomass recovery stage, as the latter are recycled back into the algal cultures. The biomass then undergoes extraction to remove its lipid content. This lipid content is converted into biodiesel. Spent biomass can be used as animal feed or digested anaerobicly to generate gas for electricity while recycling CO2 emissions back into the biomass production stage.</p><!><p>In this subsection we will provide an overview of the main steps of the biodiesel production process: choosing the species of algae, growing considerations, algae farm designs, processes for algal biomass recovery, extraction and conversion techniques, and current and future directions for research in this area to improve efficiency or productivity of the process.</p><!><p>Many species of algae have been researched with the intention of using these species as a potential feedstock for biodiesel. Of these species, Botryococcus braunii, has appeared in the literature as a laboratory favourite, although has not been commercially cultivated on a industrial scale. Table 4 lists some species of algae and their associated lipid content, but the table is not intended by any means to be comprehensive. In addition to the four species provided, there was also been a certain amount of interest in other algal species such as Scenedesmus dimorphus, Euglena gracilis, Tetraselmis chui, various Spirulina species, and many others that have been profiled as part of the ASP.</p><!><p>Species of algae included lipid content and current cultivation [22-24]</p><!><p>The concept of an open pond system is relatively self-explanatory. The design requires for the carbonation of large pools (lagoons) of medium. In this regard, the name "open pond" may be misleading as the pond may be at least partially covered to maintain high enough CO2 concentrations. A 1987 report by the Solar Energy Research Institute [8] mentions several factors affecting the overall productivity of open pond systems including, but not limited to water resources, carbonation systems, mixing systems and harvesting systems. A basic design of an open pond can be found in Figure 4.</p><!><p>"Raceway" Open Pond from Campbell [21], Tubular Photobioreactor from Christi [10]. The open pond design pictured represents a simplified and fairly common design for an open pond system. The photobioreactor system shows one possible flowpath by which harvesting, degassing, and nutrient replenishment might occur in a closed system.</p><!><p>With respect to water resources, much as been made about the ability to farm algae in salty or polluted waters, as it is common to see algae growing in polluted streams or ponds outside of an agricultural setting. With regard to open pond farming, however, since there is a relatively high cost associated with carbonation, the consideration of the properties of water can have an effect on the system costs. It was reported that in many cases hard water would require the addition of sodium carbonate, lime, or both depending upon the Ca and Mg content. The goal of selection was to minimize the dissolved CO2 levels at low pH while maximizing CO2 levels at the highest pH, where the former limit is set by the algae's ability to tolerate increasingly alkaline growth media, and the latter limit is set by solubility constraints that result in outgassing. Obviously selection criteria are quite dependent upon the species of algae chosen.</p><!><p>According to the Weissman report, carbon is also among the most expensive inputs for an open pond system, its importance, however, should not be overlooked. A 1985 study by Chirac, et al. found that air that was enriched 1% CO2 lead to a 3.5x increase in the mean doubling time of growth of B. braunii as well as a 5-fold increase in its hydrocarbon production. This effect was not observed when bicarbonate was merely added to the growth medium [9]. As mentioned before, one possible solution is to cover the pond holding a high concentration of CO2 at the surface, thereby allowing the gas to passively diffuse into solution. Even so, the desorption of oxygen and nitrogen gases under the cover prevents us from having a high percent of coverage. Alternatively, it is also possible to inject CO2 from shallow stumps below the surface of the growth medium.</p><p>Once possibility for reducing carbon costs into the system is carbon recycling. The Weissman report estimates that in an open pond 60% of the algal biomass will be lipid, and only 90% of this biomass will be harvested. The remainder of the carbon-products will degrade either into gaseous products in the form of methane or CO2, or settle to become sludge or dissolve in the lagoon water. The gaseous products can be recollected and combusted to create a 35% CO2 mixture that can then be reinjected into the ponds. Christi further adds that surplus electricity generated by the combustion process can be sold to the grid.</p><!><p>Constant agitation is also necessary in order to keep the cells in suspension, to disperse nutrients and prevent thermal stratification. The authors focused on two major means of mixing: the use of either paddle wheels or airlift mixing systems. Although the former was associated with greater initial capital costs, it was determined that there was insufficient data to properly assess the overall costs of the latter.</p><!><p>Other factors important to algal growth, but not considered extensively in the Weissman report, include light intensity, temperature control, and the costs resulting from contamination, which open systems are relatively more vulnerable to, as opposed to closed systems.</p><!><p>There are different types of photobioreactor types available, of which the tubular variety is among the most commonly described in the literature, primary because this type of reactor has been used on a small scale for numerous laboratory assays. Figure 4 presents a diagram of such a design. Nevertheless, there is some doubt as to how well this system would work on an industrial scale. Christi [10] reports that this design calls for a solar collector consisting of an array of tubes containing the cell suspension, each 0.1m in diameter. During daylight hours, microalgae broth must be circulated throughout the system, and a high turbulence flow must be maintained at all times to prevent biomass sedimentation.</p><p>Algal growth is sensitive to levels of dissolved gases such as oxygen and carbon dioxide. Concentrations of oxygen much higher than air saturation values will inhibit photosynthesis, and at very high levels in combination with sunlight, could potentially damage the cells. Furthermore, algal growth is pH sensitive, an important consideration since the process of photosynthesis will naturally cause the tube pH to rise as greater amounts of dissolved CO2 are removed from solution as oxygen is introduced. Unlike in the open system, since the medium is enclosed within tubes, it is impossible for the gas to escape under ordinary circumstances. It is therefore impossible to run the tube reactors continuously, as the tubes must be periodically emptied for aeration and degassing.</p><p>Christi further reports that the sensitivity of the cells toward temperature often requires the introduction of cooling systems. Since the optimal growth temperature for the cells is 20-30°C, especially during daylight hours when constant exposure to sunlight heats the broth and could potentially damage the cells, cooling is essential. A heat exchanger or, in drier environments, evaporative cooling from water sprayed on the tubes, was deemed sufficient.</p><p>In a 1998 study by Sanchez Miron the comparative performance of several photobioreactor designs were reported for the culture of the microalga Phaeodactylum tricorntum. The microalgae were cultivated for the production of eicosapentaenoic acid, a potential treatment for certain cancers and heart disease reported in 1996. The report also includes a mention that a commercial horizontal tubular bioreactor facility in Cartagena, Spain was abandoned by its owner, Photobioreactors Ltd., after it failed to perform [11]. In general, photobioreactors appear to require a considerably larger compared with open pond systems.</p><!><p>According to Weissman 1987, the standard protocol for the harvesting of algae from a dilute suspension of 0.05-0.1% consists of a concentration to reduce the volume of the sample by 20-50 fold followed by centrifugation, which, in turn reduces the remaining sample volume by 5-10 fold. Due to the near prohibitively high capital costs associated with centrifuges, however, it is necessary to examine other methods. A one-step separation of algae using an inclined or vibrating screen is also possible provided that packing of biomass on the screen continues to allow a high flow rate to be maintained. This latter process would allow for the effluents to be returned to the pond, but in this case, the harvesting process must not only remove the desired biomass, but also all potential contaminants. Other possible devices are dissolved gas floatation units, microstrainers, belt filters, and settling ponds. Of these devices dissolved gas floatation units had the highest capital cost (25 million 1987 USD/million gal of suspension/day), followed by belt filters (0.12), microstrainers (0.09), and settling ponds (0.05) although these latter methods are had costs of the same order of magnitude as of 1987. Any requirement to pre-treat the suspension prior to harvesting, however, will increase costs substantially, so as to trump any of the differences in capital costs associated with the price differences between these devices.</p><!><p>Once the biomass has been dried two processes must occur in order to create biodiesel: the lipids must be extracted from the biomass and they must undergo a transesterification reaction (Figure 5). Although successful protocols have been established for these processes in the laboratory, it remains that these laboratory techniques are not particularly successful on an industrial scale with regard to algal biodiesel, although several reaction techniques have been used commercially for the transesterification of tallow and soybean.</p><!><p>The Transesterification Reaction. During transesterification triglycerides obtained from biological products are processed with a three times excess of alcohol to generate glycerol and alkyl esters. Biodiesel consists largely of these alkyl esters; in the U.S. these must be monoalkyl esters.</p><!><p>The current technique for algal biodiesel production obtains lipids from biomass by means of standard grinding or sonication of the algal cells in order to lyse them, followed by extraction using organic solvents. Although such methods are common for harvesting biochemical products in the laboratory, the industrial scale equivalent usually requires the use to batch reactors, in which sonication and extraction take place in large vats. The harvested lipids are then reacted in a similar batch method using a dissolved or liquid catalyst and alcohol for the transesterification reaction. Generally, a standard reaction consists of methanol in a 6:1 molar ratio with oil input, 1:100 molar ratio of NaOH to oil input, and a 1:1 volume ratio of organic solvent to methanol [12].</p><p>However, there are several problems with batch reactor processes. The batch method does not allow for the lipids to be processed continually, as the vats must be emptied and refilled. The process has a requirement for a large amount of flammable organic solvents to be used, which could also pose some danger to workers. The use of the liquid catalyst also poses the problem that at the reaction's end the catalyst is mixed with the products, and must be separated. It is for these reasons that batch reactors do not appear to be heavily used on an industrial scale, although they are heavily used in the laboratory. There are several references to commercial transesterification plants using a continuous flow system; however, there is a notable lack of information on the specifics of how these systems operate.</p><p>Some information is presented by Ben Wen, an investigator at United Environment & Energy (UEE), who is working on an improved continuous flow tubular reactor [13]. This is filled with a solid catalyst that does not leave the reactor. Oils are flowed through the reactor, undergoing transesterification as they pass the solid catalyst. Biodiesel can be generated at a greater rate using such a design since the tube does not need to be emptied and refilled. Furthermore, since the design of the tubular reactor is not vat-like, it can be smaller and, therefore, easier to transport. The great cost reduction within this system, however, is the elimination of the need to separate product and catalyst following the reaction. Although this design is currently in Phase I and being operated only on a small scale, with algal oil samples provided by outside producers, UEE reports that the design demonstrates greater scalability compared with traditional reaction processes and has partnered with other firms to design a complete algae biodiesel production process from algal growth to extraction and transesterification. It is highly likely that similar systems are utilized in existing plants.</p><!><p>To date, publications in the scientific literature has indicated that much of the research into algal biofuels has focused on the treatment of algae and the optimization of its growth conditions varying factors such as nutrients in the growth medium, light, and gas content. Most of these optimization scenarios are designed to increase the algae productivity by increasing the algal growth rate or the lipid content. They take advantage of a highly controlled laboratory environment, but as one of the major obstacles facing the farming of algae in more cost effective open ponds is the threat of contamination, it is doubtful whether such research would be of direct usefulness to industrial production of algae. Other groups have characterized the different lipid compounds produced by various species or strains of algae, allowing for speculation on the characteristics of the products produced from these lipids.</p><p>Since the late 1980's and early 1990's, papers on algae farming have discussed the future use of genetic engineering as a means to greater productivity, whether of EPA or of lipids. Due to the great difficulty in genetic engineering of algae, however, this field is still in its infancy and little progress is evident from the literature.</p><p>Some inquiries have focused more on either the chemical features of the process or the industrial design aspect. We have already mentioned UEE in this regard, which in addition to reactor design, is attempting to optimize the form of algal biodiesel produced for performance in a standard diesel engine by reducing the biofuel's sensitivity to oxidation and increasing its chemical stability. Research along these lines is rarer to find in journals, thus we speculate that most of this research is funded as part of private ventures. As a result, apart from press releases, such as those mentioned in the previous section, we do not have an estimate as to the extent that research has made inroads into these subjects.</p><!><p>As noted before, there is very little publicly available research into algae farming. What little is available, however, is sufficient to form the basis of an economic analysis of algae farming for the purpose of producing biodiesel for both an open and closed system. For the closed system, we use a 2003 paper to form the basis of our evaluation, and a 1987 U.S. Department of Energy engineering report for the open system. For the analysis of the transesterification process, we reference several different sources. For the annualized capital costs of these systems, we discount them over ten years with a discount rate of 7%.</p><p>Molina Grima's 2003 paper [14] estimates the cost for a closed bioreactor system based off direct experience and vender quotes. This system would produce 26.2 tons of algae biomass per year for the purpose of extracting a separate algae product, EPA. However, their process also produces oil as an intermediate product. Molina Grima assumes a 10% oil yield, leading to production of 2620 tons of oil a year, or about 787 gallons of oil. Note this yield is much lower than would be expected from a closed system built for producing biodiesel. Their costs are summarized in Table 5 and Table 6. Producing those 26.2 tons of algae biomass requires a capital investment of over three million dollars and a total annual cost of $933,995 – leading to a price of $35,649 per ton of algae biomass. There is a mistake in their setup which is corrected here.</p><!><p>Capital costs of Grima closed system</p><p>*Mistake in original paper</p><p>Yearly operating costs of Grima closed system</p><!><p>This is notably conservative in its estimations of non-input expenses. For instance, construction costs are ~70% of the total capital cost, while "general overhead", about ~13% of total annual expenses, is fairly significant, along with labor taking ~17% of annual cost as well as various other miscellaneous costs that all add up. These are quite significant relative to the cost of consumable inputs (CO2, culture mediums, etc), which only make up ~13% of annual production cost. Construction costs, for one, can likely be reduced for later plants, similar to the cost savings that nuclear power plants encounter. In addition, although the algae farming systems that Molina Grima et al were studying were particularly complex, and it is possible that other bioreactor designs will be cheaper to put together. Finally, labor and general overhead can also be reduced when scaled past one plant, since a worker can then cover multiple plants.</p><p>Weissman and Goebel's 1987 U.S. Department of Energy engineering report has their basic costs inflation adjusted to 2003. In addition, their CO2 price is greatly far out of line even after adjusting for inflation, since one of their central assumptions is cheap CO2. These are thus replaced with unit costs from the Molina Grima paper, which are close to current market prices, and recalculated. While other input prices have also fluctuated since 1987, they are not far off from current price quotations. In addition, electricity costs are equalized to the Molina Grima paper as well. The DOE open system specifies 192 hectares of ponds on a total of 384 hectares of land, producing 112 metric tons of biomass per hectare per year. Again assuming a 10% oil yield, this produces 11,200kg of oil/ha/year, or 3362 gallons/ha/year. Its cost per hectare is specified in Table 7 and Table 8. Capital costs are considerably lower for this system, totaling just over $100,000 per hectare, with a total annual cost of $147,769, or $1,319 per ton of algae biomass. The greatest expense for this system is the CO2 input, which makes up an impressive ~80% of the annual cost.</p><!><p>Capital costs of DOE open system per hectare</p><p>Operating costs of DOE open system per hectare</p><p>*Cost per unit was $0.035/kg in original report</p><!><p>Unfortunately, there have been few public analyses of large scale industrial transesterification plants, despite the fact that several commercial biodiesel plants exist in various locations across the United States. Molina Grima 2003 does provide an analysis of a small-scale esterification process for both open and closed inputs. However, this paper was unclear about the specifics of the reaction despite detailed itemization, and furthermore is quite clearly designed as a small-scale operation. As such, we provide our own framework. A 1994 paper estimated that a large plant that can produce 30 million gallons of biodiesel annually, fueled by tallow, can be constructed for ~$15 million [15]. This ballpark number is supported by another analysis based off a soybean input [16]. This is surprising, since research into the actual capital costs of ethanol plants, which operate on a simpler reaction, revealed an average cost of $1.53 per gallon of capacity [17].</p><p>We present this analysis of the transesterification process economics in Table 9. Given the uncertainty of capital costs, we use a more conservative estimate, so our 30 million gallon plant will cost $46 million – equal to the cost of a similar sized ethanol plant. This is annualized to $6.55 million. Our inputs are methanol in a 6:1 molar ratio with oil input, NaOH in a 1:100 molar ratio with oil input, and hexane in a 1:1 volume ratio with methanol – a rather standard setup [12]. Production of 30 million gallons of biodiesel requires an input of 6.7 million to 25 million kilograms of oil by weight [15,16]. We assumed a requirement of 15 million kilograms. Requirements for other inputs are scaled from Molina Grima 2003. Finally, we assume an oil yield of 40% for a closed system and 15% for an open system. Capital costs are not a significant fraction of the operating cost, and the cost of biomass make up the bulk of the price of $45.12 or $4.99 per gallon for inputs from closed and open systems respectively. These are adjusted to $49.39 and $5.46 per gallon for energy equivalence to regular diesel.</p><!><p>Costs of oil extraction</p><p>115,000 tons of oil at 40% yield</p><p>215,000 tons of oil at 15% yield</p><p>* http://www.icispricing.com/il_shared/Samples/SubPage135.asp</p><p>** http://www.icis.com/StaticPages/a-e.htm#C</p><p>*** http://www.icispricing.com/il_shared/Samples/SubPage90.asp</p><!><p>These numbers can be improved by relaxing some of the assumed costs. For closed system, the greatest cost of biodiesel production is in the capital outlay required to build out photo-bioreactors. Capital and fixed input costs are the most likely to be improved given with improved technology, experience, and economies of scale. On the other hand, the requirements for variable inputs such as CO2 are unlikely to be reduced without massive advances in algae engineering (which may yet happen); regardless, the variable inputs are not a major percentage of the total cost. In addition, photo bioreactors can likely reach much higher yields. Finally, hexane is used as a solvent in the transesterification reaction, and thus can be recycled for reuse. Sensitivity analysis for the closed systems is presented in Table 10. Given that the major cost of biodiesel is the cost of algae, and thus the capital cost of constructing photo bioreactor systems, it is difficult to imagine closed system sourced biodiesel being viable.</p><!><p>Scenarios for closed system</p><!><p>On the other hand, the major cost involved in biodiesel sourced from open pond systems is the cost of CO2 input, while the capital costs are very low. While yield can never be as high as in closed systems – 50% would appear to be a pipe dream for open ponds – they certainly can be improved. Finally, as before, hexane recovery could also reduce costs. Scenarios are presented in Table 11. Improved yields greatly help algae biofuels to nearly achieve the cusp of economic feasibility. However, it is somewhat difficult to envision improvements past 30% yields for open systems. Nevertheless, combined with reductions in CO2 costs, open pond sourced biodiesel is at the cusp of feasibility. Essentially, for algae to be close to economically feasible as a biofuel simply requires little to no CO2 cost and an open pond system with reasonable lipid yields. We will now go over possible policies that may tip the balance.</p><!><p>Scenarios for open system</p><!><p>The following section is an overview of the programs currently in effect in the United States that could incentivize the production of algae biodiesel. The incentives provided by these programs provide a template for the types of policies that could allow algae biodiesel to become profitable.</p><!><p>Administered by the Environmental Protection Agency, the Renewable Fuel Standard (RFS) was established by the Energy Policy Act of 2005 and was expanded upon in the Energy Independence and Security Act of 2007. The RFS is a provision that requires all transportation fuel to be blended with a certain amount renewable fuel, including bioethanol and biodiesel. Fuel producers were to blend 9 billion gallons of renewable fuel into the nation's gasoline in 2008, with quotas increasing annually to 36 billion gallons in 2022. Notably, the expanded RFS mandates that an amount of this renewable fuel must be "advanced biofuels," defined as biofuel produced with non-corn feedstocks that have at least 50% lower lifecycle greenhouse gas emissions than petroleum fuel. Of the 36 billion gallons mandated in 2022, for example, at least 21 billion gallons must be advanced biofuel. In terms of making algae more viable, the RFS does not directly incentivize its production. It does, however, guarantee a market for algae biodiesel, as it falls under the category of advanced biofuel [18].</p><!><p>Biodiesel producers can claim a tax credit depending on the type of biodiesel produced. The credit is set at $1 per gallon produced of "agri-biodiesel," which is defined as biodiesel produced from virgin agricultural products such as soybean oil or animal fats. Alternatively, producers of biodiesel from previously used agricultural products such as recycled fryer grease can claim a 50 cent per gallon tax credit [18]. Algae biodiesel would be likely to fall under the former category of agri-biodiesel, allowing it to claim the $1 per gallon tax credit. It should be noted that this tax credit is set to expire in December of 2009, but a bill to extend it for a further five years is currently under consideration [19].</p><!><p>The Small Agri-Biodiesel Producer Credit is valued at 10 cents per gallon produced. It can only be claimed by a producer of agri-biodiesel with a production capacity of less than 60 million gallons of fuel per year, and can only be claimed on the first 15 million gallons produced in a given year. It is unlikely that algae biodiesel production facilities would produce at a low enough level to be considered "small," but in the event that smaller production facilities are ideal, this tax credit could still be beneficial [18].</p><!><p>Introduced by the Food, Conservation, and Energy Act of 2008 and administered by the United States Department of Agriculture, the Biorefinery Assistance program offers loan guarantees and grants for the construction of biorefineries, facilities specializing in the creation of advanced biofuels. The program has received $75 million in mandatory funding for FY2009 and $245 million in FY2010 for loan guarantees. In addition, $150 million has been authorized annually for FY2009-FY2012 [18]. It is unclear how much of this funding would be available to the construction of a proposed open system facility for the production of algae biodiesel, but a grant is certainly possible.</p><!><p>Another program established by the Food, Conservation, and Energy Act of 2008, the Bioenergy Program for Advanced Biofuels provides payments to producers of advanced biofuels. The program has received annual funding through FY2012: $55 million for FY2009, $55 million for FY2010, $85 million for FY2011, and $105 million for FY 2012, with authorization for an additional $25 million each year from FY2009-FY2012 [18]. Again, it is not clear how much of this funding would go to an algae production facility, but at the very least, programs such as this show that the U.S. government is willing to devote large amounts of money to advanced biofuel programs. As research into algae biodiesel continues in the private sector, if it proves successful, it will be quite likely that government funding will be available to it in the future.</p><!><p>This import duty is comprised of a 2.5% ad valorum tariff and a most-favored-nation duty of $0.54 per gallon of fuel ethanol imported into the United States from most countries. Ethanol imported from the Caribbean Basin Initiative countries may be exempt from these trade restrictions [18]. Like all import duties, it is unclear whether its effects on the world's ethanol market are for the best, but its existence certainly shows that the United States government is interested in protecting the domestic biofuels market. It is likely that similar actions might be taken to protect domestic producers of algae biodiesel from foreign markets in the event that the method becomes a profitable way to produce biofuel.</p><!><p>Our model clearly shows that closed system method of production of algae biodiesel, despite its immunity to contamination, is prohibitively expensive. The policies for incentivizing biofuel production that are currently in place, most notably the monetary assistance of the Biodiesel Tax Credit, could potentially allow algae biodiesel to be produced profitably using an open pond system given certain assumptions about the costs of algae biodiesel production (Table 12). In addition, the market created by the Renewable Fuel Standard offers profitability even if algae biodiesel does not meet these conditions.</p><!><p>Scenarios with biodiesel tax credit</p><!><p>The most important of these conditions would be if the cost of CO2 becomes negligible or substantial increases in yield are observed. The simplest way of reducing CO2 costs would be through carbon trading. Since algae biodiesel is carbon neutral overall and consumes CO2 in the production process, it is in the prime position of being able to sell emissions credit. However, given current futures prices from the European Climate Exchange (Table 13), this method of offsetting CO2 costs is currently not feasible. Carbon trading schemes must become more robust, i.e. expensive, to allow an algae biodiesel producer to sell carbon offsets. Alternatively, the CO2 cost problem could be alleviated if the cost of commercial CO2 drops significantly. Increases in yield could result from advances in genetic engineering of algae so that they are better able to compete with contaminants, though this technology is currently far from implementation. Given the assumptions presented above, open-pond algae as a biodiesel fuel is close to feasibility as a full replacement for diesel, and currently can work well as a blend in petro diesel. Nevertheless, it cannot do so without subsidies, considerable technology improvements, or increases in the price of fuel. Thus the likeliest impacts on feasibility will depend on government policy towards carbon emissions and as always, future research.</p><!><p>European climate exchange EUA futures (12/7/09)</p><!><p>The authors declare that they have no competing interests.</p><!><p>YG provided technical insight and writing on the overall process, and helped with the numbers. CG provided research and writing on policy impacts. YL provided research on the costs involved and ran the numbers. DT & CT provided background research and drafted the initial manuscript. All authors contributed to writing the final manuscript.</p><!><p>This article has been published as part of Chemistry Central Journal Volume 6 Supplement 2, 2012: Roles for chemistry in the world's energy problems. The full contents of the supplement are available online at http://journal.chemistrycentral.com/supplements/6/S1.</p>

PubMed Open Access

Substituted Lactam and Cyclic Azahemiacetals Modulate Pseudomonas aeruginosa Quorum Sensing

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence that is critical for establishing infection. The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones. The development of non-native acylhomoserine lactone (AHL) ligands has emerged as a promising new strategy to inhibit QS in Gram-negative bacteria. In this work, we have synthesized a set of optically pure \xce\xb3-lactams and their reduced cyclic azahemiacetal analogues, bearing the additional alkylthiomethyl substituent, and evaluated their effect on the AHL-dependent Pseudomonas aeruginosa las and rhl QS pathways. The concentration of these ligands and the simple structural modification such as the length of the alkylthio substituent has notable effect on activity. The \xce\xb3-lactam derivatives with nonylthio or dodecylthio chains acted as inhibitors of las signaling with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent was found to strongly inhibit both las and rhl signaling at higher concentrations while the propylthio analogue strongly stimulated the las QS system at lower concentrations.

substituted_lactam_and_cyclic_azahemiacetals_modulate_pseudomonas_aeruginosa_quorum_sensing

INTRODUCTION<!>Design and synthesis<!>Screening against las signaling<!>Screening against the rhl pathway<!>CONCLUSIONS<!>METHODS<!>5-(Propylthiomethyl)pyrrolidin-2-one [7a(5S)]. Procedure A<!>5-(Hexylthiomethyl)pyrrolidin-2-one [7b(5S)]<!>N-tert-Butoxycarbonyl-5-(propylthiomethyl)pyrrolidin-2-one [8a(5S)]. Procedure B<!>N-tert-Butoxycarbonyl-5-(hexylthiomethyl)pyrrolidin-2-one [8b(5S)]<!>5-(Propylthiomethyl)pyrrolidin-2-ol [10a(5S)]. Step a. Procedure C<!>5-(Hexylthiomethyl)pyrrolidin-2-ol [10b(5S)]<!>4-Amino-N-(tert-butoxycarbonyl)-4-deoxy-2,3-O-isopropylidene-5-O-methanesulfonyl -D-ribono-1,4-lactam (16)<!>4-Amino-N-(tert-butoxycarbonyl)-4-deoxy-5-S-hexyl-2,3-O-isopropylidene-5-thio-D-ribono-1,4-lactam (17)<!>4-Amino-4-deoxy-5-S-hexyl-5-thio-D-ribono-1,4-lactam (19)<!>4-Amino-4-deoxy-5-S-hexyl-5-thio-\xce\xb1/\xce\xb2-D-ribofuranose (23)<!>Anti-Quorum Sensing Assay (\xce\xb2-Galactosidase Assay)

<p>Quorum sensing (QS) is a type of bacterial cell-to-cell signaling pathway mediated through the production, release and detection of the small signaling molecules called autoinducers (AIs).1 Such communication allows bacterial control of crucial functions in united communities for enhancement of symbiosis, virulence, antibiotic production, biofilm formation, and many other processes. The recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of a new generation of antibacterial agents. As QS is utilized by number of pathogenic bacteria to direct virulence and biofilm formation, inhibitors/modulators of QS may serve as tools to study or intercept such community behaviors and might be beneficial as antibacterial agents.2 The most common QS signaling molecule used by Gram-negative bacteria are acylhomoserine lactones (AHLs), which are detected by their cognate regulator (R) proteins.1</p><p>Pseudomonas aeruginosa is an important human opportunistic pathogen affecting immunocompromised individuals, cancer patients, burn victims, cystic fibrosis patients and patients with impaired lung function. It uses two AHL systems called, las and rhl to mediate QS. LasI/R synthesizes and detects N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-AHL) while RhlI/R synthesizes and detects N-butanoyl-L-homoserine lactone (C4-AHL) (Figure 1a). In addition, P. aeruginosa has a third QS-dependent pathway, Pseudomonas quinolone signal (PQS) that uses 2-heptyl-3-hydroxy-4-quinolone as an autoinducer.3 Although certain genes appear to be regulated by one pathway, for example regulation of genes involved in rhamnolipid synthesis by the rhl pathway,4 there is much overlap and crosstalk between the pathways and what was once thought to be hierarchical regulation, with las activating rhl, is now known to be much more complex.5 Accumulated evidences clearly indicate the importance of P. aeruginosa QS in disease.6</p><p>Over two decades, several small molecules have been identified by many research groups as inhibitors of the AHL:R protein complex.7–9 These are mostly AHL-based structures with moderate changes on the acyl side chain and amide linkage. Some of the most potent inhibitors prepared by Geske and Blackwell (1a and 2, Figure 1b).10 Recently, Meijler and co-workers designed a ligand, 3, which covalently modified LasR.11 Since AHL is the pharmacophore present in the natural substrates, AHL-based inhibitors are likely to modulate R protein activation.</p><p>Studies of structural features other than the AHL scaffold as tools to understand the R type protein interaction with AHLs are limited,12 although the recent X-ray crystal structure of LasR with its native ligand and triphenyl mimcs ought to facilitate the rational design of QS inhibitors.14,15 Only a few examples of inhibitors with the altered lactone ring structure of AHL have been reported.12,13,16,17 For example, Smith et al. reported 3-oxo-C12-(2-aminocyclohexanone) (4, Figure 1c) as a strong antagonist of LasR system,12 while Muh et al identified two LasR inhibitors having a phenyl and tetrazole ring (e.g., 5), with IC50 in nM range.13 It is noteworthy that in the LuxR system γ-thiolactone analogue 1b showed inhibition while the corresponding ε-lactam (caprolactam) analogue was reported to lack LuxR binding.18 To explore further effects of non-native AHL scaffold on QS, we have designed novel lactam ligands. Here, we report optically pure γ-lactams and cyclic azahemiacetals, bearing alkylthiomethyl substituents with different carbon chain lengths (C3–C12), which are capable of either inhibiting or, in some cases, inducing P. aeruginosa QS pathways. The lactam ring was chosen because it is a more stable isoster of lactone ring present in AHL inhibitors. Moreover, the γ-lactam and cyclic azahemiacetal ligands were further modified in a such way that they resemble S-ribosyl-L-homocysteine, which is known to regulate QS through the LuxS-mediated biosynthesis of AI-2,1,19–21 in which the ribose oxygen is replaced with a nitrogen atom and the homocysteine unit is substituted with a simple alkylthiol chain. Although, P. aeruginosa does neither harbor LuxS nor produce AI-2, AI-2 does alter P. aeruginosa gene expression.22</p><!><p>The (S)-5-(bromomethyl)pyrrolidin-2-one (6), a key substrate for the synthesis of γ-lactam analogue 7 was conveniently prepared from L-pyroglutamic acid.23 Displacement of bromide in 6 with sodium propanethiolate produced 5(S)-(propylthiomethyl)pyrrolidin-2-one (7a, 96%; Scheme 1). Since it was previously demonstrated that the length of the side chain is crucial for determining the agonistic and antagonistic activity,24 lactams containing C6, C9 and C12 alkylthio chain lengths (7b–d) were also analogously prepared. A set of the cyclic azahemiacetals (N,O-acetals or hemiaminals) 10 with a hydroxyl group instead of a carbonyl oxygen at C2 was synthesized as well. Thus, although, attempted reduction of lactams 7 with LiBEt3H was unsuccessful, reduction25 of the N-Boc protected lactams 8a–d proceeded smoothly to afford azahemiacetals 9a–d. Subsequent deprotection with trifluoroacetic acid afforded 5(S)-(alkylthiomethyl)pyrrolidin-2-ols 10a–d as a mixture of isomers in equilibrium25–28 with the corresponding imines 11a–d in addition to the open form aldehydes 12a–d. Structure of the hemiaminal 10a was additionally confirmed by conversion to the correponding O-benzyloxime derivative 13a with benzylhydroxylamine hydrochloride in anhydrous pyridine.</p><p>To increase the polarity/solubility of the lactam and azahemiacetal analogues in the testing media, we also prepared aza analogues with hydroxyl groups at C3 and C4. The N-Boc protected 1,4-dideoxy-1,4-imino-D-ribitol (14),29–31 conveniently prepared from D-gulonic-γ-lactone, served as a suitable starting material for the synthesis of dihydroxy γ-lactams 17 and 18. Thus, mesylation of ribitol 14 followed by the selective oxidation32 of the resulting 15 afforded 16. Displacement of the mesylate 16 with sodium hexane- and nonanethiolate produced 5-alkylthiomethyl lactams 17 and 18 in high yields which were deprotected with TFA to give (5S)-(hexyl- or nonylthiomethyl)-3,4-dihydroxypyrrolidin-2-ones 19 and 20 (Scheme 2). Reduction of 5-alkylthiomethyl lactams 17 and 18 with LiBEt3H afforded cyclic azahemiacetals 21 and 22. Subsequent deprotection with TFA provided (5S)-(alkylthiomethyl)-3,4-dihydroxypyrrolidin-2-ols 23 and 24 as a complex mixture of azahemiacetals existing in equilibrium with dehydrated form (imine) as well as with open aldehyde and dimeric forms, as reported for such class of 4-azaribofuranoses.25,28,33 These azahemiacetals can be considered as aza analogues of S-ribosyl-L-homocysteine28 (SRH) in which (a) ribose oxygen is replaced by nitrogen atom and (b) the homocysteine moiety is substituted with n-alkylthiols with different length of carbon chain.</p><!><p>To determine the effect of the lactams (7, 19, and 20) and the cyclic azahemiacetal derivatives (10, 23 and 24) on the P. aeruginosa las AHL-mediated pathway, a las-dependent β-galactosidase reporter (PlasI-lacZ) was expressed with lasR in E. coli.34,35 Almost no activity was observed in absence of exogenous AHL demonstrating the AHL-dependence of the system (0.21 ± 0.31 Miller units). As expected, and in agreement with published data,34,35 addition of exogenous 3-oxo-C12-AHL activated the PlasI-lacZ (55.4 ± 1.5 Miller units). The lactams and their azahemiacetal counterparts were screened at a concentration of 0.2 to 1.2 mM for their activity against the las system (Table 1). At 0.29 mM concentration, the propylthio lactam 7a slightly but insignificantly inhibited LasR activity and its corresponding azahemiacetal 10a significantly stimulated (approximately by 2.3-fold) las reporter activity (p-value <0.01). This was dependent upon the addition of 2 μM of 3-oxo-C12-AHL as 10a did not stimulate β-galactosidase activity in the absence of exogenous AHL (data not shown). At the concentration of 0.57 mM, the azahemiacetal 10a was found to enhance las reporter activity by 15% (p-value <0.01). However, las reporter activity was inhibited by 69% (p-value <0.05) and 86% (p-value <0.01) at 0.87 and 1.14 mM, respectively. In contrast, the lactam analogue 7a inhibited las activity at all concentrations tested. Cell growth was not inhibited by the addition of lactam and azahemiacetal compounds at the tested concentrations (data not shown).</p><p>Among other lactam analogues tested, the percent inhibition of las promoter activity increased in a concentration dependent manner (Table 1). Inhibition potency also increased as the alkylthio chain length increased. Specifically, nonylthio lactam 7c and dodecylthio lactam 7d were found to possess greatest inhibition at all concentrations tested. At the lowest concentration tested (0.19 and 0.17 mM, respectively), nonylthio lactam 7c modestly inhibited while dodecylthio lactam 7d significantly inhibited las activity (48%; p-value <0.01). On the contrary, among the cyclic azahemiacetals analogues, no general trend was observed between chain length and percent inhibition. Unlike the propylthio azahemiacetal 10a, hexylthio azahemiacetal 10b did not significantly stimulate QS at the lowest concentration tested (0.23 mM) but inhibited las activity 100% at all higher concentrations used (p-value <0.01). In comparison, azahemiacetals containing nonyl side chain 10c and dodecyl chain 10d showed moderate but significant inhibitory activity (Table 1).</p><p>The ribolactam analogues 19 and 20 and their cyclic azahemiacetal counterparts 23 and 24 were found to significantly inhibit las activity at all concentrations tested but only with moderate potency. The azahemiacetals (23 and 24) appears slightly more active (Table 1).</p><!><p>To determine the effect of the lactams (7, 19, and 20) and the cyclic azahemiacetal derivatives (10, 23 and 24) on the P. aeruginosa rhl AHL-mediated pathways, a rhl-dependent β-galactosidase reporter (PrhlA-lacZ) was expressed with rhlR in E. coli.4 As expected, and in agreement with published data,4 exogenous C4-AHL activated the rhl β-galactosidase reporter (266 ± 20 Miller units). There was minimal activity in the absence of C4-AHL (0.12 ± 2.72 Miller units). In general, except for the cyclic azahemiacetals with shorter alkylthio chain 10a and 10b, the rest either had no effect or stimulated rhl expression (Table 1). The lactam analogues 7a, 7c and 7d did not modulate rhl activity, while hexylthio lactam 7b significantly stimulated (p-value <0.01) rhl QS activities at higher concentrations. In contrast to lactams, cyclic azahemiacetals with shorter alkylthio chain 10a (at higher concentration) and 10b (at all concentration) significantly inhibited rhl activity, while analogues 10c and 10d with longer alkyl chain were inactive. The hexylthio azahemiacetal 10b completely inhibited rhl signaling at concentrations of 0.46 mM and higher (p-value <0.02). The strong inhibition observed with propylthio 10a and hexylthio 10b azahemiacetal analogues having side chain lengths similar to C4-AHL is in agreement with the structure activity relationship reported for various synthetic AHL mimetics targeting RhlR.36</p><p>The hexyl-ribolactam analogue 19 significantly stimulated (p-value <0.01) rhl activity, while its cyclic azahmiacetal counterpart 23 had no effect (Table 1). The nonyl-azahemiacetal analogue 24 had significant stimulatory activity at 0.34 mM (p-value <0.05; Table 1).</p><!><p>We have designed and synthesized a set of optically pure γ-lactams with alkylthiomethyl substitution at carbon γ and their N,O-acetal counterparts. These ligands were evaluated for their effect on P. aeruginosa AHL-dependent las and rhl QS pathways isolated in E. coli. Lactam analogues 7 showed selectivity between two QS systems, acting as inhibitors against las signaling and weak activators against rhl signaling, possibly due to differences in the active sites of their cognate R proteins or transport of the native signaling molecule. Antagonism of las activity increased with the length of the alkylthio chain. Interestingly, the cyclic azahemiacetal derivative with shorter propylthio chain (10a) significantly stimulated las signaling at lower concentrations while strongly inhibiting both QS systems at higher concentrations. At least with 10a, the stimulation of the las system only occurs in the presence of exogenous AHL. It is possible that heterodimeric LasR is more active as compared to homodimers. The ribolactam (19 and 20) and cyclic azahemiacetal (24) analogs inhibited las and stimulated rhl moderately. Of all the compounds tested, the 5-(hexylthiomethyl)pyrrolidin-2-ol (10b) appears most potent inhibitor against both las and rhl systems. The mechanism of inhibition is still unknown. Since the effect tested utilized a whole cell assay, the QS inhibition could occur at multiple steps in the pathway. For example, the compound could affect the import of the natural ligand, compete with the natural ligand for binding to the regulator, LasR or RhlR, or alter binding of the QS regulator to the promoter. Alternatively, it is possible that the compounds with longer side chains affect the membrane and that the las pathway is more sensitive to these changes. Future experiments can address these possibilities. For example, quantification of extracellular AHL would reveal if the compounds affect AHL import. If this were the mechanism of action, it is expected that there would be less extracellular AHL in the absence of compound than in the presence. If the compound competes for binding to the regulator, inhibition should be overcome by increased AHL concentration. Given the central role the las and rhl QS pathways play in P. aeruginosa virulence, inhibitors such as the ones described here, have significant potential as therapeutics.</p><!><p>General experimental methods are described in Supporting Information Section. The 1H and 13C NMR spectra were determined with solutions in CDCl3 unless otherwise noted</p><!><p>Propanethiol (50 μL, 42 mg, 0.55 mmol) was added dropwise to a stirred suspension of NaH (35 mg, 0.875 mmol, 60%/mineral oil) in dry DMF (1 mL) under Ar atmosphere at 0°C. After 10 min (till gas evolution has ceased), solution of compound 623 [(5S), 82 mg, 0.46 mmol] in dry DMF (1 mL) was added dropwise, and after 15 min the reaction mixture was allowed to warm to ambient temperature. After 12 h the resulting mixture was quenched with water at 0°C, volatiles were evaporated, and the residue was column chromatographed (EtOAc → 10% MeOH/EtOAc) to give 7a(5S) (77 mg, 96%) as a colorless oil: 1H NMR δ 0.98 (t, J = 7.3 Hz, 3H), 1.60 (sx, J = 7.3 Hz, 2H), 1.76–1.87 (m, 1H), 2.25–2.34 (m, 1H), 2.34–2.45 (m, 2H), 2.52 (t, J = 7.3 Hz, 2H), 2.54 (dd, J = 7.7, 13.2 Hz, 1H), 2.68 (dd, J = 5.5, 13.2 Hz, 1H), 3.80 ('quint', J = 5.5 Hz, 1H), 6.73 (br. s, 1H); 13C NMR δ 13.4, 23.1, 26.6, 30.2, 34.7, 38.6, 53.9, 178.0; MS (APCI) m/z 174 (MH+). HRMS (AP-ESI) m/z calcd for C8H15NNaOS [M+Na]+ 196.0772; found 196.0779.</p><!><p>Treatment of 622 [(5S), 823 mg, 4.62 mmol] in dry DMF (6 mL) with a thiolate solution in dry DMF (6 mL) generated from hexanethiol (682 μL, 573 mg, 4.86 mmol), and NaH (204 mg, 5.09 mmol, 60%/mineral oil) by Procedure A [column chromatography (80% EtOAc/hexane → 5% MeOH/EtOAc)] gave 7b(5S) (932 mg, 94%) as a colorless oil: [α]D = +40.7 (c = 0.03, CHCl3); 1H NMR δ 0.90 (t, J = 7.0 Hz, 3H), 1.24–1.33 (m, 4H), 1.33–1.42 (m, 2H), 1.58 ('quint', J = 7.4 Hz, 2H), 1.78–1.87 (m, 1H), 2.27–2.46 (m, 3H) 2.53 (dd, J = 8.0, 13.4 Hz, 1H), 2.54 (t, J = 7.3 Hz, 2H), 2.70 (dd, J = 5.3, 13.2 Hz, 1H), 3.81 ('quint', J = 6.6 Hz, 1H), 6.47 (br. s, 1H); 13C NMR δ 14.0, 22.5, 26.8, 28.5, 29.7, 30.1, 31.4, 32.7, 38.7, 53.8, 177.7; MS (ESI) m/z 216 (100, MH+); HRMS (TOF MS-ESI) m/z calcd for C11H21NOSNa [M+Na]+ 238.1236; found 238.1252.</p><!><p>DMAP (114 mg, 0.93 mmol), and (Boc)2O (398 mg, 1.82 mmol) were added to a stirred solution of compound 7a (77 mg, 0.445 mmol) in CH2Cl2 (2 mL) at ambient temperature under Ar atmosphere. After 48 h, the reaction mixture was quenched with H2O (5 mL) and partitioned between CH2Cl2//NaHCO3/H2O. The organic layer was washed (brine), dried (MgSO4) and evaporated. The residue was column chromatographed (30 → 40% EtOAc/hexane) to give 8a (5S) (107 mg, 88%) as a colorless oil: 1H NMR δ 0.95 (t, J = 7.3 Hz, 3H), 1.50 (s, 9H), 1.58 (sx, J = 7.3 Hz, 2H), 1.96–2.04 (m, 1H), 2.06–2.17 (m, 1H), 2.40 (ddd, J = 2.6, 9.6, 17.9 Hz, 1H), 2.50 ("dt", J = 4.9, 7.3 Hz, 2H), 2.58–2.67 (m, 1H), 2.60 (dd, J = 9.2, 13.5 Hz, 1H), 2.86 (ddd, J = 0.5, 2.8, 13.5 Hz, 1H), 4.20–4.27 (m, 1H); 13C NMR δ 13.3, 21.9, 23.1, 28.0, 31.2, 34.8, 35.4, 57.5, 83.1 , 149.8, 174.2; MS (ESI) m/z 274 (10, MH+), 215 (100, [MH-59]+).</p><!><p>Treatment of 7b (311 mg, 1.45 mmol) in CH2Cl2 (6 mL) with DMAP (185 mg, 1.52 mmol), and (Boc)2O (746 mg, 3.42 mmol) by procedure B [column chromatography (20 → 40% EtOAc/hexane)] gave 8b(5S) (429 mg, 94%) as a colorless oil: 1H NMR δ 0.89 (t, J = 7.0 Hz, 3H), 1.25–1.33 (m, 4H), 1.34–1.42 (m, 2H), 1.55 (s, 9H), 1.59 ('quint', J = 7.4 Hz, 2H), 2.01–2.08 (m, 1H), 2.10–2.21 (m, 1H), 2.45 (ddd, J = 2.5, 9.6, 17.9 Hz, 1H), 2.56 ('dt', J = 2.9, 7.3 Hz, 2H), 2.62–2.72 (m, 1H), 2.63 (dd, J = 9.3, 13.5 Hz, 1H), 2.91 (dd, J = 2.7, 13.5 Hz, 1H), 4.24–4.31 (m, 1H); 13C NMR δ 14.0, 22.0, 22.5, 28.1, 28.4, 29.8, 31.2, 31.4, 32.9, 35.5, 57.5, 83.1, 149.8, 174.1; MS (ESI) m/z 315 (15, M+), 256 (100, [M-59]+); HRMS (TOF MS-ESI) m/z calcd for C16H29NO3SNa [M+Na]+ 338.1760; found 338.1752.</p><!><p>LiEt3BH (1M soln in THF, 0.98 mL, 0.98 mmol) was added to a stirred solution of 8a (107 mg, 0.39 mmol) in CH2Cl2 (3 mL) at −78 ºC under N2 atmosphere. After 30 min, the reaction mixture was quenched with MeOH (4 mL) and was allowed to warm to ambient temperature. Volatiles were evaporated and the residue was partitioned (EtOAc//NaHCO3/H2O), washed (brine) and dried (MgSO4). The resulting oil was chromatographed (30 → 40% EtOAc/hexane) to give N-tert-butoxycarbonyl-5-(propylthiomethyl)pyrrolidin-2-ol [9a(5S); 104 mg, 96%] as a colorless oil of the mixture of anomers/rotamers: MS (ESI) m/z 274 (10, [M-1]+), 258 (100, [M-17]+). Step b. Procedure D. Compound 9a (104 mg, 0.37 mmol) in TFA (4.0 mL) was stirred at room temperature for 2 h. Volatiles were evaporated to give 10a (62 mg, 96%) as a light yellow oil of a mixture of isomers accompanied by ~25% of the aldehyde 12a [1H NMR δ 8.89 (s, ~0.25H); and 13C NMR δ 180.8]; MS (ESI) m/z 158 (100, [M-17]+).</p><p>A solution of crude 10a (5S; 8 mg, 0.046 mmol) and O-benzylhydroxylamine hydrochloride (48 mg, 0.3 mmol) in anhydrous pyridine (1 mL) was stirred under an atmosphere of nitrogen at room temperature for 12 h. Pyridine was evaporated to afford 4-amino-5-(propylthio)pentanal O-benzyloxime [13a(4S)] of sufficient purity (~90%) for spectroscopic characterization together with the excess of BnONH2 used: MS (ESI) m/z 281 (60, MH+), 158 (100, [M-BnONH]+), (APCI) m/z 281 (100, MH+).</p><!><p>Step a. Treatment of 8b (178 mg, 0.56 mmol) in CH2Cl2 (3 mL) with LiEt3BH (1M soln in THF, 1.41 mL, 1.41 mmol), by procedure C [quenched with MeOH (4 mL) at low temp., column chromatography (30 → 40% EtOAc/hexane)] gave N-tert-butoxycarbonyl-5-(hexylthiomethyl)pyrrolidin-2-ol [9b(5S); 170 mg, 95%)] as a colorless oil of a mixture of isomers: MS (ESI) m/z 316 (100, [M-1]+), 300 (20, [M-17]+); HRMS (TOF MS-ESI) m/z calcd for C16H31NO3SNa [M+Na]+ 340.1926; found 340.1955. Step b. Compound 9b (38.5 mg, 0.12 mmol) in TFA (0.8 mL) was stirred at 0 ºC (ice-bath) for 3 h. The reaction mixture was diluted with excess of ice-cold CH2Cl2 and neutralized with solid NaHCO3. Resulting mixture was stirred for 20 min at ambient temperature and was decanted. The residual slurry was extracted with fresh portion of CH2Cl2 and the combined extracts were dried (Na2SO4) and concentrated to give crude 10b (25 mg) as a colorless oil. Crude product was column chromatographed to give first (0 → 0.25% MeOH/CHCl3) anomeric mixture of azahemiacetals 10b (α/β, 9:20; 2.0 mg, 8%) as a colorless oil: 1H NMR δ 0.89 (t, J = 7.0 Hz, 4.35H), 1.26–1.45 (m, 8.7H), 1.47–1.72 (m, 3.9H), 1.75–1.85 (m, 0.45H), 1.92–2.07 (m, 3.45H), 2.10–2.23 (m, 0.9H), 2.46 (dd, J = 9.5, 13.0 Hz, 1H), 2.52–2.65 (m, 2.9H), 2.93–3.02 (m, 0.9H), 3.23 (dd, J = 2.5, 13.0 Hz, 1H), 3.60–3.68 (m, 1H), 3.67–3.75 (m, 0.45H), 4.04–4.10 (m, 1H), 4.20–4.27 (m, 0.45H); MS (ESI) m/z 200 (100, [M-17]+). Further elution (0.25 → 0.5% MeOH/CHCl3) gave imine 11b (5.8 mg, 22%) as a colorless oil: 1H NMR δ 0.91 (t, J = 7.0 Hz, 3H), 1.27–1.46 (m, 7H), 1.55–1.65 (m, 2H), 2.02–2.11 (m, 1H), 2.49–2.63 (m, 5H), 2.95 (dd, J = 5.3, 12.8 Hz, 1H), 4.21–4.29 (m, 1H), 7.63 (br t, J = 1.1 Hz, 1H); 13C NMR δ 14.0, 22.5, 26.1, 28.6, 29.8, 31.4, 33.0, 37.0, 38.1, 72.9, 167.0; MS (ESI) m/z 200 (100, MH+).</p><p>Note: The composition of crude products after step b depends strongly on the work up conditions. For example, the reaction mixture contained also ~22% of the aldehyde 12b [1H NMR δ 8.91 (s, ~0.22H)] at pH lower than 7.</p><!><p>Step a. Triethylamine (93 μL, mg, 67 mg, 0.66 mmol) and MsCl (25 μL, 38 mg, 0.33 mmol) were added dropwise to stirred solution of 1430 (60 mg, 0.22 mmole) in anhydrous CH2Cl2 (6 mL) at 0 ºC (ice-bath). After 5 min, ice-bath was removed and the reaction mixture was allowed to stir at ambient temperature for 30 min. The reaction mixture was quenched with saturated NaHCO3/H2O and was extracted with CH2Cl2. The organic layer was washed (brine), dried (MgSO4) and evaporated to give 1-amino-1,4-anhydro-N-tert-butoxycarbonyl-1-deoxy-2,3-O-isopropylidene-5-O-methanesulfonyl-D-ribitol 15 (73 mg, 96%) as a mixture (~3:2) of two rotamers of sufficient purity to be directly used for next step: 1H NMR δ1.28 (s, 3, CH3), 1.42 (s, 12H, t-Bu, CH3), 2.96 (s, 1.2, Ms), 2.98 (s, 1.8, Ms), 3.39 (dd, J = 12.5, 4.8 Hz, 0.4H), 3.46 (dd, J = 12.5, 4.8 Hz, 0.6H), 3.69 (d, J = 12.5 Hz, 0.6H), 3.82 (d, J = 12.5 Hz, 0.4H), 4.10–4.14 (m, 0.4H), 4.22–4.30 (m, 1H), 4.22–4.29 (m, 1.4H), 4.45 (dd, J = 10.1, 4.1 Hz, 0.6H), 4.65 ('d', J = 5.9 Hz, 1H); 4.72 ('t', J = 5.3 Hz, 1H); 13C NMR (major) δ 24.9, 26.9, 29.6, 37.1, 52.5, 62.4, 68.9, 79.2, 80.4, 81.7, 112.1, 154.2; 13C NMR (minor) δ 24.9, 26.9, 29.6, 37.5, 53.1, 62.6, 68.6, 78.5, 80.6, 82.5, 112.1, 153.6; MS (APCI) m/z 352 (10, MH+), 252 (100, [MH2-Boc]+). Step b. RuO2×H2O (8.5 mg, 0.064 mmol) was added to a stirred solution of NaIO4 (172 mg, 0.96 mmol) in H2O (1 mL) at ambient temperature. After 5 min, a solution of 15 (80 mg, 0.32 mmol) in EtOAc (1 mL) was added dropwise and the reaction mixture was continued to stir for 12 h. H2O (20 mL) and EtOAc (20 mL) were added and the separated aqueous layer was furthermore extracted with EtOAc (2 × 20 mL). The combined organic layers were washed (brine), dried (MgSO4) and evaporated. The residue was column chromatographed (EtOAc) to give 16 (78 mg, 95%) as a colorless oil: 1H NMR δ 1.37 (s, 3H), 1.44 (s, 3H), 1.54 (s, 9H), 3.01 (s, 3H), 4.39–4.43 ('m', 2H), 4.58 (d, J = 5.45 Hz, 1H), 4.64 (dd, J = 11.2, 3.1 Hz, 1H), 4.70 (d, J = 5.45 Hz, 1H); 13C NMR δ 25.6, 27.0, 28.0, 37.7, 59.2, 67.0, 74.5, 77.5, 84.7, 112.8, 149.7, 170.2; MS (APCI) m/z 298 (100, [MH2-Boc+MeOH]+).</p><!><p>Treatment of 16 (60 mg, 0.16 mmol] in dry DMF (0.5 mL) with sodium hexathiolate [generated from hexanethiol (46.8 μL, 0.33 mmol)/NaH (14 mg, 0.35 mmol, 60%/mineral oil) in dry DMF (0.5 mL)] by Procedure A [column chromatography (5% → 10% MeOH/EtOAc)] gave 17 (25 mg, 40%) as a colorless oil and N-Boc deprotected 17 (24 mg, 38%) as a white crystalline solid. Compound 17 had: 1H NMR δ 0.81 (t, J = 7.0 Hz, 3H), 1.16–1.27 (m, 6H), 1.30 (s, 3H), 1.39 (s, 3H ), 1.44–1.59 (m, 11H), 2.36–2.50 (m, 2H), 2.76 (dd, J = 6.2, 14.4 Hz, 1H), 2.82 (dd, J = 2.7, 14.4 Hz, 1H), 4.31 (dd, J = 2.7, 6.2 Hz, 1H), 4.38 (d, J = 5.5 Hz, 1H), 4.78 (d, J = 5.5 Hz, 1H); 13C NMR δ 14.0, 22.5, 25.5, 27.0, 28.0, 28.3, 29.6, 31.3, 33.7, 33.9, 60.8, 76.1, 77.6, 83.9, 112.3, 149.8, 171.0; MS (APCI) m/z 288 (100, [MH2-Boc]+). N-Boc deprotected 14 had: 1H NMR δ 0.88 (t, J = 7.0 Hz, 3H), 1.25–1.36 (m, 6H), 1.38 (s, 3H), 1.48 (s, 3H), 1.56.–1.62 (m, 2H), 2.52.–2.75 (m, 3H), 2.73 (dd, J = 5.9, 13.4 Hz, 1H), 3.81 ('t', J = 6.1 Hz, 1H), 4.50 (d, J = 5.9 Hz, 1H), 4.69 (d, J = 5.9 Hz, 1H), 5.94 (s, 1H); 13C NMR δ 14.0, 29.7, 22.5, 28.5, 31.4, 25.6, 26.9, 33.2, 33.7, 58.0, 76.6, 79.2, 112.7, 173.2; MS (APCI) m/z 288 (100, MH+).</p><!><p>TFA/H2O (1 mL, 9:1) was added to 17 or N-Boc deprotected 17 (22 mg, 0.07 mmol) and the resulting solution was stirred at 0 ºC for 3 h. Evaporation of volatiles gave light yellow oil that was column chromatographed (5 → 10% MeOH/EtOAc) to give 19 (12 mg, 63%) as a colorless oil: 1H NMR δ 0.87 (t, J = 7.0 Hz, 3H), 1.24–1.39 (m, 6H), 1.52–1.59 (m, 2H), 2.50–2.55 (m, 3H), 2.73 (dd, J = 5.5, 13.6 Hz, 1H), 3.71 ('t', J = 6.4 Hz, 1H), 4.21 (d, J = 5.0 Hz, 1H), 4.44 (d, J = 5.0 Hz, 1H), 7.11 (s, 1H); 13C NMR δ 14.0, 29.6, 14.1, 22.5, 31.4, 32.7, 35.3, 59.9, 69.8, 71.8, 176.0; MS (APCI) m/z 248 (100, MH+); HRMS (TOF MS-ESI) m/z calcd for C11H21NO3SNa [M+Na]+ 270.1134; found 270.1137.</p><!><p>Treatment of 17 (40 mg, 0.1 mmol) in THF (1 mL) with LiEt3BH (1M/THF, 0.26 mL, 0.26 mmol), by procedure C [column chromatography (10 → 20% EtOAc/hexane)] gave 4-amino-N-(tert-butoxycarbonyl)-4-deoxy-5-S-hexyl-3,4-O-isopropylidene-5-thio-α/β-D-ribofuranose 21 (39 mg, 97%)] as a colorless oil of the mixture of isomers: MS (ESI) m/z 389 (100, M+); HRMS (TOF MS-ESI) m/z calcd for C19H35NO5SNa [M+Na]+ 412.2128; found 412.2117. Deprotection of 21 (39 mg, 0.1 mmol) with TFA/H2O (0.9:0.1 mL) by Procedure D gave a light yellow oil that was column chromatographed (5 → 10% MeOH/EtOAc) to give 23 (22 mg, 88%) as a light yellow oil. 1H NMR showed a mixture of isomers accompanied by the open aldehyde form. MS (APCI) m/z 230 (40, MH+), 232 (100, [M-17]+).</p><!><p>An overnight (O/N) culture of Escherichia coli DH5α harboring the plasmids pSC11, which contains a PlasI-lacZ translational fusion,34 and pJN105L, which contains a PBAD-lasR expression plasmid35 grown in LB media (10 g tryptone, 5 g yeast extract, 5 g sodium chloride per liter) supplemented with ampillicin (100 μg/ml) and gentamycin (15 μg/ml), was diluted to an OD600 of 0.150. At this time, arabinose (0.2 % w/v), N-3-(oxododecanoyl)homoserine lactone (3-oxo-C12-AHL; 2 μM), and either the compound under analysis or solvent (DMSO), was added to the culture (1.5 mL). A negative control containing only solvent, antibiotic, and arabinose (0.2 % w/v) without 3-oxo-C12-AHL was also assayed (data not shown). The cultures were incubated with shaking for three hours at 37 °C.</p><p>The conditions for the rhl biomonitor Escherichia coli DH5α harboring pECP61.5 plasmid, which contains Ptac-rhlR and PrhlA-lacZ4 were essentially same except that the LB medium was only supplemented with ampillicin (100 μg/ml), the O/N culture was diluted to an OD600 of 0.150, induced with 1 mM IPTG, 2 μM C4-HSL and the compounds or the controls added when the OD600 reached 1.0. A negative control containing only solvent, antibiotic and IPTG (1 mM) without C4-AHL was also assayed (data not shown). After incubation at 37°C for 4 hours with shaking, β-galactosidase activity was assayed as described previously.37 Miller units were calculated as described.38 Assays were repeated at least twice. For each biological replicate, experimental triplicates were performed and the average percent activity calculated by dividing the average Miller units from the samples containing compound or extract by the average Miller units from the sample containing solvent and multiplying by 100. Significance of inhibition was determined using a paired two-tailed Student t-test.</p>

PubMed Author Manuscript

Discovery and Optimization of Potent and CNS Penetrant M5-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold

The pharmacology of the M5 muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M5 positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M5 PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M5 PAM EC50 values <100 nM and rat brain/plasma Kp values of ~0.40. Interestingly, unlike M1 and M4 PAMs with unprecedented mAChR subtype selectivity, this series of M5 PAMs displayed varying degrees of PAM activity at the other two natively Gq-coupled mAChRs, M1 and M3, yet were inactive at M2 and M4.

discovery_and_optimization_of_potent_and_cns_penetrant_m5-preferring_positive_allosteric_modulators_

INTRODUCTION<!>Toward the Next Generation of M5 PAMs.<!>Chemistry and Limited Structure\xe2\x80\x93Activity Relationships (SARs).<!>Molecular Pharmacology Studies.<!>CONCLUSIONS<!>Chemical Synthesis and Purification.<!>tert-Butyl 4-[[(1R)-Indan-1-yl]carbamoyl]piperidine-1-carboxylate (11a).<!>tert-Butyl 4-[Ethyl-[(1R)-indan-1-yl]carbamoyl]piperidine-1-carboxylate (12a).<!>N-Ethyl-N-[(1R)-indan-1-yl]-1-(1H-indazol-5-ylsulfonyl)-piperidine-4-carboxamide (8, VU0488129).<!>Cell Lines and Calcium Mobilization Assay.<!>Radioligand Binding Assay.<!>In Vitro.<!>In Vivo.<!>Liquid Chromatography/Mass Spectrometry Analysis.

<p>Of the five muscarinic acetylcholine receptors (M1–M5), M5 has the lowest expression level and distribution as well as the fewest chemical tools to probe the receptor's biology.1–10 Until recently, M5 knockout (KO) mice and resultant phenotypic observations afforded the only insight into the physiological role of and therapeutic potential for M5 modulation.11–14 From these studies, M5 emerged as a potentially ideal target for drug addiction,15–17 and recent work with the M5 negative allosteric modulator (NAM) ML375 (1)18 recapitulated the M5 KO mouse data, displaying robust efficacy in models of cocaine use disorder,19 ethanol seeking,20 and opiate abuse.21 Beyond ML375, several other highly selective M5 inhibitors, such as the short half-life NAM 222 and the highly selective orthosteric M5 antagonist 323 (Figure 1) have been reported. Observations from the M5 KO mice also suggest that selective activation of M5 would be beneficial for cognition, Alzheimer's disease, schizophrenia, and ischemic stroke, by enhancing dilation of CNS vasculature and increasing cerebral blood flow.11–14 However, to validate this hypothesis, potent, selective, and CNS penetrant M5 positive allosteric modulators (PAMs) are required. Efforts with M5 PAMs have been more limited (Figure 1). We reported the first M5 PAM, ML129 (4),24 derived from a pan-M1,3,5 PAM,25 which then led to the discovery of a more potent congener, ML326 (5).26 However, neither of these isatin-based PAMs possessed acceptable CNS penetration.24–26 A new high-throughput screen (HTS) then afforded a new M5 PAM-preferring chemotype, exemplified by ML380 (6),27 yet M5 PAM potency and pharmacokinetics precluded this PAM from serving as an in vivo tool. Here, we report of the further optimization of ML380 leading to the discovery of a new chiral indanyl core with enantiospecific activity that provided highly potent M5 PAMs (at both rat and human M5) and new insights into the origins of muscarinic acetylcholine receptor (mAChR) PAM subtype selectivity.28</p><!><p>Initially, optimization of ML380 (hM5 EC50 = 190 nM) used a calcium mobilization assay with a 96 well-format FlexStation II microplate reader (Molecular Devices, LLC). When the recent optimization campaign initiated, we had developed a rat M5 cell line and were routinely assessing functional activity at both human and rat M5 on an industry-standard 384-well-format Hamamatsu FDSS7000 screening system.22 As a benchmark, we re-evaluated ML380 under these new optimal screening conditions and noted an ~5-fold rightward shift in M5 PAM potency (hM5 EC50 = 1.1 μM); thus for a robust in vivo tool, we also needed to increase M5 PAM activity ~10-fold. As all of our M5 NAMs displayed enantiospecific activity,18,22,23 we were driven to identify locations within the ML380 scaffold where we could introduce a chiral center and hopefully engender a significant increase in functional potency that might also afford new avenues for productive, tractable SAR. As ML380 possessed a lipophilic CF3 moiety in the 2 position and in close proximity to the benzylic position,27 we envisioned the introduction of a cyclic constraint in the form of an indanyl ring system providing racemic 7 (Figure 2), which proved to be an equipotent M5 PAM (hM5 EC50 = 1.06 μM, pEC50 = 5.98 ± 0.03, 89% ± 1% ACh Max). Synthesis of the two single enantiomers of 7 did afford enantiospecific M5 PAM activity, with the (R)-enantiomer, VU0488129 (8), displaying improved potency (hM5 EC50 = 481 nM, pEC50 = 6.34 ± 0.08, 71% ± 4% ACh Max; rM5 EC50 = 409 nM, pEC50 = 6.42 ± 0.06, 63% ± 3% ACh Max) relative to ML380, while the (S)-enantiomer (9) was inactive at both hM5 and rM5. To further evaluate 8 as a putative new lead, we determined several in vitro and in vivo DMPK properties. PAM 8 displayed attractive free fraction in rat and human plasma (fu,p = 0.043 and 0.019, respectively) as well as rat brain (fu,br = 0.017). However, predicted hepatic clearance in both rat and human was high (CLhep = 69.3 and 20.8 mL·min−1·kg−1, respectively, based on microsomal intrinsic clearance (CLint)), but with CNS penetration (rat brain/plasma Kp = 0.17, Kp,uu = 0.07) rivaling centrally active M1 PAMs. Thus, PAM 8 became the lead for further optimization for which we would survey functionalized indanyl systems, ring-expanded congeners, piperidine mimetics, and alternate sulfonamides. In parallel (vide infra), we initiated in vitro metabolite experiments to understand the high predicted hepatic clearance.</p><!><p>The chemistry to access this new series of M5 PAMs was straightforward (Scheme 1).29 From commercial 10, a standard HATU coupling with (R)-indanyl amine (or the tetrahydronaphthyl amine) provides secondary amides 11 in yields ranging from 82% to 89%. N-Alkylation of 11 to form the tertiary amides 12 proceeds in moderate to good yields (33–97%), followed by a Boc removal to deliver the free piperidine, which was then converted directly to the sulfonamides 13 (17–74% from the HCl salt). Piperidine mimetics (such as azetidine or [3.3.0] and [3.1.0] ring systems) utilized starting materials analogous to 10, but all were devoid of M5 PAM activity.</p><p>Initial SAR was robust, and we were pleased to see that this series did not fall into "flat" SAR (Table 1).6 The indazole moiety of 8 could be replaced with either a piperonyl group (13b), a cyclic carbamate (13c), or a 4-acetamide derivative (13f) without any significant loss in M5 PAM potency. This was unanticipated, and there appears to be remarkable tolerance for a wide range of hydrogen bond donors and acceptors. A quinoline congener (13g) lost about 2.5-fold in potency relative to 8, but a naphthalene analog (13h) surprisingly lost all activity. Finally, ring-expanded analogs 13k and 13l proved more potent than 8, with EC50 values of 256 nM and 255 nM, respectively. Overall, remarkable tolerability of changes to both the eastern and western portions of this new M5 PAM scaffold and a 4-fold improvement in potency over the prototypical M5 PAM 6 (ML380)27 were observed.</p><p>Based on the high predicted hepatic clearance of 8, we evaluated several of the new analogs 13 (13a, 13b, 13c, 13f, 13k, and 13l) and found that all were predicted to be cleared at hepatic blood flow rates in both rat and human (CLhep >68 and >20 mL·min−1·kg−1, respectively) despite possessing unique structural motifs. In rat iv PK cassette studies, there was a good in vitro/in vivo correlation (IVIVC) with all analogs showing high clearance (CLp > 65 mL·min−1·kg−1) and short half-lives (t1/2 < 15 min). In parallel, we conducted metabolite identification (MetID) studies in both rat and human hepatic microsomes with PAM 8 in an attempt to discern metabolic "hot spots" that could then be addressed via chemistry to provide PAMs with suitable PK for in vivo studies. The MetID studies quickly rationalized the high in vitro and in vivo clearance values, as PAM 8 was subject to extensive oxidative metabolism (Figure 3). M1 was the major metabolite in rat, resulting from N-dealkylation of the tertiary amide and oxidation of the indane ring. In human, there were three major metabolites: M2 (oxidation of the indane ring), M3 (presumed further oxidation of M2 to a ketone), and M5 (N-dealkylation of the indane ring).</p><p>Thus, we elected to chemically modify analogs 13 based on the rat microsomal MetID data, and we first directed attention to the tertiary amide moiety. Analogs in this series were prepared using similar alkylation chemistry as shown in Scheme 1, from additional secondary amides 11 (see Supporting Information for details). As shown in Table 2, the secondary amide 14a (also metabolite M6) was inactive, while the truncated methyl congener lost ~5-fold PAM activity. Homologation increased M5 PAM potency, with an n-propyl derivative, 14c, being the most potent M5 PAM thus far (hM5 EC50 = 112 nM). Finally, a deuterated version of 8, 14e, was of comparable potency to 8, but the reliance on the kinetic isotope effect in this instance had no impact on reducing clearance.30 As pretreatment with a pan-CYP450 inhibitor, such as 1-ABT, has previously increased Cmax and AUC for other PAMs,27,31 we explored this option with a representative compound to potentially enable target validation studies. However, this approach was not successful as 1-ABT pretreatment had negligible impact on Cmax and only afforded <10% increase in AUC following a single 10 mg/kg intraperitoneal (ip) administration to male Sprague–Dawley rats (data not shown). In this case, dosing via the ip route to partially avoid the hepatic first pass metabolism provided plasma Cmax levels approaching 2 μM (total), but estimated unbound concentrations were below the compound's in vitro EC50.</p><p>As modifications to the tertiary amide were not productive, we turned our attention to the indane ring. Here, we developed a synthetic route to enable a "fluorine walk"6 around the indane ring system, as well as allowing for fluorine incorporation in the 4-position of the piperidine ring (Scheme 2). In cases when the chiral amine starting materials were not commercially available, Ellman sulfinimine chemistry32 on substituted indanones 15 provided (R)-16 after NaBH4-mediated sulfinimine reduction, which were then hydrolyzed to key fluorinated (R)-indanes 17. Following the sequence described for Scheme 1 (HATU coupling, alkylation, Boc deprotection, and sulfonylation), analogs 18 were prepared. In cases where direct alkylation of the secondary amide proved difficult, commercially available primary chiral amines could also first be alkylated under reductive amination conditions to give secondary amine intermediates 19 (see Supporting Information for details) prior to amide coupling.29 As shown in Table 3, fluorine incorporation on the indane ring had a major impact on M5 PAM activity, with only the 4-F congener 18a retaining activity (EC50 = 500 nM); all other isomers decreased 5–10-fold in M5 PAM activity, which once again highlights the value of the fluorine walk in allosteric modulator optimization.6 Fluorine incorporation at the 4-position of the piperidine, as in 18e and 18f, led to only a slight increase in potency, but CNS penetration improved (18f, rat brain/plasma Kp = 0.40, Kp,uu = 0.13).</p><p>However, homologation of the ethyl side in 18a to an N-propyl analog (18g) afforded >10-fold increase in M5 PAM potency (EC50 = 41 nM), and this held true for alternate sulfonamides as well (18h, EC50 = 58 nM). Ring expansion (18j) provided comparable potency (EC50 = 161 nM) to 14c, yet expansion to a seven-membered ring further increased M5 PAM activity (EC50 = 76 nM). Incorporation of an oxygen atom into 18j led to a slight loss in potency (EC50 = 298 nM). Despite the significant structural changes across analogs 13, 14, and 18, both predicted hepatic clearance and in vivo clearance in rat were high (CLhep > 65 mL·min−1·kg−1 and CLp > 60 mL·min−1·kg−1) with rat brain/plasma Kp values in the 0.15 to 0.40 range (and favorable CNS MPO scores33). Thus, this new series appears to be relegated for use as in vitro tools (or electrophysiology tools); however, more extensive PK studies employing alternative routes of administration may enable in vivo utility. Prior to these decisions, we elected to take a more detailed examination of the molecular pharmacology and ensure comparable activity at rat M5 and overall mAChR selectivity.29</p><!><p>We were pleased to find that there were no major species disconnects in potency for this series of M5 PAMs, especially for the more potent analogs 18 (Table 4) between human and rat M5. However, when we assessed selectivity at human and rat M1–M4, we were surprised to find that all of these analogs showed varying degrees of PAM activity at the Gq-coupled mAChRs M1 and M3 yet were very weak to inactive at M2 and M4. As an example, the most potent M5 PAM in this set, 18g (VU6007678) proved to be M5-preferring, but was only 10–20-fold selective versus M3 and M1, respectively (Figure 4). We had encountered pan-Gq M1,3,5 PAMs previously24 but were able to develop highly selective M1 PAMs34 and M5 PAMs25 from that lead; however, selective M3 PAMs remained elusive.</p><p>Within this new series of M5 PAMs, we were unable to completely dial-out activity at human and rat M1 and M3. In radioligand competition binding assays with [3H]NMS, there is no binding interaction with the orthosteric site at either M5 (or M3), though there is a hint of positive cooperativity with the antagonist at higher concentrations (see Supporting Information).29 What could be the cause for the lack of subtype selectivity for this series of M5 PAMs? In contrast to the historical dogma of allosteric modulators, are there conserved allosteric sites across the Gq-PAMs? Or, could there be varying degrees of cooperativity that give rise to varying selectivity profiles at M1, M3, and M5? Emerging data suggest the lack of mAChR selectivity may arise from differential cooperativity via a "common" allosteric site, as opposed to pure differences in allosteric site sequence divergence; our current findings are in support of this hypothesis.28</p><!><p>We have reported a novel series of M5 PAMs, based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M5 PAM EC50 values <100 nM, rat brain/plasma Kp values of ~0.40 and enantiospecific activity. Once again, the fluorine walk proved essential in the optimization effort to develop the most potent M5 PAMs to date, for example, 18g (VU6007678), EC50 = 41 nM. However, the series was plagued with significant metabolic liabilities that could not be abrogated by either pretreatment with a pan-CYP inhibitor or utilization of the kinetic isotope effect. Interestingly, unlike M1 and M4 PAMs with unprecedented mAChR subtype selectivity, this series of M5 PAMs displayed varying degrees of PAM activity at the other two Gq-coupled mAChRs, M1 and M3, yet were inactive at M2 and M4; moreover, molecular pharmacology studies suggest the lack of selectivity is due to differential cooperativity. Thus, while compounds from this series will not likely become useful as in vivo tools, they may prove highly valuable in facilitating understanding of mechanisms underlying mAChR allosteric modulator selectivity.</p><!><p>All reactions were carried out employing standard chemical techniques under inert atmosphere. Solvents used for extraction, washing, and chromatography were HPLC grade. All reagents were purchased from commercial sources and were used without further purification. Analytical HPLC was performed on an Agilent 1200 LCMS with UV detection at 215 and 254 nm along with ELSD detection and electrospray ionization (ESI), with all final compounds showing ≥95% purity and a parent mass ion consistent with the desired structure. All NMR spectra were recorded on a 400 MHz Brüker AV-400 instrument. 1H chemical shifts are reported as δ values in ppm relative to the residual solvent peak (MeOD = 3.31, CDCl3 = 7.26). Data are reported as follows: chemical shift, multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant (Hz), and integration. 13C chemical shifts are reported as δ values in ppm relative to the residual solvent peak (MeOD = 49.0, CDCl3 = 77.16). When visible, minor rotamer peaks are denoted with an * in 1H NMR spectra. Low resolution mass spectra were obtained on an Agilent 1200 LCMS with electrospray ionization, with a gradient of 5–95% MeCN in 0.1% TFA water solution over 1.5 min. High resolution mass spectra were obtained on an Agilent 6540 Ultra High Definition (UHD) Q-TOF with ESI source. Automated flash column chromatography was performed on an Isolera One by Biotage. Preparative purification of library compounds was performed on a Gilson 215 preparative LC system. Optical rotations were acquired on a Jasco P-2000 polarimeter at 23 °C and 589 nm. The specific rotations were calculated according to the equation [α]D23 = (100∝)/(l × c) where l is path length in decimeters and c is the concentration in g/100 mL. For full experimental procedures, please see the Supporting Information.</p><!><p>To a solution of 10 (2.5 g, 10.9 mmol) and (R)-(–)-1-aminoindane hydrochloride (2.77 g, 16.4 mmol, 1.5 equiv) in DCM (45 mL) was added DIPEA (4.75 mL, 27.3 mmol, 2.5 equiv). After 5 min stirring, HATU (8.29 g, 21.8 mmol, 2 equiv) was added. The resulting mixture was stirred at rt overnight, after which time sat. NaHCO3 was added. Aqueous layer was extracted with DCM. Combined organic extracts were washed with brine and dried with MgSO4. Solvents were filtered and removed, and crude residue was purified by column chromatography (12–100% EtOAc in hexanes) to give product as a white solid (3.08 g, 82%). 1H NMR (400 MHz, CDCl3) δ 7.25–7.22 (m, 4H), 5.83 (d, J = 8.2 Hz, 1H), 5.49 (q, J = 7.8 Hz, 1H), 4.15 (br, 2H), 3.02–2.95 (m, 1H), 2.92–2.84 (m, 1H), 2.74 (br, 2H), 2.64–2.56 (m, 1H), 1.85–1.64 (m, 6H), 1.47 (s, 9H). 13C NMR (101 MHz, CDCl3) δ 174.17, 154.66, 143.40, 143.16, 128.02, 126.82, 124.86, 123.87, 79.62, 54.44, 43.40, 34.09, 30.21, 28.85, 28.61, 28.44. LCMS (215 nm) RT = 0.978 min (>98%); m/z 289.4 [M + H]+, minus t-butyl. HRMS (TOF, ES+) C20H29N2O3 [M + H]+ calcd mass 345.2178, found 345.2173. Specific rotation [α]D23 = +67.7° (c = 0.91, MeOH).</p><!><p>To a dry flask was added NaH (45.3 mg, 1.13 mmol, 2 equiv, 60% dispersion in mineral oil). DMF (5 mL) was then added, and the flask was cooled to 0 °C under an inert atmosphere. Compound 11a (195 mg, 0.57 mmol) in DMF (3 mL) was then added dropwise. The flask was warmed to rt and stirred for 30 min, after which time it was cooled back to 0 °C, and iodoethane (0.18 mL, 2.26 mmol, 4 equiv) was added dropwise. The resulting solution was warmed to rt and stirred for 2.5 h, after which time the reaction was quenched with the addition of sat. NH4Cl and extracted with EtOAc. Combined organic extracts were washed with H2O (3×) and brine (1×) and dried with MgSO4. Solvents were filtered and removed to give product as a white solid, which was pure by LCMS and used without further purification (202 mg, 96%). Note: substitutions with longer alkyl halides do not proceed to completion under these conditions and require purification by column chromatography (hex/EtOAc). 1H NMR (1.2:1 rotamer ratio, asterisk denotes minor rotamer peaks where separable 400 MHz, CDCl3) δ 7.30–7.17 (m, 3H), 7.12, 7.06* (d, J = 7.2 Hz, 1H), 6.20*, 5.45 (t, J = 7.9 Hz, 1H), 4.18 (br, 2H), 3.42–3.31, 3.28–3.20* (m, 1H), 3.13–2.86 (m, 3H), 2.85–2.63 (m, 3H), 2.50–2.42 (m, 1H), 2.17–1.70 (m, 5H), 1.49*, 1.48 (s, 9H), 1.14*, 1.10 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 175.50, 174.53, 154.72, 143.71, 142.90, 142.10, 141.32, 128.21, 127.69, 126.85, 126.55, 125.29, 124.91, 124.05, 123.64, 79.58, 79.55, 62.84, 59.18, 43.47, 39.79, 38.54, 31.34, 30.46, 30.32, 29.97, 29.34, 29.05, 28.82, 28.65, 28.45, 17.37, 14.58. LCMS (215 nm) RT = 1.135 min (>98%); m/z 317.4 [M + H]+, minus t-butyl. HRMS (TOF, ES+) C22H33N2O3 [M + H]+ calcd mass 373.2491, found 373.2484. Specific rotation [α]D23 = +57.9° (c = 0.74, MeOH).</p><!><p>Compound 12a (109 mg, 0.29 mmol) was dissolved in 4 M HCl in 1,4-dioxane solution (10 mL) and stirred at rt for 1 h, after which time solvents were removed under reduced pressure, and the resulting amine was used directly as the HCl salt (white solid) (87 mg, 96%); m/z 273.4 [M + H]+. To a solution of the HCl salt (31.2 mg, 0.101 mmol) in DCM (1 mL) was added DIPEA (0.035 mL, 0.202 mmol, 2 equiv), followed by 1H-indazole-5-sulfonyl chloride (32.8 mg, 0.152 mmol, 1.5 equiv). The resulting solution was stirred at rt for 1 h, after which time solvents were concentrated. Crude residue was purified by RP-HPLC, and fractions containing product were basified with sat. NaHCO3. Aqueous layer was extracted with 3:1 chloroform/IPA. Solvents were filtered through a phase separator and concentrated to give product as a colorless oily solid (11.9 mg, 26%). 1H NMR (1.4:1 rotamer ratio, asterisk denotes minor rotamer peaks where separable, 400 MHz, CDCl3) δ 8.27*, 8.24 (s, 1H), 8.19*, 8.16 (s, 1H), 7.75–7.69 (m, 1H), 7.59 (t, J = 10.1 Hz, 1H), 7.19–7.11 (m, 3H), 7.00–6.97 (m, 1H), 6.09*, 5.25 (t, J = 7.8 Hz, 1H), 3.90–3.80 (m, 2H), 3.33–2.74 (m, 4H), 2.51–2.31 (m, 4H), 2.07–1.68 (m, 5H), 1.02, 0.98* (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 175.03, 174.01, 143.80, 142.90, 141.85, 141.39, 141.00, 135.94, 129.03, 128.81, 128.35, 127.87, 126.93, 126.65, 125.37, 125.24, 125.03, 124.11, 123.54, 122.76, 110.75, 110.69, 62.90, 59.39, 45.78, 45.66, 38.68, 38.47, 31.32, 30.53, 30.36, 29.96, 28.92, 28.67, 28.42, 28.35, 17.34, 14.59. LCMS (215 nm) RT = 0.999 min (>98%); m/z 453.4 [M + H]+. HRMS (TOF, ES+) C24H29N4O3S [M + H]+ calcd mass 453.1960, found 453.1950. Specific rotation [α]D23 = +51.0° (c = 0.32, MeOH).</p><!><p>Chinese hamster ovary (CHO) cells stably expressing human and rat M5 were maintained in Ham's F-12 growth medium containing 10% FBS, 20 mM HEPES, antibiotic/antimycotic, and 500 μg/mL G418 in the presence of 5% CO2 at 37 °C. To determine the potency and efficacy of M5 PAMs, calcium flux was measured using the Functional Drug Screening System (FDSS7000, Hamamatsu, Japan). Briefly, the M5-CHO cells were plated in black-walled, clear-bottomed 384 well plates (Greiner Bio-One, Monroe, NC) at 20 000 cells/well in 20 μL of growth medium without G418 the day before assay. The following day, cells were washed with assay buffer (Hank's balanced salt solution, 20 mM HEPES, and 2.5 mM probenecid) and immediately incubated with 20 μL of 1.15 μM Fluo-4-acetomethoxyester (Fluo-4 AM) dye solution prepared in assay buffer for 45 min at 37 °C. During the incubation time, all compounds were serially diluted (1:3) in DMSO for 10-point concentration–response curves (CRCs) and further diluted in assay buffer at starting final concentration 30 μM using Echo liquid handler (Labcyte, Sunnyvale CA). Dye was removed and replaced with assay buffer. Immediately, calcium flux was measured using the FDSS7000 as previously described.24,27 The compounds or vehicle was added to cells for 2.5 min, and then an EC20 concentration of acetylcholine (ACh) was added and incubated for 1 min. ECmax concentration was also added to cells that were incubated with DMSO vehicle to ensure the EC20 calcium response. Using a four-point logistical equation in GraphPad Prism 5.0 (GraphPad Software, Inc., La Jolla, CA), the concentration–response curves were generated for determination of the potency and efficacy of the PAM.</p><!><p>Competition binding assay was performed using [3H]-N-methylscopolamine ([3H]NMS, PerkinElmer. Boston, MA) as previously described.18 Compounds were serially diluted 1:3 in DMSO for 11-point CRC, then further diluted at starting final concentration of 30 μM in binding buffer (20 mM HEPES, 10 mM MgCl2, and 100 mM NaCl, pH 7.4). Membranes from either human M5-CHO cells or M3-CHO cells (10 μg) were incubated with the serially diluted compounds in the presence of a Kd concentration of [3H]NMS, 0.376 nM, at room temperature for 3 h with constant shaking. Nonspecific binding was determined in the presence of 10 μM atropine. Binding was terminated by rapid filtration through GF/B Unifilter plates (PerkinElmer) using a Brandel 96-well plate Harvester (Brandel Inc., Gaithersburg, MD), followed by three washes with ice-cold harvesting buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl). Plates were air-dried overnight, 50 μL of Microscint20 was added to the plate, and radioactivity was counted using a TopCount Scintillation Counter (PerkinElmer Life and Analytical Sciences).</p><!><p>Plasma protein binding, brain homogenate binding, and hepatic microsomal intrinsic clearance assays with the M5 PAMs were conducted using the same methods described previously.30,31 Metabolite identification experiments were also performed essentially as described previously using rat and human hepatic microsomes.23,31</p><!><p>Select M5 PAMs were administered to male Sprague–Dawley rats via single IV or IP administrations of cassette doses (0.20–0.25 mg/kg; n = 1 animal per cassette) formulated in ethanol/PEG400/DMSO vehicles (varying concentrations of excipients to afford solutions, dependent upon the compounds' solubilities; 0.5 mL/kg iv or 3 mL/kg ip dose volumes). For determination of pharmacokinetics and brain distribution, serial sampling of plasma and nonserial sampling of plasma and brain with subsequent quantitation of analytes via LC-MS/MS were conducted essentially as described previously.31,35 All animal studies were approved by the Vanderbilt University Institutional Animal Care and Use Committee. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International.</p><!><p>Samples from in vitro and in vivo assays or studies with M5 PAMs were analyzed via electrospray ionization (ESI) on an AB Sciex API-4000 or Q-Trap 5500 (Foster City, CA) triple-quadrupole mass spectrometer instrument that was coupled with Shimadzu LC-10AD or LC-20AD pumps (Columbia, MD) and a Leap Technologies CTC PAL autosampler (Carrboro, NC). Analytes were separated by reverse-phase gradient elution and monitored by analyte-specific multiple reaction monitoring (MRM) utilizing a Turbo-Ionspray source in positive ionization mode (5.0 kV spray voltage), essentially as described previously. All raw data were analyzed using AB Sciex Analyst (v. 1.4.2 or later) software, and pharmacokinetic noncompartmental analysis (NCA) of time–concentration data was performed using Pharsight Phoenix WinNonLin (v. 6.0 or later; Certara L.P., Princeton, NJ).</p>

PubMed Author Manuscript

Direct quantification of nanoparticle surface hydrophobicity

Hydrophobicity is an important parameter for the risk assessment of chemicals, but standardised quantitative methods for the determination of hydrophobicity cannot be applied to nanomaterials. Here we describe a method for the direct quantification of the surface energy and hydrophobicity of nanomaterials. The quantification is obtained by comparing the nanomaterial binding affinity to two or more engineered collectors, i.e. surfaces with tuned hydrophobicity. In order to validate the concept, the method is applied to a set of nanoparticles with varying degrees of hydrophobicity. The technique described represents an alternative to the use of other methods such as hydrophobic interaction chromatography or water-octanol partition, which provide only qualitative values of hydrophobicity.

direct_quantification_of_nanoparticle_surface_hydrophobicity

E<!>Results<!>illustrates typical examples of transmission electron microscopy<!>Discussion<!>Methods

<p>ngineered nanomaterials (NMs) are widely used in many consumer and healthcare products, as well as novel nanomedicines. 1 To enable a quick and reliable NMs risk-benefit assessment, there is an urgent need for robust, standardised and reliable methods for their characterisation and toxicity screening. For this purpose, the understanding of the behaviour and fate of these NMs when in contact with biological systems is important. Physical and chemical properties such as size, surface chemistry and surface charge have been identified as essential parameters to determine, because they affect the NMs' mode of action in a given environment (water, buffer, biological fluid, etc.) through different surface molecular interactions.</p><p>In particular, hydrophobicity is considered as an important property since it has a critical role in various biological processes such as protein adsorption, 2 interaction with biological membranes, 3 cellular uptake, 4,5 immune response, 6 and haemolytic effect. 7 It is recognised that hydrophobicity is a key property to be controlled for nanomedicine applications since it has a direct influence on the stability and bio-distribution of nanovectors. 3,6,8 Only a few methods are currently available for characterisating the hydrophobicity of NMs 9 -for example surface adsorption assays, 10 NMs relative affinity for reference phases, and hydrophobic interaction chromatography. 11 None of those methods enables, for all NM types, a full characterisation and quantification of the NMs hydrophobicity and they all involve expensive and time-consuming analytical techniques. The development of a fast and reliable general method for NMs hydrophobicity characterisation is therefore of great interest.</p><p>We described in a previous publication 12 a new method for the separation of NMs according to their hydrophobicity based on a set of collectors composed of fluorinated hydrophobic surfaces whose surface energy components can be modified and finetuned by the layer-by-layer (LbL) deposition of polyelectrolytes. A proof-of-concept study of the method was carried out with hydrophobic and hydrophilic polystyrene nanoparticles. The experimental results were qualitatively supported by the eXtended Derjaguin Landau Verwey Overbeek (XDLVO) theory, 13 enabling the assessment of the different forces in play.</p><p>Here we extend this method to quantitatively determine the surface energy components of the NMs by measuring their binding affinity to the collectors' surfaces, via analysis of their adsorption kinetics. The adsorption kinetics is calculated by measuring the number of nanoparticles binding to the different collector as a function of time, by Dark-Field microscopy. The method is particularly suitable for nanoparticles with large light scattering capability (for example noble metals with typical size >50 nm), but it is also applicable to other materials such as SiO 2 with typical size >200 nm. On the other hand, by using a special dark-field condenser 14 or other single-particle microscopy techniques 15 it is in principle possible to detect NMs of any material and down to sizes of the order of 20 nm.</p><p>The surface energy potential acting between each NM and the collector is then calculated using the XDLVO theory. The energy barrier potential between the NMs and the collector represents the potential energy at which the NMs are repelled by the collector surface. 16 This energy barrier potential is inversely proportional to the binding affinity: the larger the energy barrier potential, the lower the affinity. In some conditions, the NMs may be completely repelled by the collector due to very high energy barrier potential. Electrostatic forces are mainly responsible for the formation of the energy barrier; hence for an accurate calculation, the Z-potential for both the surfaces and the NMs should be rigorously measured.</p><p>The NM's surface energy and in particular the degree of hydrophobicity is then calculated by comparing the NM binding affinity of two or more collectors. The experiments were performed with Au and SiO 2 nanoparticles (NPs) with different degrees of hydrophobicity to test the validity of the approach. An advantage of this method is that it can cover all the surface energy range with only one set of collectors, and a quantitative determination of the surface energy is possible thanks to several measurements on different collectors.</p><!><p>Surface characterisation of collectors. The first step of the method is to determine the binding affinity of the AuNPs with the different collector surfaces. Each collector prepared as described above is characterised by distinct surface energy components that control the binding affinity of the nanomaterial. First, the binding kinetics of AuNPs was measured on each collector, in a buffer solution (Phosphate Buffer, PB 10 mM, pH = 7). The buffer composition has been chosen in order to partially neutralize the charges present on both the surface and the AuNPs, which would otherwise lead to a long-range repulsion. The principle of the method is described in Fig. 1.</p><p>The selectivity and specificity of the AuNPs binding to the surfaces strongly depend on characteristic of the interaction forces such as interaction strength as a function of distance and attractiveness and repulsiveness.</p><p>The collector and nanoparticles characterisation, the study of the binding affinity of the AuNPs on the different collectors, calculation of the acting potential between the AuNPs and the collector surfaces, and extraction of the surface energy component of the AuNPs from the adsorption rates of the AuNPs on the collectors surfaces are presented below.</p><p>In order to fabricate the collectors with different surface properties, a standard Si wafer has been coated first with a plasma deposited layer of polytetrafluoroethylene PTFE (hydrophobic) and then with several layers of polyelectrolytes (PE) (poly (diallyldimethylammonium chloride, PDDA and polysodium 4styrene sulphonate, PSS) for giving the surface a more hydrophilic character. The subsequent adhesion of PEL layer also permits the modification of the surface free energy components of the collectors. Five surfaces denominated T0 (PTFE), T1, T2, T3 and T4 are thus produced with the numerical component of the name corresponding to the number of PEL layers deposited.</p><p>According to the Owens-Fowkes-Wendt (OFW) theory, 17,18 the total surface energy of a solid is the sum of the dispersive component (taking into account the non-polar interactions), called γ LW (Lifschitz-van der Waals component) and of a polar component γ AB (acid-base component). Solid materials with low γ AB are called "hydrophobic". The increase of the γ AB of a solid corresponds to an increase of its hydrophilicity. The PTFE was chosen because of its very low γ AB . The surface free energy components have been determined by measuring the contact angle of the solid surfaces with a polar (water) and a dispersive solution (Bromonaphtalene).</p><p>The surface energy values of the as-deposited PTFE were 0.9 mJ/m 2 and 19.3 mJ/m 2 for respectively polar and dispersive components. Then, PEL layers were deposited on the collector to increase both surface energy components. The deposition of the cationic PEL leads to a decrease of the initial PTFE z-potential (−60 mV) to values close to zero, while the anionic PEL deposition does not change the negative z-potential (−60 mV). Surface properties of the PTFE and of the T2 collectors present very similar z-potential values while the T2 collector has a higher surface free energy component value, i.e. polar component increasing by a factor 5 and the dispersive components increasing by only 1.4. 12,19 The increase of the surface free energy has been directly measured by the F-D curves using different functionalized gold-coated tips (Supplementary Figure 1).</p><p>The adhesion force between the PFT (polyfluorotetraethylene) modified Au-coated silicon tip and respectively the PTFE and T2 collector were measured as 642 ± 200 nN and 248 ± 100 nN (Supplementary Figure 2). This higher adhesion force between the PFT-modified Gold on the tip and the hydrophobic PTFE is attributed to the hydrophobic interacting forces resulting from their very low polar surface energy component (the tip-surface new interface is energetically more favourable than the two surfaces alone). The average adhesion force of a polyethylene oxide (PEO)-modified Au-coated tip on both the PTFE and the T2 collector (not shown) was 100 ± 76 nN indicating that the hydrophilic surface functionalization reduces the strength of interaction between the surfaces, consequently reducing their force of adhesion.</p><p>The atomic force microscopy (AFM) analysis indicates that collector surfaces are rather homogenous with roughness increasing from 0.29 nm to 0.85 nm showing that the morphology of the surfaces is not dramatically affected by the formation of the polyelectrolytes layers. The z-potential was measured for different pH. A negative z-potential was obtained for the whole range of pH, especially for the PTFE non-modified and the PSS layers, and closer to neutral for the different PDDA layers. This result can be explained by the fact that the PDDA is positively charged and the PSS and PTFE negatively charged.</p><p>A more comprehensive overview of the surface characterisation of the collectors is given in ref. 12 .</p><p>Finally, the set of collectors presents the following features:</p><p>1. Relatively low surface roughness, root mean square (RMS) roughness <2 nm. 2. Surface chemical homogeneity, as indicated by the X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (Tof-SIMS) analysis. 12 3. Surface homogeneity at the nanoscale level in terms of the adhesion properties as indicated by the force spectroscopy measurements. 4. A surface Zeta-potential which is negative (for the PSS terminated surfaces) or slightly negative (for the PDDA terminated surfaces). 5. Different values of LW and AB components.</p><p>Nanoparticles characterisation. Supplementary Figure 3 (TEM) images obtained by spotting 4 μl AuNPs stock solution onto a C/Formvar TEM grid and dried overnight in air. Although the morphologies observed were mainly spherical, a certain degree of faceting characteristic of large AuNPs was also observed. Size distributions determined by TEM image analysis on over than 100 objects were narrow with an average value of 60.2 ± 7.9 nm. The AuNPs were functionalized with different amounts of PEO-ligands in order to produce different surface coverage on the surface i.e. different hydrophobic characters. The selected ligand chain was functionalised with a SH-group at one terminus to provide a strong affinity for gold, a central non-polar alkyl chain to provide to the structure the ability to self-assemble in dense layer that excludes water due to the hydrophobic core and a PEO sequence to enhance stability in water.</p><!><p>The PEO surface coverage has been first characterised by UVvis adsorption measurements (Supplementary Figure 4). The results show that the increase of PEO content in the solution results in a red-shift of the surface plasmon resonance wavelength from 543 nm for uncoated to approximately 546 nm for all the functionalized PEO-Au NP samples (Table 1). The shift from the uncoated and the PEO coated surfaces might be due to slight differences in the bulk refractive index of the dispersant.</p><p>Dynamic light scattering (DLS) also confirms the successful functionalization of AuNPs 60 nm with an increase of the hydrodynamic diameter from 62.7 nm for pristine AuNPs to 68.7 nm for the highest PEO concentration (Table 1). Z-potential measurements show a slight decrease in the negative surface charge from −45.7 mV for pristine AuNPs to −39.4 mV for the AuNPs coated at the highest PEO concentration. Differential centrifugal sedimentation (DCS) shows that the sediment time increases with the PEO concentration in the solution. This shift toward higher sedimentation times is the net result of (1) the increase of NP diameters and (2) the decrease of the NP apparent densities due the PEO binding. By combining the sedimentation times measured by DCS and the DLS diameter, 20 the mass of absorbed PEO molecules on the AuNPs as a function of the PEO concentration in solution and therefore the number of molecules per NP can be calculated. The values are reported in Table 1.</p><p>A theoretical full coverage of the AuNPs by the PEO molecules can be calculated by dividing the available gold surface area of the particles by the footprint of a alkanethiolate molecule (21.4 Å 2 ), i.e. the minimum space occupied by an absorbed molecule as as estimated by electron diffraction studies of monolayers of alkanethiolates on Au(111) surfaces. 21 We must underline that the obtained value of the coverage represents a rough approximation, since it corresponds to a perfect packing of the ligand on the surface and doesn't take into account physical effects such as the steric repulsion of the PEO chains, the gold surface inhomogeneity, the possibility different orientation due to the PEO chains interaction with the gold surface, occupying more space, etc. Using the TEM diameter (60.2 nm) and considering the AuNPs as perfect spheres, we calculated a maximum number of 55 × 10 4 PEO molecules per AuNP. According to Table 1 this number is reached for a volume of PEO added to the AuNPs of 3.2 µl. Any additional PEO added to the solution would potentially lead to the formation of an excess of PEO on the surface of the AuNPs, i.e. multilayers.</p><p>More interestingly, the PEO functionalized AuNPs were characterised by two other techniques: protein adsorption by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and affinity to the water phase by water/octanol partition.</p><p>The amount of proteins detected by the SDS-PAGE on the AuNPs was very low, since the total amount of AuNPs available was very small. However, it was found that a detectable quantity of Albumin was found on both the pristine AuNPs and the AuNPs treated with low volumes of PEO (Supplementary Figure 5, Supplementary Note 2). By plotting the SDS-PAGE Albumin band intensity as a function of the volume of added PEO, it is clear that the intensity decreases and it goes to zero for a PEO volume larger than 3 µl. This means that the PEO surface coverage obtained by adding a volume of 3 µl to the solution was sufficient to prevent any protein adsorption on the surface of the particles.</p><p>A similar trend is observed in the water/octanol partition coefficient. Briefly, experiments were carried out both with pristine AuNPs of 60 nm and the differently PEO-functionalized AuNPs at a concentration of 0.5 mM of gold. As expected, pristine hydrophobic AuNPs coated with weakly bound citrate surfactant interacted strongly with octanol resulting into complete aggregation clearly visible in the interphase (Supplementary Figure 6). By increasing the degrees of PEO-functionalization, a progressive decrease in the amount of aggregation together with higher amount of NPs in the aqueous solution can be observed (increases of red colour intensity). For volume of added PEO larger than 3 µl, the AuNPs did not interact at all with Octanol as a result of their hydrophilic character. These results confirm the decrease of hydrophobicity as the PEO coverage increases and a saturation of the surface for 3 µl of thiolate added to the suspension. The proportion of NPs in water was calculated by measuring the intensity of the red colour component in Supplementary Figure 6 for each vial and normalizing to the intensity of the red colour of the original solution of NP with the same concentration in water.</p><p>These two results suggest that, as calculated by DCS/DLS combined measurements, a full monolayer of PEO is reached by adding a volume of PEO larger than about 3 µl to the pristine AuNPs solution. The addition of larger volumes of PEO did not further modify the surface properties of the AuNPs. The results from these three experiments are summarized in Fig. 2. Furthermore, we characterised the hydrophobicity/hydrophilicity degree of the Au-Citrate stabilised and of the Au-PEO NPs, by contact angle measurements performed on a dried film of NPs.</p><p>Contact angle measurements show that Au-citrate NPs are hydrophobic with a corresponding contact angle with water of about 95°. The AuNPs with the addition of 7.2 µl of PEO are on the other hand very hydrophilic, with a contact angle of 23°. A similar behaviour was observed on the flat Au surface, where the pristine Au surface showed a contact angle of 84°with water and the PEO-functionalized Au flat surface a contact angle of 40°.</p><p>The previously described combination of characterisation techniques of the AuNPs allowed us to classify the AuNPs according to their effective PEO surface coverage. The surface coverage is linearly proportional to the amount of PEO added to the pristine AuNPs solution between 0 and 3.2 µl of volumes. The proportionality factor, k, between volumes was added and the PEO surface coverage was calculated as follows:</p><p>The AuNPs samples A, B, C, D, F, G and L, corresponding to the PEO surface coverage indicated in Table 1 were used to study the binding affinity with the different collectors.</p><p>Nanoparticles binding study. The AuNPs affinity has been calculated by directly measuring the real-time binding kinetics of the NPs to the different collectors. The binding curves are built by using a script created in the software ImageJ allowing to automatically detecting the NPs as round objects in the video sequence. Typical sequence images of the adsorption of NPs on the T0 collectors are shown in Fig. 3, for the Au-citrate NPs (hydrophobic) and for the Au-100% PEO coated NPs (hydrophilic). Each round object in each frame corresponds to one AuNP; for each frame, the NPs are counted and the position of each NP is recorded. It is either possible to detect the total number of NPs for each frame or to track the NP motion on the surface. Only the NPs perfectly in focus with the surface plane are detected with a focal depth of 4000 nm. Taking this into account the nanoparticles were detected in the volume above 4000 nm from the surface: these particles are moving much faster that the time resolution of the camera (>ms). Hence we are able to observe only the nanoparticles slowed down by the contact with the surface, which are moving or rolling. The number of NPs detected is then related to the affinity of the NPs to that particular surface.</p><p>It should be noted that AuNPs with different PEO coverage are characterised by the same core size and a similar negative Zpotential. The choice of negatively charged collectors (terminated by the anionic polyelectrolyte) and buffer conditions was made in order to minimize any possible electrostatic interaction between the nanoparticles and the surfaces while keeping the stability of the colloids. Uncoated Au-citrate NPs dispersion stability is maintained also thanks to their very negative value of the zpotential while the increasing PEO coating coverage contributes to the NPs stability by steric repulsion. In the absence of an energy barrier, the number of NPs reaching the surface as a function of time Γ Ads ðtÞ is determined by the diffusion constant, D e via Eq. 2:</p><p>Where Σ is the area of measurement and C n is the bulk concentration of NPs. The maximum velocity of adsorption v max of the NPs of radius r and bulk concentration C n on an area Σ is then</p><p>Eq. 3 shows that, in the absence of an energy barrier, the NPs adsorption velocity is determined by the radius of the NPs, through their diffusion constant. 22 If an energy barrier, ΔG max , is present, the local concentration of NPs, C loc at a distance lower than the energy barrier distance is related to the bulk concentration by Eq. 4:</p><p>Eq. 4 is the classical Maxwell-Boltzmann distribution which determines the distribution of objects in the presence of an energy barrier. 22 The generalized NPs adsorption velocity is then</p><p>That substantially means that the NP adsorption velocity is reduced by a factor e ÀΔG max =kT in the presence of an energy barrier as compared to the maximum velocity.</p><p>The v max can be calculated by Eq. 3, knowing the size of the NPs or it can be measured directly by observing the rate of arrival of the negatively-charged NPs to a positively-charged surface (such as T1 or preferably T3 since one layer of polyelectrolyte on the PTFE is not sufficient to guarantee full surface coverage).</p><p>On the other hand, by measuring the velocity in the presence of an energy barrier one can calculate ΔG max =kT as</p><p>The ratio v v max can vary between 0 and 1 and it is a direct For the 60 nm AuNPs the maximum adsorption rate is obtained when the activation energy barrier is zero, and the exponential in Eq. 5 is equal to 1.</p><p>In the cases where the gravitational transport is not negligible for the timescale observed (dense and large NPs) Eq. 2 should be corrected with a factor proportional to the time and the sedimentation velocity.</p><p>where</p><p>with ρ NP and ρ f being the NP and fluid mass density respectively, and η the fluid viscosity. Eq. 7 takes into account that the local concentration of the NPs is increasing with time due to the sedimentation of the NPs.</p><p>The adsorption curves are fitted by a modified Eq. 9:</p><p>where α 1 and α 2 are respectively the linear and quadratic factors (for the polynomial function in t 1/2 ). The maximum adsorption rate which depends on the geometrical properties of the NPs is plotted in Fig. 4 in comparison with the kinetics obtained for the different NPs for the different collectors. The rate of adsorption of the NPs on the different collectors is decreasing while increasing the acid-base (polar) component of the collectors. This is more evident for the non-PEO-coated NPs. Then the rate of adsorption is found decreasing drastically with the increasing PEO% coating on the NPs. For a nominal coating of PEO larger than 25% of the NPs, the adsorption rate is close to zero.</p><p>The principal information that can be extracted from Table 2: on the T4 collector, the rate of adsorption is zero for all the NPs, the rate of adsorption is larger on the collector T0 than on the collector T2 for all the NPs and the difference of the rate of adsorption between T0 and the collector T2 is decreasing by increasing the PEO coating.</p><p>Calculation of the surface free energy component of the nanoparticles. According to the XDLVO theory 23 , the total interaction energy G tot can be expressed as</p><p>where G el G AB and G LW are energies relative to the electrostatic, acid-base and Lifshitz-Van der Waals interactions respectively. The three potential depends on the distance between the NP and the surface. Electrostatic interaction energy is</p><p>where d is the separation distance between the NP and the surface, ζ N and ζ S are the surface charge of the nanoparticle and the collector surface respectively. 1/κ is the double layer thickness, which is expressed from Eq. 12.</p><p>κ ¼ e 2 εkT</p><p>where ε is the permittivity of the medium, e is the charge of electron, k is the Boltzmann constant, T is the temperature, z i is the valency of the ions i, and n i is their number per unit volume.</p><p>The Lifshitz-Van der Waals ΔG LW components to the free energy of interaction between a nanoparticle and surface are calculated following the extended DLVO theory:</p><p>where d is a separation distance between the NP and the surface, and r is the radius of the nanoparticle.</p><p>H the effective Hamaker constant for the NP-collector-water system, which can be expressed as</p><p>Looking at the dependence of the G LW on the distance d, the Lifshitz-Van der Waals force then active for very short distances (d < 1 nm), only for the particles that are able to overcome the energy barrier.</p><p>While the analytical expressions for the electrostatic potential and the Lifshitz-Van der Waals potentials are well known and commonly accepted, the acid-base interaction potential is mainly represented as an empirical formulation based on experimental observations 24 and direct measurements of the interaction potential between two surfaces (sphere-sphere, sphere-plane, plane-plane) in a polar medium or in an electrolytic solution. The G AB is including all forces involving the structural reorganization of the water molecules around two surfaces, depending on the degree of wettability of the surfaces involved. For a sphere-plane system</p><p>where d 0 is the minimum separation distance between the NP and the surface, taken generally as 0.158 nm for many different kinds of substrates and d the separation distance in nm. G AB is defined as a short-range acting potential, having an exponential decrease with the distance. The field of interaction of the potential is mainly determined by the correlation length λ, expressed in nm. Various values for λ have been reported in literature, ranging from 0.2 nm to up to 13 nm 24 .</p><p>The nature of the two interacting surfaces intervenes in the AB potential with the term ΔG AB , which can be expressed as</p><p>where the term ffiffiffiffiffiffiffi γ AB i p refers to the polar component of the surface free energy for N = nanoparticle, W = water and S = surface.</p><p>The interaction energy maps were calculated using the function wizard included in the software OriginPro 2015. The values for the interaction energy are given in kT units (1 kT = 4 × 10 −21 J). The total interaction energy is expressed in Eq. 10 for the interaction of a NP and a surface as a function of the distance and of the hydrophobic correlation length λ. Even if the range of possible and measured value in literature for λ is broad (between 0.6 nm and 13 nm), we decided to keep it in the range between 0.6 nm and 2 nm, in order to take into account possible influences of the radius of curvature of the NPs and of the roughness of the collectors 24 . The heat-maps for the 15 nm, 60 nm and 200 nm hydrophobic NPs on hydrophobic collector are shown in Supplementary Figure 7 and illustrate the influence of the value of λ on the potential profile as a function of the NP size (Supplementary Note 3). The blue colour map indicates that the interaction energy between the NPs and the surface is equal to or lower than 0 kT. When interaction energy between the NP and the surface is equal to or larger than 5 kT the colour map is marked as red. 5 kT is considered as the threshold value for an energy barrier that would inhibit completely the adsorption of NPs to the surface. The corresponding adsorption velocity would be v _5 kT = 0.0067. v_max.</p><p>The heat maps for 60 nm bare AuNPs with T0 hydrophobic and T2 and T4 hydrophilic collectors are presented in Fig. 5.</p><p>To illustrate the variation of the energy barrier, in assuming a value of 1.65 nm for λ, (dashed line in Fig. 5) the energy barrier maximum is found lower for the T0 (about 0.5 kT) than for the T2 and T4 collectors. The position of the maximum in z is also depending on the collector, being larger for T0 (about 13 nm) and smaller (or closer to the surface) for T2 and T4.</p><p>For each collector, the energy barrier γ AB N must verify the following system of non-linear equations:</p><p>where the first equation in bracket corresponds to the value of the maximum of the energy barrier and the second equation corresponds to the condition of the maximum (first derivative equals to zero). According to Eq. 6, the maximum of energy barrier determines the reduction of the velocity of adsorption with respect to the maximum velocity (when the energy barrier is zero). The varying parameters to verify the system in Eq. 17 are: the NPs-surface distance z i which depends on the collector. The z i is the NP-surface distance at which the energy barrier has a maximum, as a function of the collector, the hydrophobic interaction characteristic distance, λ and the polar component of the surface free energy of the NPs, γ AB N .</p><p>In order to solve the non-linear system of equations in Eq. 17 we need at least two collectors, in order to have four equations with four unknowns, namely z 1 , z 2 , λ and γ AB N . The system of equations, for each couple of collectors has been numerically solved using the value boundaries listed in Supplementary Table 1. The boundaries were chosen as follows: z i is the typical range of interaction of the electrostatic forces; the γ AB N values were chosen between the value corresponding to PTFE (hydrophobic) and the one of water (the maximum hydrophilicity). The tolerance for the G tot was set at 10% of the value calculated between a hydrophilic particle and a hydrophilic collector.</p><p>The numerical solution for the T0-T2 couple of collectors for the different AuNPs is listed in Table 3.</p><p>The results obtained from the solution of the system of equations for the T0-T2 couple of collectors are physically meaningful. The fitted value for λ is very similar for the different NPs and it is very close to the one obtained graphically from Fig. 5. A value for each NP of γ AB N is then determined with this method. According to what was expected and also measured by the contact angle on the dried particles pellets, the γ AB N is the lowest for the Au-citrate NPs (1.57 mN/m 2 ) and increases for the PEO coverage percentage. For coverage larger than 25% the AuNPs are largely hydrophilic with a large value for the polar component of the surface free energy (30.95 mJ/m 2 ). The graphical evolution of the γ AB N with the PEO coverage is shown in Fig. 6.</p><p>A second type of model NPs has been used, to verify the validity of the proposed method. 200 nm hydrophilic SiO 2 NPs were modified with different chemical groups to increase the surface hydrophobicity. Three functional groups were used to modify the NP surface, consisting of alkyl chain of different length: propyl, butyl and dodecyl groups. The SiO 2 NPs modified with dodecyl groups were not stable and started to aggregate, forming dimers and trimers as shown by the DLS measurement. The propyl and butyl functionalized SiO 2 NPs were stable for days as shown by DLS measurement.</p><p>The adsorption rate of pristine SiO 2 and epoxy modified NPs on T0 (and consequently on T2 and T4) was zero.</p><p>The adhesion of propyl and butyl functionalized SiO 2 NPs to T0, the hydrophobic collector, was slow but different from zero, and it was evaluated as α 1 = 2.15 t −1/2 and α 1 = 2.17 NP/t −1/2 , respectively. The maximum adsorption rate, calculated with Eq. 2, is 232 NP/t 1/2 , meaning that the potential barrier is relatively large, around 6.97 kT and 6.98 kT. The results are summarized in Table 4.</p><p>This result is confirmed by the contact angle measurement with water of a silica surface functionalized with propyl and butyl groups: the contact angle is about 10°and 15°for the propyl and butyl, respectively, confirming the highly hydrophilic nature of these surfaces.</p><!><p>In this work a method is presented for the quantitative characterisation of nanoparticles hydrophobicity by measuring their affinity towards specifically functionalized surfaces.</p><p>The determination of the affinity of NPs towards substrate surfaces with different hydrophobicity degrees enables the direct characterisation of the NPs having unknown surface functionalization and residual hydrophobicity in a direct manner. The quantification of the surface energy of the NPs is possible by comparing the evaluation of the affinity of the NPs with different collectors and the comparison with the XDLVO theory, which takes into account the hydrophobic forces.</p><p>The general method is highly sensitive to variation of the surface free energy polar components and could be adapted for the direct evaluation of the stability of the anti-fouling coatings which are usually used to prevent the agglomeration of the NPs in biological complex media and living bodies and which, through the formation of the protein corona, may lead to inflammatory responses and/or uncontrolled clearance of the NPs. The proposed method allows the quantitative characterisation of NP hydrophobicity in solution and thus is potentially highly relevant to important applications in the field of nanomaterial safety assessment in consumer and industrial products.</p><!><p>Surface preparation. Silicon wafers (Si(100); diameter, 50 mm; resistivity, 1−20 Ω cm) supplied by ITME (Warsaw, Poland) were used as the substrate for the whole study. Before modification the wafers were washed with ethanol and water and dried under nitrogen flow.</p><p>Surface modification. The silicon substrate was modified by different deposition processes in order to tune the surface hydrophobicity. First, polytetrafluoroethylene (PTFE) coating was plasma deposited to generate a hydrophobic surface. The deposition was performed using pure octofluorocyclobutane (C 4 F 8 ) as the gas precursor at a pressure of 3.5 Pa, applying a power of 142 W for 5 min 12 .</p><p>In order to tune the surface hydrophobicity, a layer-by-layer deposition of two polyelectrolytes (PELs) was then performed. The PTFE modified substrates were incubated for 2 min in poly(diallyldimethylammonium chloride) (PDDA) 2% solution in water or in poly(sodium 4-styrene sulphonate) (PSS) 2% in water for the self-assembly deposition of each PEL layer, starting from PDDA (positively charged) and alternating with PSS (negatively charged). After each step, the substrate was rinsed with milliQ water and dried under nitrogen flow. The surfaces obtained are referred to as T1, T2, T3 and T4 depending on the number of PEL layers deposited.</p><p>Collector surface characterisation. In order to fully characterise the collector surfaces, several techniques were used. The thickness and refractive index of each deposited layer were measured by Ellipsometry (Vase VUV TM J.A. Woollam Co.). The contact angle and surface energy of the surfaces were determined by using a Digidrop TM goniometer with 2 liquids (water and 1-bromonaphtalene). The surface topography and roughness of the surfaces were measured using AFM (NT MDT Russia). Finally, Z-potential measurements were performed for a range of pH from 3 to 10 with a step of 0.5 in order to characterise the surface charge. The Zpotential was measured for independent samples just after adjusting the pH of the dispersions with either 1 M NaOH or 1 M HCl and at a total NaCl concentration of 1 mM to keep the conductivity at approximately 1 mS/cm. AFM was also used to directly measure the different adhesion forces present between the collector surfaces and functionalised tips to ensure that the surface properties evaluated were constant over a 1 μm range. A conventional silicon tip for contact mode (NT MDT C-Probe, spring constant k = 0.02 N/m and nominal radius of curvature of 15 nm) was employed. The instrument returns the values for the deflection of the cantilever in nA (the difference of current at the 4-quadrant optical detector of the AFM). This value was translated in force values (nN) by calculating the actual deflection of the cantilever in nm and by multiplying the value for the elastic constant of the cantilever used.</p><p>The cantilever was coated with a 20 nm-thick layer of gold by magnetron sputtering (a 1 nm layer of titanium was previously deposited to ensure the adhesion between the gold and the silicon). The gold-coated tip was then immersed in 1 mM ethanol solutions of thiolated alkyl group terminated with PEO (Polyethylenoxide). The tip was then rinsed in ethanol and ultrapure water and gently dried in flowing N 2 . The gold-coated and PEO-functionalised tip was then used to perform so-called "Force-distance curves" (F-D curves). Briefly the tip was brought in contact with the surface in a certain location, and then the tip was retracted by a few micrometers by means of a piezoelectric crystal. Then a series of approaching-retracting curves were acquired in an area surrounding the contact position and the deflection of the cantilever was recorded as a function of the piezo-position. The measurements were performed in ultrapure water. A minimum of 100 curves was acquired. The adhesion force between the tip and the surface was measured for each curve as the difference between the zero-force line and the minimum of the "snap-off" force. The adhesion forces were then averaged for all the curves (see Supplementary Note 1).</p><p>Nanoparticle synthesis functionalisation and characterisation. Two sets of NPs, Au and SiO 2 were used for the experiments. AuNPs were synthesised (see below) using gold (III) chloride trihydrate (>99.9%), and trisodium citrate dihydrate (>99.9). Surface modifications of SiO 2 particles was done using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) (GPTMS, >98%), propylamine (>99%), butylamine (>99%) and dodecylamine (>99.5%). All reagents were purchased from Sigma-Aldrich and used as received without further purification. Ligand 2-(2-[2-(11-mercapto-undecyloxy)-ethoxy]-ethoxy)-ethoxy-ethoxy-ethoxy-ethoxy-acetic acid (PEO-ligand) was purchased from ProChimia and kept under N 2 and in the freezer at −20 °C. SiO 2 NPs of approximately 200 nm (NS-0200A) at a concentration of 7.8 × 10 12 NP/mL were purchased from MSP Corp. Centrifugal filter units (Amicon, USA) were washed three times with milliQ-water at 3500 rcf 10 min before their use. All solutions were prepared with ultrapure water (Millipore Milli Q system, resistivity, 18.2 Ω cm). The synthesised AuNP dispersions were stored in the fridge at 4 °C.</p><p>Synthesis of the AuNPs. AuNPs of 60 nm in diameter were prepared by a seeding-growth four-step procedure. Initially, AuNP seeds of approximately 15 nm in diameter were synthesised with a modified Turkevich method 25 using a specialised microwave Discover apparatus (CEM Corporation, USA). Briefly, 5 mL of aqueous gold (III) chloride trihydrate (10 mM) were added to 95 mL of milliQwater in a single-necked 100 ml round bottom flask equipped with a magnetic stirrer and a glass condenser column. The flask was mounted in the microwave, heated rapidly (<1 min) to 97 °C while stirring and then held for 5 min using a maximum microwave power of 150 W. Under vigorous stirring, 2.5 mL of sodium citrate (100 mM) was injected and the reaction mixture was maintained at 97 °C for 20 min after which the reaction vessel was rapidly cooled to 60 °C with compressed air and then allowed to cool to room temperature. The nominal concentration of gold in the AuNP dispersion was 0.5 mM. The 15 nm AuNP seeds were first grown to 30 nm, and then from 30 nm to 45 nm.</p><p>The last stage of the synthesis to 60 nm AuNPs was carried out by the regrowth method from the 45 nm AuNPs. The as-synthesised seeds (25 mL) of AuNPs of 45 nm were diluted in milliQ-water (60 mL) and mixed with 2.8 mL of sodium citrate (100 mM) and 1.25 mL of aqueous gold (III) chloride trihydrate (10 mM). The pH of the resulting solution was adjusted to a value of 9.0 with aqueous NaOH (200 mM, 0.42 mL) and the mixture was heated to 60 °C for 48 h in order to produce AuNPs of approximately 60 nm in diameter. The nominal concentration of gold in final AuNP dispersion was 0.24 mM.</p><p>The 60 nm AuNP dispersions were purified twice by centrifugation (2500 rcf, 20 min, 4 °C) followed by redispersion in the same volume of MilliQ water, and Functionalisation of SiO 2 NPs with different hydrophobic ligands. Functionalisation of commercial SiO 2 NPs was performed as described in the literature 27 . Briefly, a sample of nano-silica NS-0200A (0.5 mL) was diluted in water (1.5 mL) and the pH increased with the addition of 1 M NaOH (1 µL). GPTMS (0.5 µL, 2.3 μmol) was immediately added and the reaction mixture was stirred at room temperature for 24 h. Modifications of epoxy-functionalised SiO 2 NPs were obtained by the addition of 100 mM solutions in water of propylamine, butylamine and dodecylamine (final concentrations of 2.24 mM) at pH = 9. The mixtures were left to react for 24 h and all the samples were then purified using centrifugal filter units of 10 kDa MWCO Amicon Ultra-15 (3500 rcf, 5 min) via two washing steps with water in order to eliminate the excess of non-conjugating molecules. DLS measurements showed no major differences in the mean hydrodynamic size for any of the samples, which was a good indicator of the colloidal stability of these solutions. Surface charges of pristine and functionalised SiO 2 NPs were determined by Z-potential measurements at a value of pH = 7.5 after adjusting with 1 M HCl and at a conductivity of 1 mS/cm after adjusting with 1 M NaCl. The final nanoparticle dispersions were diluted in a buffer (Phosphate buffer (PB) 10 mM, pH = 7.5) to a final concentration of 1.4 × 10 11 NP/mL (i.e. 55 times less than the original concentration).</p><p>Nanoparticle characterisation. AuNPs were imaged using a transmission electron microscope (TEM, JEOL 2100, Japan) at an accelerating voltage of 200 kV. The samples were prepared by placing a drop (4 µL) onto ultrathin Formvar-coated 200-mesh copper grids (Tedpella Inc.), followed by drying in air at room temperature. For each sample, the size of at least 100 particles was measured to obtain the average and the size distribution. Digital images were analysed with the ImageJ software, using a custom macro to perform smoothing (3 × 3 or 5 × 5 median filter), a manual global threshold and the automatic particle analysis of ImageJ. The programme can be downloaded from http://code.google.com/p/psa-macro. A circularity filter of 0.8 was used to exclude agglomerates that could occur during drying. UV-vis adsorption spectra were recorded with an Evolution 300 UV-vis spectrophotometer (Thermo Scientific, USA) at room temperature.</p><p>DLS and Zeta-potential measurements were obtained using a Zetasizer Nano ZS instrument (Malvern Instruments, UK). Hydrodynamic diameters were calculated using the internal software analysis from the DLS intensity-weighted particle size distribution.</p><p>Differential centrifugal sedimentation. DCS measurements (instrument model DC24000UHR, DCS Instruments Inc, USA) were performed in an 8-24 wt% sucrose density gradient with a disc speed of 22,000 rpm. Each sample injection of 100 µl was preceded by a calibration step using certified polyvinyl chloride (PVC) particle size standards with a weight mean size of 280 nm.</p><p>Contact angle. The contact angle with water was measured using a Digidrop Contact Angle metre (GBX, France). Contact angles of the differently functionalised AuNPs were measured by spotting a drop of sample solution (10 µL) on a silicon surface to form of confluent film. Then, a 0.5 μL water droplet was deposited onto this film and the contact angle was measured.</p><p>Contact angles were also measured on flat Au-surfaces functionalised with PEO-ligand. Au deposition on silicon wafers (3 min, ~100 nm Au) was performed by physical vapour deposition. The surfaces were cleaned with sonication in EtOH, followed by several washing steps with EtOH and MilliQ H 2 O. They were then functionalised using 10 mM aqueous solution of PEO-ligand, and dried. A 0.5 μL droplet of either water or 1-bromonaphtalene was deposited on the surface and the contact angle measured.</p><p>For measurements of contact angles of flat SiO 2 -surfaces with hydrophobic ligands, silicon wafers were treated with O 2 plasma (treatment time = 5 min) and then exposed to 1 mM aqueous solution of GPTMS at room temperature for 24 h. After washing with MilliQ H 2 O, wafers were exposed to 2.24 mM solutions in water of propylamine, butylamine and dodecylamine at pH = 9 for 24 h at room temperature before further washing with MilliQ H 2 O. After drying, a 0.5 μL water droplet was deposited on the surface and the contact angle measured. SDS-PAGE gel electrophoresis. AuNPs of 60 nm diameter, both pristine and PEO-functionalised were incubated with human serum (Sigma-Aldrich) for 24 h at 37 °C. The mixture was centrifuged (10,000 rcf, 5 min, 4 °C) and the supernatant carefully removed. The NP pellet was subsequently washed with 1× PBS (Gibco). This washing procedure was repeated three times. The final pellet was suspended in 20 µL Pierce Lane Marker reducing sample buffer (ThermoFisher) and incubated at 95 °C for 5 min. After a short spin down, the supernatant was loaded in 12% SDS polyacrylamide gel and run at 110 V, 25 mA in 1× SDS running buffer. After electrophoresis, the gel was Coomassie stained. Scanned gel images were analysed with the ImageJ software using sharp + smooth + brightness/contrast adjustment, followed by splitting of colour channels (best contrast for Red and Green). Quantification of the proteins was made by calculating pixel intensity in the central band (lower intensity = dark = more proteins) and plotted as the inverse of the pixel intensity.</p><p>Nanoparticles adsorption study by dark-field microscopy. Dark field microscopy (DFM) videos were recorded to measure the NP adsorption rates on the different collectors. Image analysis was performed using the open source graphics software ImageJ (http://rsb.info.nih.gov/ij/). A special data processing protocol was developed to enable automatic frame-by-frame analysis of the collector surface coverage by the NPs. The coverage boundaries were identified as red and green stains on a black background and only those with a size between 7 and 200 pixel units and circularity value between 0.20 and 1.00 were taken into account in calculating the degree of surface coverage (using the Analyse Particles function of ImageJ). After background correction, noise reduction (Despeckle, Kalmann Stack Filter with values 0.05-0.80), brightness, contrast and colour balance adjustment, the Unsharp Mask filter (radius 4.0 and mask weight 0.7) was applied. The image was then converted to an 8-bit file, this being required to adjust the threshold and analyse the particles with the provided function. This procedure allowed real-time kinetics analysis with a microfluidic chip, calculating the coverage degree of each substrate as a function of time. The association rate could then be determined, providing a quantitative measurement of the affinity of the NPs for the different collectors. These real-time measurements were carried out in static mode by using commercial slides provided by Ibidi (Sticky-Slide IV, Germany) with a channel volume of 30 µl and cell height of 0.4 mm. The channel was filled with the NPs solution using a syringe and the subsequent analysis of the collector surface was done with DFM.</p><p>Numerical solver. The Solver of Microsoft Excel © was used for finding the numerical solution of a system of non-linear equations using the GRG nonlinear solving method. The equations and the boundaries chosen are described in the results and discussion section, since they are part of the developed method.</p>

Nature Communications Chemistry

The study of pH-dependent stability shows that TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin\xe2\x80\xa0

Ankyrin repeat (AR) proteins possess a distinctive modular and repetitive architecture, and their global folds are maintained by short-range interactions in terms of primary sequence. In this work, we extended our previous study on the highly conserved TPLH tetrapeptide and investigated the impact of a solvent-exposed histidine residue on the pH-dependent stability of gankyrin, providing further insight into the contribution of the TPLH motif to the tertiary fold of AR proteins. Consisting of seven ARs, gankyrin has five histidine residues in TPLH motifs or its variants, all of which adopt a H\xce\xb52-tautermeric form and are shielded from solvent. By truncating the C-terminal ankyrin repeat (AR7), we exposed H177 in the 174TPLH177 of AR6 (the second C-terminal AR) to aqueous environment. We showed that this truncated gankyrin mutant, namely, Gank1-201, was well-folded at a neutral pH with a slightly lower stability with respect to gankyrin wild type (WT). However, unlike gankyrin WT, the stability of Gank1-201 was markedly decreased together with a loss of conformation at a pH slightly below 6.0. It was rationalized that the protonation of the H177 imidazole ring triggered the disruption of the TPLH-mediated hydrogen-bonding network, which in turn led to the loss of conformation and stability. These results together with the work on Q210H mutant nicely explain that the C-terminal AR7 has a 207TPLQ210 variant, and are in support of the notion that a string of TPLH/variant, which may arguably act like a zip lock to the elongated AR proteins via intra-/inter-repeat hydrogen-bonding, is important in maintaining the tertiary fold. Additionally, we made rational mutagenesis to introduce extra surface charge on AR7 of gankyrin and demonstrated that G214E and I219D mutations increased the stability of gankyrin while the function remained intact. Taken together, our results indicate that TPLH-mediated hydrogen-bonding network is important for the conformation and stability of human gankyrin, and the C-terminal AR contributes to the conformational stability of gankyrin (AR proteins in general) through shielding this TPLH network from solvent as well as making the surface area more accessible to solvent.

the_study_of_ph-dependent_stability_shows_that_tplh-mediated_hydrogen-bonding_network_is_important_f

<!>Protein expression and purification<!>Pull-down assays<!>Circular dichroism (CD) analyses<!>NMR experiments<!>Q210H mutant tends to aggregate and precipitate in solution<!>Gank1-201 shows a transition point around pH 6.0 for a significant loss of conformational stability by CD analysis<!>Gank1-201 retains a global fold around neutral pH but loses some structures at a pH below 6.0 evidenced from NMR studies<!>Introducing charged residues into the C-terminal AR of gankyrin stabilized the global structure<!>Mutations and truncation of the C-terminal AR7 did not impair gankyrin\xe2\x80\x99s CDK4 binding but truncation abolished its MDM2 binding<!>DISCUSSION<!><!>Ribbon diagram of gankyrin showing the side chains of Thr (Ser) and His (Gln) in TPLH motifs as well as the heavy atoms of His (+30) in cyan color<!>Evaluation of the conformational stabilities of WT, Gank1-201, and Q210H under different pHs<!>Homology and structure comparisons of human gankyrin and Notch ARD<!>1D 1H NMR spectra on mutant Q210H at pH 5.4 and pH 7.4, respectively<!>NMR studies showing that the removal of the C-terminal AR destabilized the tertiary structure of Gank1-201 at acidic pH<!>Evaluation of the conformational stabilities of gankyrin missense mutants<!>Pull-down assays to assess the mutagenic effect on gankyrin\xe2\x80\x99s function<!>

<p>Ankyrin repeat (AR) proteins are one of the most abundant repeat protein classes in nature and play vital roles in numerous physiological processes through mediating protein/protein interactions (1–4). Consisting of 33 amino acid residues, an ankyrin repeat exhibits an L-shaped conformation in which two anti-parallel α-helices are followed by a relatively flexible β-loop roughly perpendicular to the helical axes. Multiple L-shaped units in an AR protein are stacked contiguously side-by-side in a nearly linear fashion to form helix-turn-helix bundles (3, 4). Such elongated fold, unlike prevalent globular ones, is maintained by short-range interactions in terms of primary sequence, and the conformational stability is the accumulation of the intrinsic stability of each AR and the interfacial stability between neighboring ARs (4).</p><p>One salient feature in an ankyrin repeat is the presence of the conserved TPLH tetrapeptide motif (5, 6). In this motif, the proline residue initiates the first α-helix of the L-shaped unit, while the pair of Thr and His act in concert to form intra-/inter-repeat hydrogen bonds (Figure 1). It is noteworthy that the conserved histidine adopts a Hε2-tautermeric form and plays a unique role in maintaining the global conformation of an AR protein (6). Briefly, Nδ1 of this histidine is engaged in bifurcated hydrogen bonds with the Thr in the same TPLH motif, whereas protonated Nε2 acts as a donor to the backbone carbonyl oxygen of the residue immediately preceding the next TPLH motif (6). The occurrence of a string of TPLH motifs, such as in human ankryinR (7) and human gankyrin (8, 9), could conceivably lead to the formation of a large hydrogen-bonding network throughout the repeat stack, thus playing crucial roles in stabilizing the tertiary fold. This notion is supported by our previous studies showing that the TPLH motif contributes substantially to the conformational stability of human gankyrin (10).</p><p>Gankyrin is an oncogenic protein involved in cell cycle progression, apoptosis, and proteasomal degradation through binding and modulating the cyclin-dependent kinase 4 (CDK4), pRb (the retinoblastoma susceptible gene product), MDM2 (a protein encoded by murine double minute gene, mdm2), and S6 ATPase (11). Among its seven AR units (namely, AR1 to AR7), all but the N-terminal one (AR1) have TPLH motif or a close variant, such as 207TPLQ210 in the C-terminal AR unit, AR7. In this example, the highly conserved histidine is replaced with a glutamine residue (6). Unlike the counterpart in the TPLH motif of an internal or N-terminal AR, histidine in the C-terminal AR does not have a hydrogen-bonding acceptor for the protonated Nε2, and the imidazole ring is expected to be largely solvent exposed, which may not be favored with regard to the conformational stability (10). In this work we investigated the potentially deleterious effect by such a solvent-exposed histidine on the global conformation and stability, aiming at further understanding the structural role of the TPLH motif. We anticipated that the likelihood protonation of the imidazole ring at physiological pH may destabilize the protein via impairing the TPLH-mediated hydrogen-bonding network. To address this premise, we generated Q210H mutant, and most importantly, the truncated gankyrin mutant, Gank1-201, in which the entire C-terminal AR (AR7) is removed thus rendering H177 in the 174TPLH177 motif of AR6 exposed to the aqueous environment (12). The mutants were then subjected to pH-dependent studies. This work also intertwined with another specific aim that is to investigate the structural and functional role of AR7. Our work is significant in the following. (1) Participation of a Hε2-tautermeric histidine is a prerequisite for forming a large TPLH-mediated hydrogen-bond network; and this network may act like a zip lock to the elongated fold of AR proteins; (2) Protonation of the participating histidine residue likely disrupts the above network and triggers perturbations in the conformation and stability; (3) Unlike in Drosophila melanogaster Notch ankyrin repeat domain (ARD) (13), the C-terminal AR7 of gankyrin does not have a significant contribution to the global conformational stability but does enhance protein's susceptibility against pH; and (4) we also showed that AR7 of gankyrin is important for MDM2 binding but not CDK4 binding, and G214E and I219D mutations in this repeat significantly increased the conformational stability.</p><!><p>Human gankyrin was expressed as a glutathione-S-transferase (GST) -fusion protein in Escherichia coli BL21 (DE3) Codon plus cells (Novagen) as previously described (14). After sonication and centrifugation, the soluble cell lysate was loaded on a reduced glutathione-agarose (G beads) column (Sigma) equilibrated with phosphate-buffered saline (PBS, pH 7.4). Bound GST-gankyrin was eluted out with reduced glutathione (20 mg/mL in PBS) and further purified by a Q Fastflow column (Amersham). To remove the GST tag, 100 units of PreScission protease (2 units/μl; Amersham) were added to GST-gankyrin in PBS. After incubation at 4 °C for 24 h, the protein solution was loaded onto a column of G beads and the flow-through was concentrated and further purified by an S100 column (Pharmacia) equilibrated with 5 mM HEPES, 1 μM EDTA, and 1 mM DTT (pH 7.4). After SDS–PAGE analysis, fractions containing free gankyrin protein were pooled, concentrated, and lyophilized for further analyses. All gankyrin mutants except Q210H were generated using PCR-based site-directed mutagenesis (Stratagene), and were expressed and purified essentially the same as gankyrin wild type (WT).</p><p>Gankyrin Q210H was generated using a pET-21d (+)-gankyrin template in which gankyrin was expressed as a C-terminal His6-tagged protein (14). The lysate from bacteria harboring the pET-21d (+)-Q210H was loaded onto a TALON Cobalt affinity column (BD Sciences) and washed with PBS-20 mM imidazole. Bound proteins were eluted using PBS containing 50, 100, 200, and 300 mM imidazole. After SDS-PAGE analysis, fraction containing Q210H were pooled and dialyzed against PBS and the aforementioned HEPES buffer (pH 7.4). Of note, there is virtually no difference in the conformational stabilities of gankyrin wild type proteins purified from both GST-tagged and his6-tagged procedures (data not shown).</p><p>Mouse mdm2 cDNA gene was cloned into pGEX-4T-1 (Amersham) and expressed as a GST -fusion protein in BL21 (DE3) Codon plus cells upon IPTG induction. GST-MDM2 was purified following the same procedure of GST-gankyrin purification as described above. Similarly, GST was purified from BL21 (DE3) Codon plus cells harboring pGEX-4T-1 and used as control in the following pull-down assays.</p><p>The CDK4/cyclin D2 holoenzyme was purified from Highfive insect cells (Invitrogen) as described previously (15). Briefly, insect cells were co-infected with baculoviruses expressing human CDK4 and cyclin D2 for 48 h. Upon centrifugation, cells were resuspended in the ice-cold sonication buffer (20 mM Tris-HCl, 100 mM NaCl, 0.1 mM Na3VO4, 1 mM NaF, 10 mM β-glycerophosphate, 5mM β-mercaptoethanol, 0.2 mM AEBSF, 5 μg/mL aprotinin, 5 μg/mL leupeptin, pH 7.4) and broken by sonication. Lysates were cleared by centrifugation and loaded on a TALON affinity resin column (BD Sciences; pre-equilibrated with the sonication buffer). After the resin was washed with the sonication buffer and the sonication buffer containing 10 mM imidazole (pH 7.4), respectively, bound CDK4/cyclin D2 holoenzyme was eluted in the sonication buffer containing 50 mM imidazole (pH 7.4) and dialyzed against the kinase buffer (50 mM HEPES, 10 mM MgCl2, 2.5 mM EGTA, 1 mM DTT, 0.1 mM Na3VO4, 1 mM NaF, 10 mM β-glycerophosphate, 5mM β-mercaptoethanol, 0.2 mM AEBSF, 5 μg/mL aprotinin, 5 μg/mL leupeptin, pH 7.4). Concentrated aliquots (at about 0.3 mg/mL) were stored at −80 °C before use.</p><!><p>To investigate the interaction between GST-gankyrin proteins (including WT and mutants) and the CDK4-cyclin D2 complex, 10 μg of the CDK4-cyclin D2 complex and 25 μg of GST-gankyrin proteins were incubated in 300 μL of PBS (pH 7.4) at 4 °C for 3 h (14). The concentration of CDK4-cyclin D2 and GST-gankyrin were 0.3 and 3.0 μM, respectively. Subsequently, 250 μL of G beads (pre-equilibrated with PBS at 4 °C) was added into the reaction mixture. After incubation at 4 °C for another 4 h, the reaction mixture was loaded onto a spin column (Fisher Scientific), centrifuged at 4 °C, 1000 rpm for 2 min, and washed with PBS three times. G beads-bound proteins were then eluted out using 150 μL of PBS containing reduced glutathione (20 mg/mL) and further analyzed with Western blot using anti-human CDK4 antibody (sc-2006; Santa Cruz Biotechnology) as previously reported (14).</p><p>A similar assay was performed to evaluate the potential interaction between MDM2 and free gankyrin proteins except: (1) each reaction mixture contained GST-MDM2 (at about 0.3 μM) and non-tagged gankyrin protein (including WT and mutants; at 3.0 μM); (2) the pull-down products were blotted against anti-human gankyrin polyclonal antibody (PW8325; BIOMOL International).</p><!><p>For guanidinium hydrochlorife (GdnHCl)-induced unfolding, lyophilized gankyrin proteins were dissolved in 10 mM sodium borate buffer (at pH 7.4, 8.4, 6.4, 5.4 as indicated) containing 40 μM DTT and dialyzed against this borate buffer at 4 °C overnight (12). Samples containing about 2.5 μM proteins were incubated with different amounts of guanidinium hydrochloride (GdnHCl, in a stock solution of 8.5 M) on ice overnight and then equilibrated at 20 °C just prior to CD analysis. The rotation at 222 nm was measured on an AVIV 62A DS far-UV spectropolarimeter using a quartz microcell (Helma) of 0.1 cm light pass length, and the exact concentrations of GdnHCl were determined using the refractive index. For each sample, three scans were averaged. The ellipticity at 222 nm, an indicator of the existence of α–helical secondary structure was taken as the measure of the degree of structure present in the protein at each GdnHCl concentration. The following values were obtained on the basis of two-state approximation: ΔGdwater (denaturation free energy in water), D1/2 (denaturant concentration at the midpoint of transition), and the slope of m (a constant related to a protein's susceptibility to chemical denaturant).</p><p>Heat-induced unfolding experiments were performed using about 10.0 μM proteins in the borate buffer (at indicated pH) with 1 nm bandwidth and a 10 second response time. Thermal melting spectra were recorded at 222 nm by heating from 5 °C to 65 °C (for gankyrin WT and a Gank1-201) or 70 °C (for gankyrin missense mutants) at the rate of 1 °C per minute and a 1 °C interval followed by cooling down to 5 °C at the same rate. Tm was defined as the temperature at the midpoint of transition.</p><!><p>Eight NMR samples were prepared containing 0.2 mM either WT or Gank1-201, 5 mM HEPES, 1 μM EDTA, 1 mM DTT in 90% H2O/10% D2O (15). The pH was adjusted to 5.4, 6.4, 7.4, and 8.4, respectively. 1D 1H NMR and 2D NOESY (200 ms mixing time) experiments were performed at 25 °C on a Bruker DRX-600 equipped with a cryoprobe using WATERGATE (16) for the water suppression.</p><!><p>Previous studies in our laboratory have demonstrated that the conformation of gankyrin is stabilized by the TPLH-mediated hydrogen-bonding network, in which the highly conserved histidine residues play pivotal roles. Interestingly, in the C-terminal AR of gankyrin (AR7), a glutamine residue replaces the conserved histidine in the aforementioned tetrapeptide motif (207TPLQ210), which is assumed to "close" the hydrogen-bonding network to stabilize the global structure. To address this premise, we introduced histidine residue in the C-terminal 207TPLQ210 and evaluated the biochemical and biophysical properties of the resultant Q210H mutant protein. Unlike gankyrin WT and other missense mutants investigated in this work as well as our previous studies (10, 17), Q210H aggregated to a considerable extent during the expression and purification process, and the protein obtained easily precipitates at a concentration of around 0.2 mM. It was noticed that precipitation became more severe at a pH lower than 6.0. While the mechanisms underlying such poor solubility remain to be elucidated, it is important to note that Q210H mutation occurs in the middle of the first helix of AR7, which is somewhat abbreviated (in comparison with its counterparts in other ARs) due to the presence of Gly tandem (G214 and G215) at the end of the first helix (Figure 3). In regard to the low solubility of Q210H, folding/unfolding experiments were performed at an extremely low concentration of about 5 μM using CD spectroscopy. All data were fitted to a biphasic model between native and completely unfolded proteins (Figure 2) (4). Table 1 lists the values of ΔGdwater, D1/2, and the slope m obtained in GdnHCl-induced unfolding. Under physiological pH (pH 7.4), the ΔGdwater value of Q210H was 2.32 kcal*mol−1, which is moderately lower than that of gankyrin WT (2.72 kcal*mol−1). Consistently, in heat-induced unfolding, the Tm value of Q210H at pH 7.4 was 49.9 °C, slightly lower than the corresponding one (51.2 °C) for gankyrin WT. These results suggest that Q210H mutation moderately destabilizes the global structure of gankyrin under physiological pH. However, further pH-dependent stability studies on Q210H were hindered most likely due to its poor solubility at acidic pHs. While the ΔGdwater and Tm values of Q210H at pH 8.4 (1.97 kcal*mol−1 and 49.0 °C, respectively) were only marginally lower than the corresponding values at pH 7.4. we failed to obtain the denaturation curves at pH 5.4 and 6.4 due to extremely poor CD signals, which may be ascribed to precipitation and/or a complete loss of the secondary structure of Q210H at acidic pHs.</p><p>Attempts to study Q210H by NMR experiments, such as 2D NOESY to tentatively assign Hε2 of H210, were also compromised by this mutant's tendency to severe aggregation at a concentration, i.e. 0.2 mM, necessitated for NMR analyses. Only 1D 1H NMR spectra of Q210H at pH 5.4, 6.4, 7.4, and 8.4 were recorded at extremely low concentrations (< 0.1 mM). As shown in Figure 4, the mutant at neutral pH does possess good chemical shift dispersion with weak signals in the 10~ 12 ppm downfield region, indicative of a preservation of tertiary structure. However, the mutant becomes less structured at pH 5.4 evidenced by the disappearance of those downfield signals, likely attributed to the unfavorable solvent exposure of H210.</p><p>Taken together, it is arguably safe to state that Q210H mutation impairs some biophysical properties of gankyrin, such as the tendency to aggregate, which may have a bearing on the stability of the global structure. However, the severe aggregation tendency of Q210H mutant complicated our endeavor to explore the pH dependency of its conformational stability. Therefore, we turned to constructing Gank1-201 mutant, in which AR7 is removed thus exposing H177 in 174TPLH177 motif of AR6 to solvent.</p><!><p>Both Gank1-201 and WT were subjected to parallel experiments of chemical- and thermal-denaturation at pH 5.4, 6.4, 7.4, and 8.4 monitored by CD spectroscopy (12). As shown in Table 1 and Figure 2, Gank1-201, the truncated mutant, only exhibited a slight or moderate change of 0.62 kcal*mol−1 in conformational stability with respect to gankyrin WT at neutral pH. Moreover, the ΔGdwater and m values of Gank1-201 at pH 6.4 and 8.4 were comparable with the corresponding values of Gank1-201 at neutral pH, indicating that Gank1-201 is relatively stable in the range of pH 6.4 – 8.4. However, the unfolding behavior of Gank1-201 changed dramatically at pH 5.4, which can be appreciated by a more sigmoidal denaturation curve (Figure 2A). Even though there is no representative baseline at the native state in the unfolding of Gank1-201 at pH 5.4, its apparent ΔGdwater value from the two-state transition approximation was around 0.5 kcal*mol−1, indicating that this mutant becomes only marginally stable at pH 5.4. Consistently, at pH 5.4, Gank1-201 is much less structured in the presence of high concentrations of the denaturant (GdnHCl) with an apparent D1/2 value of 0.42 M. In comparison, the D1/2 value of Gank1-201 at pH 6.4 is 1.44 M. Therefore, Gank1-201 likely undergoes dramatic conformational changes from pH 6.4 to 5.4. In contrast, slightly acidic pH appeared to have little influence on gankyrin WT, which did not show any significant changes in ΔGdwater, m and D1/2 in the range of pH 5.4 – 8.4 (Table 1).</p><p>The above notion is further supported by heat-induced unfolding studies (Figure 2B). First, the heating and cooling curves of gankyrin WT and Gank1-201 are virtually superimposable, and the Tm values determined from the heating and cooling curves are within 1 °C, indicating that the proteins are at thermodynamic equilibrium and the thermo-induced folding/unfolding is reversible (data not shown). Secondly, Gank1-201 and gankyrin WT have comparable thermo-stability around neutral pH, confirming the result of chemical-induced unfolding that AR7 does not have a significant impact on the conformational stability of gankyrin. However, while the Tm of gankyrin WT are within seven degree of difference in the pH range studied, Gank1-201 showed a twice larger of the Tm differences due to a significant lower value at pH 5.4, further indicating physical-chemical changes of Gank1-201 under this pH condition. Taken together, while we do not attempt to over-interpret small or modest changes of ΔGdwater and Tm in both gankyrin WT and Gank1-201, it is safe to draw the conclusion that the removal of the C-terminal AR renders the global structure more susceptible to acidic circumstances with a turning point roughly around pH 6.0.</p><!><p>We subsequently revealed that the decreased stability of Gank1-201 at pH 5.4 is associated with the loss of conformation. Firstly, the mutant was subjected to the studies of 1D 1H NMR and 2D 1H homonuclear NOESY at pH 7.4 (Figure 5). The large 1H chemical shift dispersion in the aliphatic (e.g. upfield resonances at −0.59, −0.39, and −0.13 ppm) and the amide regions (e.g. downfield resonances at 10.54, 10.60, and 10.83 ppm) together with the high density of NOE cross peaks suggested that this mutant was well folded under this pH condition. Secondly, the analysis of the downfield region of NOESY spectrum led to the tentative assignment of the five distinctive Hε2 resonances of histidine in TPLH/variants, which are sensitive to the formation of hydrogen-bonding network and have been used as a convenient measure of tertiary fold (Figure 5A) (6, 10). Consistent with the modular feature of tertiary fold, the perturbations of Hε2 chemical shifts with respect to the counterparts in gankyrin WT followed the rank order of H177 (0.17 ppm) > H144 (0.09 ppm) ~ H111 (−0.09 ppm) > H78 (0.02 ppm) ~ H45 (0.00 ppm). These resonances also displayed comparable NOE patterns to those corresponding ones in WT, indicating the preservation of TPLH-mediated hydrogen-bonding network. And lastly, 1D 1H NMR was performed to monitor those tractable Hε2 resonances at pH 5.4, 6.4 and 8.4. Data collected immediately upon titration showed little changes (Supplementary Material). However, after incubation at room temperature for a week, the sample at pH 5.4 but not at higher pH revealed dramatic changes that those distinguished downfield Hε2 signals disappeared with concurrent loss of amide 1H chemical shift dispersion (Figure 5B). Even though residual structures persist evidenced by residual upfield resonances around 0 ppm, Gank1-201 indeed is of significant less structured at a pH below 6.0. These observations on Gank1-201 are in sharp contrast with those in the parallel experiments performed on WT, which has virtually no changes in the 1H NMR, including the downfield region concerning the Hε2 signals (Figure 5C). Taken together, Gank1-201 shows vulnerable pH-dependent conformation and stability as has been anticipated.</p><!><p>Our studies on Gank1-201 as described above demonstrate that AR7 functions to shield the internal ARs from aqueous environment thus increasing the resistance of the global structure to pH. However, it is interesting to explore whether or how AR7 itself directly contributes to the conformational stability of the global structure. Even though gankyrin has the ΔGdwater value of 2.72 kcal*mol−1 and is more stable than some small AR proteins, such as P16INK4A (4 ARs) and P18INK4C (5ARs) (12), its behavior in GdnHCl-induced unfolding is significantly different from that of Drosophila melanogaster Notch ankyrin repeat domain (ARD), another well-studied AR protein consisting of seven ARs (18). Notch ARD was reported to have a conformational stability of 8.03 kcal*mol−1 in GdnHCl-induced unfolding and the C-terminal AR7 contributed significantly to the global stability as evidenced by a decrease of 3.89 kcal*mol−1 upon the removal of AR7 (19). In contrast, the removal of AR7 in gankyrin only brought about a slight or moderate change of 0.62 kcal*mol−1 in conformational stability. Based on the sequence homology analysis using ClustalW (www.expasy.org) (Figure 3), it was noticed that there are comparatively more polar residues at the C-terminus capping repeat of Notch ARD, some of which (R186, E191, R192, and D196) are present on the surface. Presumably, the considerably more surface charges enhance the stability of Notch ARD by making the C-terminal surface more hydrophilic. We therefore made the corresponding substitutions L209R, G214E, G215R, I219D, and G214E/I219D together with two histidine mutations G217H and L218H, and evaluated the mutagenic effect on protein stability.</p><p>As shown in Figure 6, all of the mutant proteins exhibit biphasic transitions in GdnHCl- and heat-induced unfolding, indicating that the aforementioned substitutions do not impair the cooperative nature of gankyrin unfolding. The fitting results are summarized in Table 1. Briefly, the values of L209R and G215R are comparable to that of gankyrin WT, whereas G214E and I219D show considerable increase of 1.9 and 2.6 kcal*mol−1, respectively, in the ΔGdwater, indicating that G214E and I219D mutations substantially increase the conformational stability. Interestingly, the ΔGdwater value of the double mutant, G214E/I219D is almost identical to that of G214E, suggesting that the effects of G214E and I219D are not additive. These findings are further supported by results from heat-induced unfolding (Table 1). Finally, Notch ARD has two histidine residues in the C-terminal AR turn. The corresponding mutations G217H and L218H would introduce a solvent-exposure histidine to the gankyrin C-terminal AR7. However, neither mutation lowers the conformational stability at neutral pH, suggesting that a solvent-exposed histidine in the terminal AR does not intrinsically destabilize the gankyrin.</p><!><p>We used the pull-down assay to assess the mutagenic effect on the CDK4-binding and MDM2-binding abilities (11). When GST-tagged gankyrin proteins, including WT, Gank1-201, and AR7 point mutants, were incubated with the CDK4-cyclin D2 holoenzyme, CDK4 was detected in all of the pull-down products but not in the negative control using GST itself (Figure 7A), indicating that all of the aforementioned gankyrin proteins are able to bind to CDK4 (14). Since the amounts of CDK4 in the pull-down products are comparable by visual inspection between each individual mutant and the WT, it is concluded that AR7 has minimal impact on the binding of gankyrin to CDK4, consistent with our previous finding that the first four ARs of gankyrin are sufficient for CDK4 binding and modulating (14).</p><p>It has been also reported that the AR7 of gankyrin may be involved in binding to MDM2, a key component in the P53 pathway (11, 20). A similar pull-down assay was performed on the reaction mixtures containing GST-MDM2 and gankyrin proteins (Figure 7B). First, under the experimental conditions, gankyrin WT was present in the pull-down product from the GST-MDM2/WT reaction mixture but absent in the one from the GST/WT mixture, indicating that gankyrin specifically interacts with MDM2. Secondly, among all of the reaction mixtures containing GST-MDM2 and AR7 point mutants, gankyrin proteins (WT or point mutants) were detected in the pull-down products, implying that these C-terminal AR7 mutants remained functional in binding to MDM2. However, we failed to detect Gank1-201 in the pull-down product from the GST-MDM2/Gank1-201 mixture. Since this truncated mutant is able to react with the polyclonal anti-gankyrin antibody used in this assay (data not shown), the result indicates that the removal of the entire C-terminal AR significantly impaired or even abolished its MDM2-binding ability.</p><!><p>Out of a variety of TPLH-containing AR proteins, gankyrin was chosen as a model in our current study for the following reasons: first, both the structure and function of gankyrin have been extensively investigated; secondly, the modular structure of gankyrin is of considerable resiliency as evidenced by previous findings that gankyrin retains the tertiary fold and CDK4-binding ability upon the introduction of missense mutations and the removal of up to three ARs at the C-terminus (14). The current study extended previous effort to address the important structural role of the conserved TPLH motif for an AR protein. The following discussion will be based on Gank1-201 since more comprehensive results were obtained with respect to Q210H.</p><p>Both gankyrin WT and Gank1-201 have close theoretical pI values (5.71 and 5.45, respectively) (12), indicating that there is no significant difference in net charge at physiological pH. As shown in Table 1, the stability of gankyrin WT increases from 2.65 kcal*mol−1 to 3.55 kcal*mol−1 as pH decreases from 8.4 to 5.4. Apparently, the correlation between the stability of gankyrin WT and pH is consistent with the Linderstrom-Lang model (21) that a protein tends to be more stable as pH approaches to the isoelectric point (pI), at which the unfavorable electrostatic interactions resulting from an excess of either positive or negative charges is minimized. Similar observation has been reported in an optimized de novo designed 4-ANK protein (22). In comparison, Gank1-201 exhibits increasing stability as pH decreases from 8.4 to 6.4 (1.95, 2.10, 2.45 kcal*mol−1 at pH 8.4, 7.4, 6.4, respectively), but undergoes dramatic decrease in stability at pH 5.4 (0.55 kcal*mol−1). Clearly, the Linderstrom-Lang model cannot fully account for the pH dependence of Gank1-201 in stability. The change of pH susceptibility of Gank1-201 strongly indicates that some significant chemical/physical changes are triggered by ionization of titratable groups around pH 6.0. Since histidine is the only amino acid with a pKa of its side chain in the physiologically relevant pH range, the candidates responsible for the aforementioned change in pH susceptibility can be narrowed down to nine histidine residues in gankyrin. Among them, H177 is the only one that is directly impacted by the truncation of AR7. In gankyrin WT, H177 is largely shielded from solvent like a typical histidine in an internal TPLH. However, the removal of the C-terminal capping repeat (AR7) would increase the solvent-accessibility of the side chain of H177 from roughly 10% to 50% (assuming that potential structural rearrangement is negligible), which may partially contribute to the discrepancy between gankyrin WT and Gank1-201 in thermodynamic properties.</p><p>The impact of H177 on conformation and stability of Gank1-201 bears structural implication. First, a newly introduced histidine in the C-terminus does not necessarily cause destabilizing effect as exemplified by two C-terminal AR7 point mutants G217H and L218H. Secondly, the change in the electrostatic status of a solvent-exposed histidine does not necessarily induce dramatic changes in the conformational stability, as H137 is present in the AR4-AR5 loop of both gankyrin WT and Gank1-201 and its side chain is solvent-exposed to the extent of more than 70%. Thus, as has been anticipated, the observation can only be rationalized in the context of the TPLH-mediated hydrogen-bonding network. One may imagine such a scenario that at a pH below 6, H177 in Gank1-201 will gradually shift to a protonated form as described by the Henderson–Hasselbalch equation, bearing Nδ1-H and Nε2-H bonds in the imidazole ring and a positive charge distributed between Nδ1 and Nε2. This change would in turn weaken the hydrogen bonds of T174 HN-H177 Nδ1 and T174 Hγ1-H177 Nδ1, and eventually trigger the loss of the hydrogen bonds mediated by 174TPLH177 (6, 10). Such perturbation will induce a ripple effect on the 141TAMH144 of the preceding repeat AR5, as the hydrogen bond between N173 O and H144 Hε2 is likely perturbed. Consequently, the perturbation propagates through the entire repeat stack and ultimately leads to the loss of the TPLH-mediated hydrogen-bonding network and even the global fold. The result on Q210H also appears to support this scenario.</p><p>Due to the lack of long-range interactions (3, 4), it is conceived that the TPLH-mediated hydrogen-bonding network plays important roles in maintaining the elongated tertiary fold of AR proteins. Presumably, the strings of TLPH might act like a "zip lock" to the repeat stack via the intra-/inter-repeat hydrogen bonds, complementing to the strong hydrophobic interaction between adjacent repeats. In such a model, the tautermeric state of histidine in the TPLH motif is critical, and relatively small shifts in pH around pKa may lead to unzipping the tertiary fold if any of participating histidines is exposed to aqueous environment. In addition, at the C-terminal AR, a variant in place of histidine would be needed to cap the "zip lock", i.e. the TPLH-mediated hydrogen-bonding network. Interestingly, there exists a glutamine (Gln) residue in 207TPLQ210 of AR7, the C-terminal AR of gankyrin, which is capable of forming reciprocal hydrogen bonds with T207 while having a pKa far from physiological range. Similar substitutions at the C-terminal AR have been observed in other AR proteins such as 252SPYQ255 in IκBα (5), 769TPLA772 in human ankyrinR (7), and TPLD or TPQD in a couple of Bcl-3 homologs (23).</p><p>The sequence variation in the TPLH motif in the C-terminal AR also implies that even though there is a consistent pattern of key residues in the consensus sequence of AR motifs to retain the characteristic helix-turn-helix conformation, there is considerable sequence divergence in AR motifs, especially terminal repeats to adapt to the aqueous environment. Unlike an internal AR that has interfacial interactions with neighboring ARs from both sides and tends to have two hydrophobic surfaces, a terminal AR is two-faced: a hydrophobic "inner" surface interacting with the penultimate AR and a hydrophilic "outer" surface accessible to the aqueous environment. Apparently, a more hydrophilic "outer" surface of the C-terminal AR may favor its "capping" effect as well as the global stability. As previously demonstrated (24), a few mutations in the C-terminal AR could significantly improve the stability a de novo designed AR protein. We here show that by introducing charged residues at appropriate position in the C-terminal AR, we could also improve the stability of a naturally occurred AR protein. The difference in stability change for the four mutants could be explained from the structural point of view. L209, G214, and I219 are positioned in the "inner" helix, the turn, and the "outer" helix of the C-terminal AR, respectively. Accordingly, L209 faces the "outer" helix of the penultimate AR and contribute to those "hydrophobic" interfacial interactions with the side chains of K220 and L221 (Figure 3C). Thus, introducing a charged residue through L209R substitution in such "hydrophobic" microenvironment is not thermodynamically favored. In contrast, I219 is directly exposed to the aqueous environment, and substitutions like I219D would enable the C-terminal AR more solvent accessible thus positively influencing the stability of the global structure. So does the substitution of G214E, which introduced a negatively-charged side chain in the turn region and increased the aqueous accessibility. In fact, the percentages of the solvent accessible surface are roughly 32% and 55%, respectively, for the bulky hydrophobic side chains of L209 and I219 (Figure 3D). Interestingly, all these four C-terminal mutants retained CDK4-binding and MDM2-binding abilities comparable to those of gankyrin WT. Therefore, C-terminal substitutions such as G214E can stabilize the global structure of gankyrin but brings about little perturbation to its physiological function. This may be of significance in protein engineering of AR proteins, that is, we can modify those residues far away from the functionally important regions in an AR protein to enhance stability without compromising its function.</p><p>It is also worthwhile to note that many AR proteins do not have a string of TPLH motifs (10). In such case, the aforementioned TPLH-mediated hydrogen-bonding network does not exist, and the stability of these AR proteins should be ascribed to the hydrophobic interaction network across the molecule as well as other undefined mechanisms. In addition, it has been reported that two designed AR proteins consisting of three and four identical repeats of consensus sequence, respectively, retain very high stabilities even under acidic pH. On one hand, consensus sequence should result in more optimized hydrophobic interactions between neighboring repeats; and these two proteins may have overcome less ideal or even the loss of TPLH-mediated hydrogen bonding network to retain a global fold. On the other hand, the crystallographic structures of these two designed AR proteins revealed a ring flip of the imidazole ring around Cβ-Cγ bond (χ2 ~ −90°) (24) in comparison to gankyrin and another de novo designed AR protein (25). The ring flip conformation precludes a hydrogen-bond between His Nε2 and the carbonyl oxygen of the residue His (+30), suggesting that the TPLH-mediated hydrogen bonding network in these two design AR proteins has been perturbed at the low pH if still existing, and may not be the primary factor contributing to the high stabilities of these two proteins at low pH. Lastly, the pH-dependence of conformational stability attributed to histidine has been observed in other proteins (26).</p><p>In conclusion, AR7 of gankyrin is not essential to maintain a tertiary fold of AR1-AR6, but it moderately stabilizes the global structure as well as increases its susceptibility against acidic pHs by shielding the internal hydrophobic interactions and TPLH-mediated hydrogen-bonding network from the aqueous environment. The current work is valuable for understanding the unique biophysical properties of gankyrin (AR proteins in general) and for novel AR protein design.</p><!><p>This work was partly supported by a research grant NIH, R01 CA69472 (J.L.).</p><p>Support Information Available</p><p>The downfield regions of 1D 1H NMR spectra were recorded on Gankyrin WT and Gank1-201 mutant under different pHs to determine the impact of pH on Hε2 signals of H45, H78, H111, H144, and H177. This material is available free of charge via the Internet at http://pubs.acs.org.</p><p>ankyrin repeat</p><p>circular dichroism</p><p>cyclin-dependent kinase 4</p><p>guanidinium hydrochloride</p><p>a protein encoded by the murine double minute (mdm2) oncogene</p><p>Nuclear Magnetic Resonance</p><p>Nuclear Overhauser effect Spectroscopy</p><p>the ankyrin repeat domain in Notch</p><p>wild type</p><!><p>The hydrogen bonds between Thr Hγ1 (Ser Hγ) and His Nδ1 and between His Hε2 and His (+30) O are highlighted by the solid line in magenta color, which are part of the TPLH-mediated hydrogen bonding network (6, 10).</p><!><p>A, Chemical-induced unfolding of gankyrin and Gank1-201. Samples were incubated with different amounts of GdnHCl on ice overnight. The ellipticity at 222 nm was monitored by far-UV CD (190–260 nm) at 20 °C. The fraction unfolded, defined as (the ellipticity at 222 nm at a denaturant concentration-the ellipticity at 222 nm at the native state)/(the ellipticity at 222 nm at the fully unfolded state-the ellipticity at 222 nm at the native state), was plotted against the GdnHCl concentration. B, Heat-induced unfolding of gankyrin, Gank1-201, and Q210H. Thermal melting spectra were recorded at 222 nm by heating from 5 °C to 65 °C with a rate of 1°C per minute and a 1 °C interval. Tm, the temperature at the midpoint of transition, was obtained through fitting the melting curve to a two-state transition model. In both A and B, solid circles, triangles, and diamonds represent experimental data for gankyrin WT, Gank1-201, and gankyrin Q210H, respectively, and different colors indicate different pHs: black, pH 7.4; green, pH 8.4; blue, pH 6.4; red, pH 5.4. The unfolding parameters were derived through a two-state transition approximation (27) and listed in Table 1. The dotted lines represent the fitting curves.</p><!><p>A, Structural comparisons of human gankyrin (PDB ID: 1TR4) and Notch ARD (PDB ID: 1OT8). Charged residues located at the C-termini of gankyrin and Notch ARD were highlighted (28). B, Sequence homology analysis using ClustalW (www.expasy.com). Residues subjected to mutagenesis are underlined. C, Ribbon diagram of gankyrin showing residues L209 (magenta), G214 (green), and I219 (blue) in the C-terminal AR. It is noted that the side chain of L209 is located between the anti-parallel helices of the 7th AR and is engaged in helix-helix packing through interacting with the side chains of K220 and L221, In contrast, the side chain of L219 is largely solvent-exposed. D, Solvent accessible surface of gankyrin with L209 and I219 painted in magenta and cyan, respectively. The percentages of the solvent-accessible side chain surface are 32% and 55%, respectively, for L209 and I219. In A, C, and D, the N-termini are located at the top and the C-termini are at the bottom.</p><!><p>The inserts are blow-up showing the downfield region that contains the signals of Hε2 from histidine residues in TPLH motifs.</p><!><p>A, Downfield region of the 2D NOESY recorded on Gank1-201 at pH 7.4. The top projection shows the assignments of the histidine Hε2 protons in TPLH and its variant. B, 1D 1H NMR of the amide region recorded on Gank1-201 at pH 5.4, 6.4, and 7.4 after a week of incubation. C, 1D 1H NMR of the amide region recorded on gankyrin WT at pH 5.4, 6.4, and 7.4 in the parallel experiments. In both B and C, the inserts are the blowup of the region between 9.00 and 12.50 ppm.</p><!><p>A, Chemical-induced unfolding. B, Heat-induced unfolding. The experiments were conducted as described in Figure 1 except that the experimental pH was 7.4 and the temperature range for heat-induced unfolding was 5–70 °C since some gankyrin mutants might have higher stabilities than gankyrin WT. In both A and B, solid circles represent experimental data and the dotted lines are fitting curves. Green, L209R; red, G214E; cyan, G215R; pink, G217H; yellow, L218H; blue, I219D; G215R/I219D, dark green.</p><!><p>A, Effect on the interaction between gankyrin and the CDK4-cyclin D2 complex (14). The reaction mixtures containing GST gankyrin proteins (~3.0 μM) and the (His)6-tagged CDK4/cyclin D2 holoenzyme (~0.3 μM) were incubated with G beads. After washing with PBS, bound proteins were eluted out using reduced glutathione and blotted with anti- human CDK4 antibody. GST was used as a negative control. B. Effect on the interaction between gankyrin and MDM2. The experiments were performed as described in A except that the reaction mixtures containing free gankyrin proteins (~3.0 μM) and GST-MDM2 (~0.3 μM) and the pull-down products were blotted with a primary antibody again human gankyrin. The control reaction contained free gankyrin WT (~3.0 μM) and GST (~0.3 μM).</p><!><p>The conformational stabilities of gankyrin proteins</p><p>ΔGdwater, D1/2, and m values were calculated according to a two-state transition model (27), and the error in ΔGdwater is estimated to be ± 0.2 kcal*mol−1. Tm was defined as the temperature at the midpoint of transition, and the error is estimated to be ± 0.5 °C (12).</p><p>ND, not determined. Tm of R210H at pH 6.4 was not determined due to the fact that after heat-induced unfolding, the refolding process was not recorded, i.e. the thermo-induced folding/unfolding of R210H at pH 6.4 was not reversible.</p>

PubMed Author Manuscript

Natural Shape-Retaining Microcapsules With Shells Made of Chitosan-Coated Colloidal Lignin Particles

Thin film coating of charged nanoparticles with oppositely charged polymers is an efficient and straightforward way for surface modification, but synthetic polyelectrolytes should be replaced by abundant biopolymers. In this study a thin film of chitosan was adsorbed onto colloidal lignin particles (CLPs) that were then systematically studied for olive oil stabilization with an objective to develop shape-retaining microcapsules that comprised of only renewable biomaterials. Full surface coverage was achieved with merely 5 wt% of chitosan relative to the dry weight of CLPs, reversing their surface charge from negative to positive. Such modification rendered the chitosan-coated particles excellent stabilizers for forming Pickering emulsions with olive oil. The emulsion droplets could be further stabilized by sodium triphosphate that provided ionic intra- and inter-particle cross-linking of the chitosan corona on the CLPs. Following the optimum conditions, the non-cross-linked microcapsules exhibited a strong stability against coalescence and the electrostatically stabilized ones additionally retained their shape upon drying and rewetting. Non-cross-linked microcapsules were used to demonstrate encapsulation and rapid release of ciprofloxacin as a model lipophilic drug in aqueous media. Overall, the combination of antimicrobial chitosan and antioxidative lignin nanoparticles hold unprecedented opportunities as biocompatible and biodegradable materials for controlled drug delivery.

natural_shape-retaining_microcapsules_with_shells_made_of_chitosan-coated_colloidal_lignin_particles

Introduction<!>Materials<!>Preparation of Chitosan Solution<!>Preparation of Aqueous Colloidal Lignin Particles<!>Preparation of Chitosan-Coated Colloidal Lignin Particles (chi-CLPs)<!>Preparation of chi-CLP Stabilized Oil-in-Water Pickering Emulsions<!>Preparation of Ionically Cross-Linked Pickering Emulsions<!>Preparation of Ciprofloxacin-Loaded Pickering Emulsions<!>Ciprofloxacin Release Study<!>Particle Diameter and Zeta Potential Analysis<!>Droplet Diameter and Uniformity of Emulsion Analysis<!>Confocal Microscopy<!>Optical Microscopy<!>Environmental-SEM<!>AFM<!>QCM-D<!>Results and Discussion<!><!>Determining the Minimum Needed Chitosan Coating on CLPs<!><!>Determining the Minimum Needed Chitosan Coating on CLPs<!>Effect of Chitosan to CLP Mass Ratio on Emulsion Formation<!><!>Effect of Chitosan to CLP Mass Ratio on Emulsion Formation<!>Effect of chi-CLP Concentration on Emulsion Formation<!><!>Stability and Surface Morphology of the Emulsion<!>Ionic Cross-Linking of the Emulsion Capsules for Enhanced Mechanical Performance<!><!>Ciprofloxacin Release From the Emulsion Capsules at Various PH Values<!><!>Conclusion<!>Author Contributions<!>Conflict of Interest Statement

<p>Lignin is the second most abundant natural biopolymer and the most abundant aromatic polymer in nature (Ragauskas et al., 2014). Annually over 70 million tons of industrial lignin is produced as a by-product in the pulp/paper industry. However, only 5% of industrial lignin is commercialized as value-added products, such as additives, dispersants, adhesives, or surfactants (Laurichesse and Avérous, 2014). The rest 95% is used as low value energy source or simply treated as waste. The low extent of commercialization of lignin is mainly due to its complex and inhomogeneous structure resulting from different sources and extraction processes (Li et al., 2015). However, recent studies have shown that fabricating spherical micro- or nanoparticles from lignin not only enables the large scale utilization of lignin (Ago et al., 2016; Leskinen et al., 2017a; Ashok et al., 2018; Lintinen et al., 2018), but also improves properties such as surface activity, antioxidative, UV shielding, and antimicrobial activity for potential high-value applications (Beisl et al., 2017).</p><p>To name a few examples, colloidal lignin particles (CLPs) have been utilized to entrap hydrophobic drugs for drug delivery purposes in biomedicine (Chen et al., 2016; Dai et al., 2017; Figueiredo et al., 2017; Sipponen et al., 2018b). By introducing CLPs in cellulose nanofilms, Farooq, and co-workers rendered the composite films waterproof, provided antioxidant activity and UV-shielding in addition to the improved mechanical properties (Farooq et al., 2019). Surface-modification of CLPs by adsorption has been demonstrated with synthetic poly(diallyldimethylammonium chloride) (Lievonen et al., 2016), proteins (Leskinen et al., 2017b), and cationic lignin (Sipponen et al., 2017). Such modifications alter the interfacial properties of the particles and broaden the window for possible high-value applications. For instance, cationic CLPs were demonstrated for binding negatively charged enzymes for enhanced catalytic activity in aqueous ester synthesis (Sipponen et al., 2018a).</p><p>In addition, micro- and nanoscaled lignin particles have been exploited as surface-active stabilizers for Pickering emulsions (Wei et al., 2012; Yang et al., 2013; Nypelö et al., 2015; Ago et al., 2016; Sipponen et al., 2017). Pickering emulsions are stabilized by solid particles at the oil/water interface (Pickering, 1907). Compared to conventional surfactant-stabilized emulsions, Pickering emulsions are more stable against coalescence, because high energy input is required to remove solid particles from the oil/water interface (Binks, 2002; Rayner et al., 2014; Berton-Carabin and Schroën, 2015). Additionally, some food-grade particles are less toxic than synthetic surfactants (Yang et al., 2017). Therefore, Pickering emulsions can be applied in a wide range of fields including biomedicine, cosmetics, and food (McClements, 2015; Tang et al., 2015; Yang et al., 2017).</p><p>To date, lignin-stabilized Pickering emulsions reported in scientific articles are mainly aliphatic or aromatic hydrocarbon oil-based, e.g., kerosene, hexadecane, styrene, and toluene (Wei et al., 2012; Yang et al., 2013; Nypelö et al., 2015; Ago et al., 2016; Sipponen et al., 2017). Such oils are often toxic, which therefore are not suitable for biomedicine, cosmetics, or food. Instead, triglyceride vegetable oils are a better choice, since they do not present any safety issues (Pouton and Porter, 2008). Furthermore, triglycerides are good solvents for many lipophilic drugs and have the ability to increase intestinal wall permeability, which makes them particularly useful for drug delivery (Kalepu et al., 2013). Nevertheless, unmodified CLPs exhibit limited capacity in stabilizing triglyceride-in-water Pickering emulsions, possibly due to insufficient hydrophilicity. Sipponen et al. modified CLPs by adsorption of cationic lignin to form more hydrophilic cationic CLPs (c-CLPs), which exhibited improved capacity for stabilizing olive oil compared to unmodified CLPs (Sipponen et al., 2017). Yet, the formed olive oil-in-water emulsions were not sufficiently stable. Additionally, alike other quaternary ammonium substances (Zhang C. et al., 2015), the cationic lignin grafted with quaternary ammonium groups may be toxic.</p><p>Chitosan, on the other hand, has shown good emulsification capacity in stabilizing triglyceride-in-water emulsions whether in the form of molecules (Schulz et al., 1998; Del Blanco et al., 1999; Rodriǵuez et al., 2002) or particles (Mwangi et al., 2016a; Shah et al., 2016; Asfour et al., 2017). Furthermore, chitosan possesses biologically relevant properties such as biocompatibility, biodegradability, bio-adhesion, and antimicrobial activity (Pavinatto et al., 2010; Croisier and Jérôme, 2013; Asfour et al., 2017). As a consequence, chitosan appears to be a plausible material for modifying CLPs to form triglyceride-based Pickering emulsions.</p><p>In this work, we systematically studied the modification of CLPs by thin film coating of chitosan and determined the emulsification capacities of the chitosan-coated CLPs (chi-CLPs) for olive oil. We used sodium triphosphate (STP) to ionically cross-link the chi-CLPs locating at the oil-water interface and compared the mechanical properties of the electrostatically stabilized chi-CLP microcapsules to those of non-cross-linked ones. Finally, we demonstrated the ability of the chi-CLP microcapsules in the encapsulation and release of ciprofloxacin at various pH values at 37°C. Overall, this work provides optimum conditions for the preparation of non-coalescent and shape-retaining microcapsules for future applications in controlled drug delivery in the field of biomedicine.</p><!><p>Kraft lignin (BioPiva 100) used in this work was isolated from softwood using the LignoBoost® technology at Domtar's Plymouth plant (NC, USA). The kraft lignin was well-characterized in the previous publication (Sipponen et al., 2018a). The number and weight average molecular weights (Mn and Mw) of the kraft lignin are 1,193 and 5,250 g/mol, respectively. The carboxyl groups, aliphatic hydroxyl groups, and total phenolic hydroxyl groups of the kraft lignin are 0.57, 1.89, and 4.05 mmol/g, respectively. Tall oil fatty acid (TOFA) "For2" was a kind gift from Forchem Oyj (Finland). Chitosan (molecular weight 100 to 300 kDa, deacetylation degree ≥90%) and sodium triphosphate (STP) (≥98%) were purchased from Fisher Scientific (Acros organics). Olive oil (highly refined, low acidity), ciprofloxacin (≥98%), poly(styrene) (M¯w: 35,000 g/mol) and acetic acid (glacial, ≥99.8%) were purchased from Sigma-Aldrich. The content of free fatty acid in olive oil was determined to be 1.9 ± 0.2 wt% (n = 7) by titration (Sipponen et al., 2017). All purchased chemicals and solvents were used without further purification.</p><!><p>1 wt% chitosan solution was prepared by dissolving 1 g of chitosan in 99 g of 0.1 M acetic acid under stirring for 24 h. The dissolution of chitosan in 0.1 M acetic acid resulted in an increase of the pH value from 2.9 to 4.5, which indicated a partial protonation of chitosan (ca. 60 to 70% protonation of the primary amine groups, calculated according to the pKa 4.75 of acetic acid and the deacetylation degree of chitosan).</p><!><p>Preparation of 0.2 wt% colloidal lignin particle (CLP) dispersion followed the procedure described in the previous publication (Sipponen et al., 2017) with the modification that in this study 2 g kraft lignin (dry weight) was dissolved in 200 g of acetone-water mixture (mass ratio: 3:1), instead of using THF-water mixture as the solvent. The final aqueous CLP dispersion (0.2 wt%) was obtained with a lignin mass yield of 85%. The particle diameter and zeta potential of the CLPs at native pH 3.9 were determined to be 97 nm (PDI 0.18) and −27 mV, respectively. The preparation of 1 wt% CLP dispersion was similar to that of 0.2 wt% CLP dispersion except that in this case rotary evaporation (40°C under reduced pressure) was used to remove acetone instead of dialysis. 0.5 wt% CLP dispersion was prepared by diluting the 1 wt% CLP dispersion with deionized water. The particle diameter and zeta potential of the CLPs (0.5 and 1 wt%) were 113 nm (PDI 0.19) and −30 mV (at native pH 3.1), respectively.</p><!><p>Chitosan-coated colloidal lignin particles (chi-CLPs) were prepared by adding CLP dispersion (0.2 or 0.5 or 1 wt%) slowly into 1 wt% chitosan solution under vigorous stirring for 30 min. The prepared chi-CLP dispersions were stored over night before use. For 0.2 wt% chi-CLP dispersions, the mass ratio of chitosan to CLP was varied from 0 to 200 mg/g. For 0.5 and 1 wt% chi-CLP dispersions, chi-CLPs were prepared at a fixed mass ratio of 50 mg/g.</p><!><p>The Pickering emulsions were prepared by ultrasonication (Branson 450 Digital Sonifier with a 3 mm-diameter microtip) under ice bath condition at a fixed volume ratio of 1:1 olive oil to chi-CLP (or CLP) dispersion. More specifically, 60 s with the cycles 10 s on and 5 s off were applied for emulsion formation, the amplitude was set at 10% for a total volume of 2 ml and 40% for 10 ml. These procedures resulted in similar size distributions of oil droplets when the concentration of the chi-CLP (50 mg/g) dispersion was 0.5 or 1 wt%.</p><!><p>Pickering emulsion formed with 1 wt% chi-CLP (50 mg/g) dispersion was used for the cross-linking study. The preparation was achieved by adding the emulsion slowly into 6 wt% sodium triphosphate (STP) aqueous solution at the volume ratio of 1:9 (emulsion: STP solution) under vigorous stirring for 30 min.</p><!><p>1 wt% chi-CLP (50 mg/g) dispersion was used for forming the emulsion with ciprofloxacin-loaded oil for release study. Ciprofloxacin was firstly dissolved (20 mg/mL) in TOFA followed by dilution with olive oil to 2 mg/mL. The emulsion formation followed the aforementioned procedure.</p><!><p>The release study was performed in three different pH buffers, pH 2 (the buffer was prepared by mixing 0.1 M KCl and 0.02 M HCl to obtain a solution at pH 2), pH 5.5 (0.05 M PBS) and pH 7.4 (0.05 M PBS) at 37°C. For each sample, 1.2 mL ciprofloxacin-loaded emulsion (50% oil phase) was injected into 60 ml of buffer solution. The aliquots were taken at various time intervals and filtered through a 0.2 μm syringe filter to separate the oil droplets and/or lignin particles. The concentration of ciprofloxacin in the buffer was calculated from the absorbance values at 277 nm, after correcting with the absorbance resulting from minor dissolution of lignin in the absence of ciprofloxacin, according to the calibration curve shown in Figure S1. The average of four ciprofloxacin-loaded samples and two reference samples were used in the analysis and reporting of data.</p><!><p>Particle diameters and zeta potentials of CLPs and chi-CLPs were analyzed using a Zetasizer Nano ZS90 instrument (Malvern Instruments Ltd., U.K.). A dip cell probe was utilized for the determination of the zeta potential. CLP, chi-CLP dispersions and chitosan solution were diluted accordingly with deionized water or pH 4.5 acetic acid prior to measurement. Mean values of three replicates of the particle diameter (Z-average, intensity mean) and zeta potential were used in the analysis and reporting of data.</p><!><p>The droplet diameter of the emulsion was determined by static light scattering (Mastersizer 2000, Malvern, UK). The emulsions were diluted with deionized water to reach the laser obscuration of 6 to 12% prior to starting the measurement. The refractive index (RI) of olive oil and water used in the calculations were 1.47 and 1.33, respectively. Mean droplet diameter was calculated over volume data (d43, De Brouckere Mean Diameter). Uniformity of the droplets was calculated according to Equation (1)</p><p>where d(v, 0.5) is the median diameter in the volume-based distribution, di is the diameter in class i and Xi is the corresponding volume fraction in %. Mean values of six replicates of mean droplet diameter (d43) and uniformity were used in the analysis and reporting of data.</p><!><p>The emulsions stabilized by 1 wt% chi-CLP (50 mg/g) were imaged with the confocal laser scanning microscope (Leica DMRXE, Germany). Emulsions were diluted 50 times with deionized water or with 6 wt% STP aqueous solution followed by staining the oil with Nile red (1 mg/mL in ethanol) (ca. 50 μl of Nile red in 1 ml emulsion) prior to measurement. For each sample, a drop (5 μl) of the Nile red-stained emulsion was placed on the glass slide for imaging at the wavelength of 488 nm, using a 10 × air objective and a 63 × oil immersion objective.</p><!><p>A Leica Zeiss (DM750) optical microscope was used for observing the emulsions without staining.</p><!><p>A Zeiss Environmental Scanning Electron Microscope (EVO HD15) was used for observing the surface morphology of the oil droplets. The emulsion capsule that was stabilized with 1 wt% chi-CLP (50 mg/g) and cross-linked with STP was selected for the observation. A drop (5 μl) of the diluted emulsion (500 times with 6 wt% STP aqueous solution) was cooled at −20°C in a Peltier cooling element in order to freeze the oil and sublimate the water. During the observation, the temperature was kept at −20°C and the pressure of the SEM chamber was set at 50 Pa, images were captured with an EPSE detector.</p><!><p>The AFM analysis was carried out with a MultiMode eight atomic force microscope equipped with a NanoScope V controller (Bruker Corporation, U.S.A.). The images for CLPs were obtained in tapping mode under ambient air condition with NCHV-A tapping mode probes (Bruker). The images for oil droplets (stabilized by chi-CLP and cross-linked with STP) were obtained in ScanAsyst mode under ambient air condition with SCANASYST-AIR probes (Bruker). Samples were prepared by dropping 5 μl of the diluted dispersion or emulsion on the mica surfaces and drying under ambient conditions. Nanoscope analysis 1.5 software was used for image analysis.</p><!><p>The lignin substrates for QCM-D studies were prepared as described by Salas et al. (2013) except that the lignin used in this study was kraft lignin. In brief, lignin was spin-coated onto gold-coated QCM crystals (Q-Sense, Sweden) that had been pre-coated with poly(styrene). Adsorption of chitosan on the thin films were carried out using QCM-D E4 (Q-Sense, Sweden) in continuous flow mode. Chitosan was firstly dissolved (1 wt%) in 0.1 M acetic acid and then diluted with pH 4.5 acetic acid to 100 μg/mL. During the measurements, the lignin films were firstly rinsed with pH 4.5 acetic acid buffer at the flow rate of 0.1 mL/min until a plateau baseline (Δf 5) was reached, followed by replacing the acetic acid buffer with the diluted chitosan solution. The temperature was controlled at 25°C throughout the experiments. The mass of chitosan adsorbed on the lignin surface was related to the change of resonance frequency Δf according to the Sauerbrey equation (Sauerbrey, 1959):</p><p>where Δm is the change of mass, Δf is the change of resonance frequency determined by the device, C is a constant (0.177 mg m−2 Hz−1) that describes the sensitivity of the device to changes in mass, n is the overtone number (n = 1 represents the fundamental frequency at 4.95 MHz). The energy dissipation due to adsorption is indicated by the dissipation factor D (Rodahl and Kasemo, 1996), which is defined as:</p><p>where Edis is the dissipated energy and Est is the stored energy during one oscillation cycle. The dissipation change is defined as, ΔD = D–D0, where D0 denotes the initial dissipation prior to adsorption. In this work, the average values (Δ f 5 and ΔD5) of two replicate samples were used in the analysis and reporting of data.</p><!><p>The overarching objective of this work was to establish a reliable approach for chitosan-coated CLPs, and application of the modified particles in stabilization of olive oil-in-water Pickering emulsion capsules. A general scheme of this work is shown in Figure 1. The CLPs were prepared by rapid anti-solvent nanoprecipitation method, followed by either dialysis or evaporation to remove the acetone that was used as solvent. In contrast to the more commonly used THF (Lievonen et al., 2016), acetone can be easily removed by evaporation, as acetone has no azeotrope with water (Smallwood, 2012). The obtained CLPs were modified with thin film coating of chitosan by physical adsorption and systematically studied for the stabilization of Pickering emulsions. Selected emulsion capsules were further stabilized by non-covalent cross-linking. Encapsulation of a model drug into the oil phase and its release from the non-cross-linked capsules was finally demonstrated in aqueous media. The first important step was to optimize the chitosan coating and emulsion formation processes, which are discussed more in detail below.</p><!><p>General scheme of this work. (A) Preparation of colloidal lignin particles (CLPs). (B) Coating of CLPs with chitosan for forming Pickering emulsion with olive oil, and demonstration of the emulsion capsules for drug delivery. Note: not drawn to scale.</p><!><p>The CLPs purified by dialysis (0.2 wt%) were used for assessing the effect of mass ratio of chitosan to CLP on the particle properties. It was determined by DLS that pure CLPs had the average particle diameter of 97 nm (PDI 0.18) and zeta potential of −27 mV (at native pH 3.9). When 10 mg/g chitosan to CLP was added into the CLP dispersion, the dispersion showed strong aggregation and sedimentation, indicating neutralization of CLPs by chitosan (Figure 2A). When 20 mg/g chitosan to CLP was added, the zeta potential of CLP was reversed from negative (−27 mV) to positive (17.6 mV), showing overcompensation of the surface charge by chitosan. The overcompensation can be explained as follows. Firstly, chitosan could easily adsorb onto the surfaces of CLPs, which was mainly driven by the large entropy gain of the released counterions from both CLPs and chitosan (Kronberg et al., 2014). Secondly, once adsorbed, columbic attraction, hydrogen bonding and van der Waals forces were the attraction forces between chitosan and CLP. The adsorption also caused conformational entropy loss of chitosan, yet this was much smaller relative to the entropy gain of the released counterions. On the basis of adsorption, the overcompensation could happen as the absolute charge density of chitosan (apparent zeta potential +54.4 mV) (Figure S2) was higher than that of CLPs (−27 mV). However, at the mass ratio 20 mg/g chitosan to CLP, the amount of chitosan was not sufficiently high to cover all the surfaces of CLPs, which was indicated by the broad particle diameter distribution and not high enough positive surface charge of chi-CLP20 (Figure S3).</p><!><p>Characterization of coating of CLPs with chitosan. (A) Photograph of the chi-CLP dispersions (0.2 wt%, with chitosan-to-CLP mass ratio varying from 0 to 200 mg/g) after storage for 2 h. (B) Particle diameter and zeta potential of chi-CLPs plotted against the mass ratio of chitosan to CLP. (C) QCM-D analysis of adsorbed mass of chitosan on lignin surface. (D) Particle diameter of chi-CLP75, chi-CLP100, and chi-CLP200 plotted against the chi-CLP concentration, chi-CLP dispersions were diluted with pH 4.5 aqueous acetic acid. (E) Zeta potential of chi-CLP75, chi-CLP100, and chi-CLP200 plotted against chi-CLP concentration, chi-CLP dispersions were diluted with pH 4.5 aqueous acetic acid. The error bars in (B,D,E) denote the standard deviations of three replicates. Note: chi-CLP10 was not measurable by DLS due to strong aggregation and sedimentation.</p><!><p>At the mass ratio of 35 mg/g chitosan to CLP, the chi-CLP35 exhibited a higher surface charge density (24.6 mV) than that of chi-CLP20 (17.6 mV), and the resulting particle diameter distribution of chi-CLP35 (PDI 0.25) was also much narrower than that of chi-CLP20 (PDI 0.43) (Figure 2B and Figure S3). Those values indicated that, at 35 mg/g, the CLPs were efficiently covered by chitosan, and thus the chi-CLP dispersion became stable. Higher mass ratios of chitosan to CLP resulted in similar particle diameter distributions (Figure S3) compared to that obtained at 35 mg/g, but a slight increase in the particle diameter as well as zeta potential was observed (Figure 2B). Such observation resulted from the presence of excess chitosan in the aqueous phase. With excess chitosan relative to the CLP surfaces, large chitosan molecules were more prone to adsorb than the smaller ones due to lower solubility/stability of large chitosan molecules in solution. This phenomenon has been described in detail elsewhere (Fu and Santore, 1998; Terada et al., 2004; Kronberg et al., 2014). The adsorption of chitosan to lignin surface was also studied by using QCM-D. It was shown in Figure 2C that, the adsorption of chitosan on lignin film was rather slow, probably due to the low negative charge of lignin. Comparing to CLPs that formed via self-assembly, lignin film that was prepared by spin-coating probably had a lower density of charged groups oriented toward the aqueous phase. In addition, the low value of sensed mass Δm and negligible change in dissipation factor ΔD detected by QCM-D indicated that the adsorbed chitosan layer on lignin surface was very thin (Rodahl and Kasemo, 1996; Naderi and Claesson, 2006).</p><p>The excess of chitosan in the aqueous phase was indicated by DLS measurement. As shown in Figure 2D, the increase of chitosan/CLP mass ratio from 75 to 200 mg/g resulted in a stronger dependency of the particle diameter as the function of chi-CLP concentration. The particle diameter of chi-CLP75 increased slightly with increased concentration, yet it increased significantly more in the case of chi-CLP200. Such phenomena can be explained by the increased viscosity of the aqueous media in the presence of excess chitosan. The more excess chitosan in the aqueous phase, the larger the overestimation of particle diameter determined by DLS. The swelling/deswelling of chi-CLPs could be neglected in this case, as the dispersions were diluted with pH 4.5 acetic acid and thus the pH and ionic strength remained constant throughout the experiments. As another evidence, the zeta potential remained essentially unchanged as a function of chi-CLP concentration (Figure 2E). The unchanged zeta potential also indicated that no detectable desorption of chitosan occurred upon dilution.</p><!><p>Olive oil-based Pickering emulsions were formed using 0.2 wt% chi-CLP dispersions, in which the mass ratio of chitosan to CLP varied from 0 to 200 mg/g. The volume ratio of olive oil to chi-CLP dispersion was fixed at 1:1. The emulsions were formed by ultrasonication and hand-shaking. It was found that, the mass ratio of chitosan to CLP had a strong effect on the properties of the emulsions that formed by ultrasonication. At low mass ratio of chitosan to CLP (≤ 20 mg/g), the formed emulsions were poor that showed broad biphasic diameter distributions of the oil droplets (Figure 3B and Figure S4). However, with a higher mass ratio of chitosan to CLP at 35 mg/g, a strong increase of the fraction of small oil droplets with diameter between ca. 10 to 100 μm and reduction in droplets between 100 to 1,000 μm occurred (Figure S4). Effectively, optical microscopic images showed that oil droplets from around 10 to 100 μm dominated the diameter distribution (Figure 3B). This transition happened in accordance with the partial to efficient coverage of CLPs by chitosan from 20 to 35 mg/g mass ratios. We can therefore conclude that a sufficient coverage of CLPs by chitosan is essential for stabilization of olive oil in water. This finding correlates well with the limited emulsification capacity of regular anionic CLPs (Sipponen et al., 2017) and good emulsification capacity of cationic chitosan for triglycerides (Schulz et al., 1998; Del Blanco et al., 1999; Rodriǵuez et al., 2002). When the mass ratio of chitosan to CLP was increased to 50 mg/g, the distribution of the droplets became nearly monophasic, the droplets mainly distributed between 10 to 100 μm and the "macro" droplets from 100 to 1,000 μm vanished (Figure S4A). Such transition can be explained from two aspects. On the one hand, the cationic charge density along with the hydrophilicity (Zhang S. et al., 2015) of the chi-CLPs increased with higher chitosan-to-CLP coating ratio, which enhanced the attraction of chi-CLP to olive oil that contained 2 wt% of negatively charged oleic acids. On the other hand, the excess chitosan in the aqueous phase was likely involved in forming the emulsion with chi-CLP and thus reduced the coalescence of the oil droplets. The involvement of chitosan in the emulsion formation was also indicated by the fact that the average diameter of oil droplets decreased slightly further with more excess chitosan from chi-CLP50 to chi-CLP200 (Figures 3B,C). The mean droplet diameter nearly plateaued at ~25 μm, which is considerably smaller size than reported for triglyceride-in-water Pickering emulsions stabilized with chitosan particles alone or as mixtures with STP (Mwangi et al., 2016a; Shah et al., 2016).</p><!><p>Effect of chitosan to CLP mass ratio and chi-CLP concentration on the properties of olive oil-in-water (volume ratio 1:1) Pickering emulsions (formed by ultrasonication). (A) Photograph of the Pickering emulsions stabilized by 0.2 wt% chi-CLPs (0 to 200 mg/g). (B) Optical microscopic images of the Pickering emulsions stabilized by 0.2 wt% chi-CLPs (0 to 200 mg/g) (Scale bars: 200 μm). (C) Mean oil droplet diameter (d43) and uniformity (according to Equation 1) plotted against the mass ratio of chitosan to CLP from 0 to 200 mg/g (0.2 wt%). The error bars denote standard deviations of six replicates. (D) Diameter distribution of the oil droplets stabilized by 0.2, 0.5, and 1 wt% chi-CLPs at a fixed mass ratio of 50 mg/g chitosan to CLP. Note: photograph, optical microscopic images and diameter measurements were done 24 h after emulsion formation.</p><!><p>Comparison of the amounts of chi-CLPs required to obtain stable emulsions to those in the literature reveals that the emulsifier-to-oil ratios obtained in the present work are significantly lower. The stable emulsions in Figure 3A contained chi-CLPs at the coating ratio of 100 mg/g and at 0.2 wt% concentration that equals 0.2 wt% emulsifier relative to olive oil. Previously, 0.5 wt% of cationic lignin-coated CLPs were used in incomplete stabilization of olive oil/water emulsion (Sipponen et al., 2017), while the values for aggregated chitosan particles range from 0.3 wt% (oleic acid-in-water) (Asfour et al., 2017) to 1.2 wt% (medium chain triglycerides-in-water) (Mwangi et al., 2016b). We expect that the lower ratio of chi-CLPs that suffices for emulsion stabilization in the present work stems from the large surface area and efficient assembly of the colloidal particles at the oil/water interface.</p><p>Interestingly, when comparing the emulsions that formed by hand-shaking. Even chi-CLP10 succeeded in forming the emulsion with olive oil, while pure CLP failed to do so. Additionally, optical microscopic images showed that all the emulsions showed broad droplet diameter distributions ranging from a few to hundreds micrometers regardless of chitosan to CLP mass ratio (Figure S5). This is reasonable, because the energy input generated by manual shaking is much lower and inhomogeneous compared to that of ultrasonication. As a consequence, hand-shaking could not produce small and uniform emulsion droplets even with the presence of excess chitosan.</p><!><p>Aforementioned results showed that the minimum coating ratio at which the emulsions became nearly monophasic was 50 mg/g chitosan to CLP. Therefore, this mass ratio was fixed for studying the effect of chi-CLP concentration on the emulsion properties. Three different concentrations of chi-CLP dispersions (0.2, 0.5, and 1 wt% chi-CLP) were used to form emulsions with olive oil (volume ratio 1:1) by ultrasonication. As anticipated, higher concentration of chi-CLP dispersion resulted in emulsions with smaller mean droplet diameter and better uniformity (Figure 3D). This phenomenon can be understood from the perspective of diffusive and adsorptive kinetics of the chi-CLPs. At higher concentration of the chi-CLP dispersion, a shorter diffusion time of the particles to the oil/water interfaces and a faster adsorption occurs due to shorter diffusion distances. As a consequence, the oil droplets were more efficiently and rapidly covered by the chi-CLPs during the transient mixture of oil and water caused by ultrasonication, which therefore resulted in less coalescence of the oil droplets during formation. Finally, an emulsion with smaller and more uniform droplets was obtained. At the highest concentration (1 wt%) of chi-CLP dispersion, the formed emulsion showed the smallest mean droplet diameter of ca. Seventeen micrometer with the most uniform diameter distribution (Figures 3D, 4A). Such uniformity value of ca. 0.3 outperforms the uniformity of 0.5 reported for palm oil/water Pickering emulsion stabilized by aggregated chitosan particles (Mwangi et al., 2016a). As another comparison, chitosan molecule alone could not result in such uniform droplets with olive oil, even at the concentration of 1 wt% (Figure S6). Furthermore, long term observation (over 6 months) found that the emulsions stabilized by chitosan molecules were much less stable than those stabilized by chi-CLPs (Figure S7). Additionally, calculated from the data in Figure 4A, the emulsion stability index (volume ratio of the emulsion layer to the total volume) was nearly one, which is obviously much higher than that obtained with cationic colloidal lignin particles (Sipponen et al., 2017).</p><!><p>Stability and surface morphology of the emulsion stabilized by 1 wt% chi-CLP (50 mg/g). (A) Photograph of the emulsion (before and after creaming) and the corresponding confocal microscopic images of the emulsion (oil was stained by Nile red). (B) Creaming behavior of the emulsion, measured with Turbiscan (height was chosen at the backscattered intensity 0.5 (Figure S9) and rescaled by starting from 0, corresponding to the height change marked by orange dash boxes in A). (C) Diameter distributions of the oil droplets on day 1, day 14, and day 60 from emulsion formation. (D) Surface morphology of a single oil droplet covered by STP cross-linked chi-CLPs, imaged with E-SEM (left, oil was solidified at −20°C) and AFM (right, measured at ambient conditions).</p><!><p>The emulsion stabilized by 1 wt% chi-CLP (50 mg/g) was selected for further stability test. In general, the emulsion exhibited a strong stability against coalescence, where the droplet diameter distribution of the emulsion did not change over 2 months (Figure 4C). Such strong stability resulted from the high energy requirement for the desorption of chi-CLPs from oil/water interface and the positive charge of the chi-CLPs that providing electrostatic stabilization (Binks and Clint, 2002; Rayner et al., 2014). In addition, the relative high viscosity (Figure 4B) also contributed to the high stability of the emulsion. As a consequence, the emulsion could be utilized for drug storage purposes, since many drugs are degradable when exposed to oxygen and UV light, which can be inhibited by the antioxidative, and UV shielding properties of the lignin nanoparticles (Farooq et al., 2019).</p><p>The surface morphology of the capsule was rather smooth when observed under environmental SEM (Figure 4D, left). A closer observation by AFM showed that the particles were partly embedded in the oil droplets (Figure 4D, right). SEM also showed that a longer exposure of the frozen droplet to the electron beam could expose the embedded chi-CLPs from the surface of the oil droplet (Figure S8). Such observations indicated that the chi-CLPs had a strong affinity to the oil.</p><!><p>The emulsion stabilized by 1 wt% chi-CLP (50 mg/g) was selected for the ionic cross-linking study. Instead of covalent cross-linking of chi-CLPs, ionic cross-linking is reversible, and can be done easily at ambient conditions. In this work, sodium triphosphate (STP) was used because it has low toxicity (Human and Environmental Risk Assessment, CAS: 7758-29-4)1 and has been used successfully in cross-linking chitosan (Mwangi et al., 2016a; Shah et al., 2016; Larbi-Bouamrane et al., 2017). The ionic cross-linking of chi-CLP by STP followed similar mechanism as the CLP coating by chitosan. Briefly, the replacement of the monovalent counterions of chitosan by trivalent triphosphate resulted in a large entropy gain. After cross-linking of the emulsion capsules, the mechanical stability of the oil droplets increased significantly. The oil could still be retained in the chi-CLP capsules even after drying and rewetting, indicating that the corona of the capsules was very strong (Figure 5A). In comparison, the non-cross-linked emulsion capsules easily broke down after drying and released the oil (Figure 5B). Similar observation was confirmed by optical microscopy, which showed that the cross-linked emulsion capsules retained their round shape after drying, yet the non-cross-linked ones were broken up to irregular shapes (Figure S10). With the exception of organic/inorganic capsules prepared by emulsion polymerization (Zhang et al., 2010; Wang et al., 2012), essentially all of the prior works have visualized shell rupture of Pickering capsules upon drying (van Rijn et al., 2011; Sipponen et al., 2017). In addition to their enhanced stability, the rewettable emulsion capsules prepared in the current work enable changing the aqueous phase or forced diffusion of oleophilic substances into the capsules by water evaporation. Besides, compared to the covalent epoxy cross-linking of lignin capsules demonstrated by Tortora et al. (2014), our approach is reversible. The ionic cross-linkers are released into aqueous phase upon dilution by water due to the deprotonation of the protonated primary amine groups (pKa 6.5), which is beneficial for dissembling of the capsules that serve as drug carriers.</p><!><p>Confocal microscopic images of sodium triphosphate (STP) cross-linked and non-cross-linked olive oil-in-water (volume ratio 1:1) Pickering emulsions stabilized by 1 wt% chi-CLP (50 mg/g) (Scale bars: 80 μm). (A) STP cross-linked emulsion in wet state (oil droplets in water), in dry state (water evaporated at room temperature), and after rewetting with deionized water. (B) Non-cross-linked emulsion in wet state, dry state and after rewetting as the reference.</p><!><p>The biocompatibility of various types of lignins is currently under intense investigation. Recent studies have indicated that kraft lignin and alkali lignin are not antiproliferative and instead mostly biocompatible (Tortora et al., 2014; Dai et al., 2017; Figueiredo et al., 2017). We selected ciprofloxacin as a model drug for the release demonstration by the emulsion capsule which was stabilized by 1 wt% chi-CLP (50 mg/g). Ciprofloxacin is an antibiotic drug (Zeiler and Grohe, 1986) that has potential synergic effect with chi-CLPs that likely possess antimicrobial activities (Croisier and Jérôme, 2013; Beisl et al., 2017). The drug release experiment was conducted under simulated physiological conditions, i.e., at pH 2, 5.5, and 7.4 at 37°C. As shown in Figure 6A, the release rate of ciprofloxacin from the capsule to the buffer was rather fast regardless of the pH. The leveling-off concentration was lower at pH 7.4 compared to that of pH 2 and 5.5, mainly because lower aqueous solubility of the drug at higher pH (Maurer et al., 1998). The fast release was related to the small diameter of the droplets, which were well-dispersed in the buffer at the initial stage. Additionally, the chi-CLP coating of the oil droplets allowed rapid diffusion and leakage of the drug from inter-particle pores. Apart from that, the reference samples (without ciprofloxacin) showed that the emulsion capsules were much more stable at acid pH compared to pH 7.4, where the absorbance at 277 nm indicated partial dissolution of CLPs (Figure 6B). From this perspective, the microcapsules can be beneficial for an intestinal drug delivery, since the dissolution of CLPs at higher pH can result in a larger porosity of the capsules and thus faster drug release.</p><!><p>Ciprofloxacin release from the emulsion capsules [stabilized by 1 wt% chi-CLP (50 mg/g)] to the buffer solutions at various pH values. (A) Ciprofloxacin release kinetics at pH 2, 5.5, and 7.4 at 37°C, mean values ± standard deviations of four replicate samples are shown. (B) Absorbance of the reference samples (without ciprofloxacin) at 277 nm measured under same conditions as the ciprofloxacin-loaded samples, mean values ± absolute deviations of two replicates are shown.</p><!><p>In this study, the important parameters for the preparation of colloidally stable chitosan-coated CLPs and their application for emulsion stabilization were studied. We found that the thin film coating of CLPs by chitosan significantly improved their emulsification capacity for olive oil. In addition, the uniformity of the emulsion droplets could be further improved at a higher concentration of chitosan-coated CLP (chi-CLP) dispersion. The obtained Pickering emulsions exhibited strong stability against coalescence, and the droplet diameter distribution remained almost unchanged for over 2 months. The positively charged chi-CLP layer locating at the oil/water interface could be electrostatically cross-linked by sodium triphosphate, which resulted in enhanced mechanical stability of the emulsion capsules. Such microcapsule with the shell that mainly comprised of CLPs are beneficial for instance for drug storage purposes, since CLPs possess antioxidative and UV-shielding properties. On the other hand, the cationic net charge of the microcapsules due to chitosan is also interesting with respect to bioadhesion and drug delivery into cells. Ongoing work in our laboratory is focused on closer investigation of these interactions of colloidal lignin particles with living cells.</p><!><p>TZ designed and carried out the experiments under the guidance of MS. The results were analyzed by TZ with input from MS and MÖ. The manuscript was drafted by TZ with contributions from all of the authors.</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. The handling editor declared a shared affiliation, though no other collaboration, with TZ, MS, MÖ at time of review.</p>

PubMed Open Access

Exploring new near-infrared fluorescent disulfide-based cyclic RGD peptide analogs for potential integrin-targeted optical imaging

We synthesized disulfide-based cyclic RGD pentapeptides bearing a near-infrared fluorescent dye (cypate), represented by cypate-c(CRGDC) (1) for integrin-targeted optical imaging. These compounds were compared with the traditional lactam-based cyclic RGD counterpart, cypate-c(RGDfK) (2). Molecular modeling suggests that the binding affinity of 2 to integrin \xce\xb1v\xce\xb23 is an order of magnitude higher than that of 1. This was confirmed experimentally, which further showed that substitution of Gly with Pro, Val and Tyr in 1 remarkably hampered the \xce\xb1v\xce\xb23 binding. Interestingly, cell microscopy with A549 cells showed that 1 exhibited higher cellular staining than 2. These results indicate that factors other than receptor binding affinity to \xce\xb1v\xce\xb23 dimeric proteins mediate cellular uptake. Consequently, 1 and its analogs may serve as valuable molecular probes for investigating the selectivity and specificity of integrin targeting by optical imaging.

exploring_new_near-infrared_fluorescent_disulfide-based_cyclic_rgd_peptide_analogs_for_potential_int

<p>The RGD (arginine-glycine-aspartic acid) tripeptide motif plays an essential role in the molecular recognition of integrin αvβ3 and some other integrin subtypes.1–6 The over-expression of integrin αvβ3 found in various types of tumors and neo-vasculatures and this dimeric protein complex is involved in regulating tumor growth, angiogenesis, and metastasis. Therefore, RGD-based integrin αvβ3 targeting has provided an effective approach for improving tumor imaging, and drug delivery. Cyclic RGD compounds such as the conventional lactam-based cyclic pentapeptide c(RGDfK) (where "f" represents D-phenylalanine) exhibit remarkable binding affinity and selectivity for integrin αvβ3.7–9 For example, [18F]galacto-RGD and cilengitide have been tested for cancer imaging and therapy in the clinic.7, 10–12</p><p>Despite some promising results, many aspects of integrin targeting, tumor imaging, and therapy that employ RGD peptides remain unclear.13 In particular, integrins have 24 subtypes through different combinations of α and β subunits.14, 15 Therefore, it is important to explore novel integrin-targeted ligands that are distinguishable from c(RGDfK) in structure as well as in receptor binding selectivity and specificity. We envision that such compounds with different structural and functional features may further enhance our understanding of integrin expressions, signal transduction, and their roles in cancer biology and pathology.</p><p>Over the past years, various types of disulfide-based cyclic RGD peptides have been reported.16–27 Recently, an internalizing disulfide bond-containing RGD peptide, called iRGD, has been developed to enhance both cancer detection and treatment by deep tissue penetration. 6, 28 Nevertheless, such cyclic RGD peptides have not been explored fully for integrin targeting and tumor imaging compared to the conventional lactam-based cyclic RGD peptide analogs. Optical imaging has emerged as a powerful modality for studying molecular recognition and molecular imaging in a noninvasive, sensitive, and real-time way. The advantages of optical imaging include cost-effectiveness, convenience, and safety. The method is also complementary to other imaging modalities such as positron emission tomography (PET), single-photon emission computed tomography (SPECT), and magnetic resonance imaging (MRI). Considerable advances have been made in tumor optical imaging by using integrin-targeting compounds in both preclinical and clinical studies.11, 29–37</p><p>In this work, we synthesized a disulfide-based cyclic penta-RGD peptides conjugated with a near-infrared fluorescent carbocyanine dye (cypate) (1) and its analogs for potential integrin targeting and optical imaging. They were evaluated for their integrin αvβ3 binding and cellular staining in comparison with a lactam-based cypate-c(RGDfK) (2),38 where the cypate moiety was connected to the ε-amino group of lysine.</p><p>Based on the previous methods for synthesis of cypate-peptide conjugates on solid support,37 we used a similar strategy to prepare the disulfide cyclic RGD peptides and their conjugates. The protected RGD peptide Fmoc-C(Acm)-R(Pbf)-G-D(OBut)-C(Acm) was first assembled on Rink amide MBHA resin (1 equiv) using conventional Fmoc chemistry (Scheme 1). The disulfide-based cyclization was realized by swirling the resin-bound peptide with a solution of Tl(F3CCOO)3 in DMF for 2 h, yielding the disulfide-containing cyclic peptide on a resin, Fmoc-c[CR(Pbf)GD(OBut)C]-Resin.39, 40 After the Fmoc protecting group was removed with piperidine/DMF, the free amino group at the N-terminus was conjugated with cypate in the presence of DIC and HOBT. Finally, the desired product, cypate-c(CRGDC)-NH2 (1) was cleaved from the resin with aqueous TFA (95%) and purified by semi-preparative HPLC.</p><p>Similarly, several analogs were also successfully synthesized by varying the glycine residue with other amino acids including Pro, Val, and Tyr (3, 4, and 5), as shown in Table 1. In addition, we synthesized the lactam-based cyclic RGD peptides: cypate-c(RGDfK) (2) and c(RGDfV) (6) for comparison.38 As reported previously,37 we also identified the simultaneous formation of their dimeric analogs such as the dimeric analog of 1 cypate-[c(CRGDC)]2 (7) (ES-MS: [MH2]2+1000.05, [MH3]3+ 667.35, and [MH4]4+ 500.70) as shown in Scheme 2. All the dimeric analogs showed shorter retention time compared to their monomeric analogs, but its yield was low compared to the monomeric analogs.</p><p>All the compounds were fully identified by both HPLC and ESI-MS after semi-preparative HPLC purification. As shown in Figure 2, all the compounds have similar UV-Vis absorption and fluorescence emission spectra ( λmaxabs783nm and λmaxem808nm) in the near-infrared region in 20% aqueous DMSO.</p><p>Integrin αvβ3 binding affinities were determined based on the competitive binding between purified integrin αvβ3 and peptide ligands using radiolabeled echistatin as a tracer. Echistatin is a polypeptide that binds irreversibly with high affinity and specificity to the integrin αvβ3. In particular, 125I-echistatin has been widely widely used as a a tracer in integrin αvβ3 binding assays.41–43 The lactam-based cyclic RGD pentapeptide, c(RGDfV), was used as a reference standard because it is known to bind integrin αvβ3 with high affinity.8, 29 The obtained IC50 values are summarized in Table 1. The disulfide cyclic penta-peptide (1) showed lower receptor binding affinity compared to the two lactam-based cyclic RGD pentapeptide 2 and c(RGDfV), both of which display similar binding affinity. This might be ascribed to the more flexible structure of 1 compared to the lactam-based analog 2, as was further confirmed by molecular modeling.</p><p>Molecular modeling performed according to energy calculation protocol described elsewhere 44 found 24 low-energy conformations of the peptide backbone for compound 6, 18 for compound 2 and 68 for compound 1. Expectedly, RGD peptide analogues with more compatible conformations to the structure of c(R1-G2-D3-f4-NMe-V5) in complex with integrin αvβ3 (as revealed by the X-ray spectroscopy15) display tighter specific binding to integrin αvβ3. Upon overlapping of Cα-atoms in the "template" X-ray structure of c(R1-G2-D3-f4-NMe-V5) to the corresponding atoms of compound 6, 19 out of 24 low-energy conformations for compound 6 (ca. 79%) showed the root mean square deviation (rms) values ≤ 1 A. For compound 2, the same percentage was also ca. 78% (14 out of 18), but it was significantly lower at ca. 62% (42 out of 68) for compound 1. These results correlate well with experimental data on integrin binding (see Table 1) showing binding affinities of compounds 2 and 6 of about one order of magnitude higher than that of compound 1.</p><p>Some of the low-energy conformations for compounds 1 and 2 compatible with the template X-ray structure displayed plausible orientation of the cypate moiety in the cavity between the two subunits of integrin αvβ3. As a representative examples, Figure 3a–c show spatial orientations of selected conformations of compounds 1 and 2 in complex with integrin αvβ3 as compared with the orientation of the template compound.</p><p>The A549 cells have been widely used for integrin-targeted tumor imaging due to their overexpression of integrin αvβ3. Compounds 1 and 2 were incubated with A549 cells for cellular staining and imaging by fluorescence microscopy (775/50 nm excitation and 845/55 nm emission filters). As shown in Figure 4, 1 stained the cells more strongly than 2.</p><p>Disulfide bridges represent important evolutionarily conserved structural motifs in many biologically important peptides and proteins. Our results demonstrated that disulfide-based cyclization may provide an efficient approach for studying receptor binding affinity and selectivity of RGD peptides as well as enzymatic and metabolic stability of RGD peptides employed for integrin targeting.1, 45, 46 As described above, we have successfully synthesized the near-infrared fluorescent disulfide cyclic RGD peptide and evaluated their integrin αvβ3 binding. The results also confirmed the importance of Gly in maintaining the binding affinity of such RGD peptides to integrin αvβ3.</p><p>Based on our results from receptor binding assay and molecular modeling, 1 could not compete efficiently with 2 in binding affinity with integrin αvβ3. However, the remarkable cellular uptake of 1 suggests the potential use of this class of RGD for imaging and treating cancer cells. These preliminary data suggest that, besides the receptor binding affinities, receptor binding selectivity and other structural factors such as lipophilicity may also play important roles in the cellular internalization. Therefore, all the disulfide-RGD compounds synthesized for this study deserve further investigation in vitro and in vivo. Further work is underway to unravel the integrin selectivity, signal transduction, and related biological activities as well as its potential in integrin-targeted tumor imaging as we reported previously. 29, 37</p><p>This work further demonstrated that the dicarboxylic acid-containing cypate can serve as a scaffold for constructing diverse near-infrared fluorescent agents for optical imaging as we reported previously.37 Especially, the compound 1 can serve as an attractive template for further molecular design and structural modification to discover some novel innovative integrin-targeted agents for tumor optical imaging, therapy, and drug delivery. For example, a library of novel diverse disulfide RGD peptides can be synthesized by solid phase peptide synthesis. The acid group at the side chain of cypate motif also provides a site for structural modification to improve the physicochemical properties and integrin targeting ability. As described above, some dimeric analogs bearing two disulfide-RGD peptide motifs such as 7 can be obtained simultaneously. All these will provide some insights into molecular design and structural modifications to further improve the integrin-targeting activities.</p><p>In summary, we have prepared and evaluated some cypate-labeled near-infrared fluorescent disulfide-based cyclic RGD peptide analogs. Although 1 has relatively lower receptor binding affinity for integrin αvβ3 compared to 2, it exhibited higher cellular staining. This class of near-infrared fluorescent RGD compounds deserve further exploration for their integrin targeting in cancer biology, optical imaging, and targeted therapy.</p><p>The two types of cyclic RGD pentapeptides bearing a near-infrared fluorescent dye, cypate.</p><p>Normalized UV-Vis and emission spectra of cypate and its conjugate cypate-c(CRGDC) (1).</p><p>Representative conformations of c(RGDf-NMe-V) [a, template conformation], cypate-c(RGDfK) [b], and cypate-c(CRGDC) [c] in complex with integrin. All hydrogens are omitted. The cypate moiety is shown in magenta. αv and β3 subunits of integrin are shown as semi-transparent surfaces in green and red, respectively.</p><p>Representative fluorescence images of live A549 cells (top) and relative fluorescence intensity of cellular staining after incubation of A549 cells (bottom) with compounds 1 and 2 at 1 μM.</p><p>Synthesis of cypate-labeling cyclic disulfide RGD peptide.</p><p>The simultaneous preparation of two compounds: the monomeric and dimeric cyclic disulfide RGD peptide analogs on solid support.</p><p>The major ES-MS peaks and integrin αvβ3 binding affinities of 1, 2, and their analogs.</p>

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Cerebral cavernous malformation is a vascular disease associated with activated RhoA signaling

Cerebral cavernous malformation (CCM) involves the homozygous inactivating mutations of one of three genes, ccm1, -2, or -3 resulting in hyperpermeable blood vessels in the brain. The CCM1, -2, and -3 proteins form a complex to organize the signaling networks controlling endothelial cell physiology including actin dynamics, tube formation, and adherens junctions. The common biochemical defect with the loss of CCM1, -2, or -3 is increased RhoA activity leading to the activation of Rho-associated coiled coil-forming kinase (ROCK). Inhibition of the ROCK rescues CCM endothelial cell dysfunction, suggesting that the inhibition of RhoA-ROCK signaling may be a therapeutic strategy to prevent or arrest the progression of the CCM lesions.

cerebral_cavernous_malformation_is_a_vascular_disease_associated_with_activated_rhoa_signaling

Introduction<!>CCM1, CCM2, and CCM3<!>RhoA GTPase<!>The emerging role of proteasomal degradation for control of RhoA signaling<!>RhoA activation, expression, and stress fiber formation in CCM<!>ROCK inhibition and RhoA geranylgeranylation as therapeutic interventions for CCM

<p>Cerebral cavernous malformation (CCM) is a genetic disease where loss of CCM1, -2, or -3 is associated with dilated, hyperpermeable blood vessels primarily in the brain (Robinson et al., 1991). The CCM lesions are less frequently found in the spinal cord, retina, liver, and skin. Many patients with CCM experience symptoms; other patients develop neurological deficits including seizures and stroke from the extravasation of blood due to an increased vascular permeability or hemorrhage. The treatments for CCM include surgical resection or radiation therapy, both are associated with substantial risks; therefore, the current standard of care is observation until the symptoms necessitate intervention (Batra et al., 2009). The goal of an intense research effort by several laboratories is to develop a pharmacological treatment to arrest and/or reverse the vascular hyperpermeability of the CCM lesions.</p><p>The CCM lesions develop upon the homozygous mutation of ccm1, -2, or -3 in endothelial cells. (Laberge-le Couteulx et al., 1999; Liquori et al., 2003; Guclu et al., 2005). Germline homozygous loss of a ccm gene is embryonic lethal, and it is estimated that 1 in 200 people in the general population harbor a mutation in one allele of the three ccm genes. The frequency may be as high as 1 in 70 in Hispanics due to a founder mutation (Gunel et al., 1996). Although a speculation at this time, the high frequency of heterozygous carriers in the general population suggests there may be a selective advantage to have one mutant allele of a ccm gene. The mRNA for the CCM2 protein is highest in macrophages, suggesting that a loss of one allele of a ccm gene could, for example, influence the innate immune response. Currently, no evidence for such a selective advantage has been discovered. The homozygous mutation of the ccm genes in endothelial cells from patient lesion samples, but not in surrounding normal brain tissue, has given rise to the hypothesis of CCM as a disease defined by the loss of heterozygosity, similar to the 'two-hit' hypothesis for neoplastic cancer progression (Knudson, 1971; Pagenstecher et al., 2009). One report suggests that the loss of the ccm3 gene expression by conditional deletion in glial cells may induce CCM disease (Louvi et al., 2011). Familial CCM, in which a patient has inherited one mutant ccm allele and the subsequent loss of heterozygosity appears to be responsible for more than 2/3 of the patients harboring lesions (Labauge et al., 1998). The familial CCM patients often present between infancy and the third decade of life. The non-familial, sporadic CCM patients often do not present with lesions and neurological deficits until later in life, presumably because of the longer time duration involved in the accumulating mutations in both alleles of a ccm gene in the brain endothelial cells. Genetic testing suggests that some patients thought initially to be sporadic are most likely familial (Labauge et al., 1998). The ability to rapidly sequence the ccm genes using the next generation deep sequencing methods should provide a more comprehensive understanding of the ccm mutations.</p><!><p>Clinically, the inactivating mutation of both alleles in any one of the three ccm genes, referred to as ccm1, -2, and -3, gives rise to phenotypically similar lesions, indicating that the three proteins are genetically in the same pathway (Faurobert and Albiges-Rizo, 2010). Consistent with this hypothesis, Hilder et al. were able to demonstrate through multidimensional protein identification technology (MudPIT) that the three CCM proteins coimmunoprecipitate in a complex from cell lysates and can be shown to form a CCM1-CCM2-CCM3 protein complex in vitro (Hilder et al., 2007). A major conclusion from the MudPIT analysis was that the CCM proteins were involved in regulating the cytoskeleton. This hypothesis has now been proven using different endothelial cell models of CCM including RNAi studies in human and mouse endothelial cells, CCM1, -2, and -3 inhibitory morpholinos in zebraflsh, and targeted ccm gene knockouts in mice (Boulday et al., 2009; Kleaveland et al., 2009; Whitehead et al., 2009; Zheng et al., 2010). Cumulatively, it is clear that a major function for CCM1, -2, and -3 proteins in endothelial cells is the regulation of cytoskeletal dynamics and the control of polarity, migration, and adherens junction stability (Glading et al., 2007; Borikova et al., 2010; Lampugnani et al., 2010; Stockton et al., 2010). It is also evident that the subcellular location of CCM1, -2, and -3 is dynamic, and the three proteins are not always found in a ternary complex, indicating that they have functions independent of each other in addition to their function as a complex.</p><p>A feature of the three CCM proteins is that none has a defined catalytic activity, and their primary function appears to be the scaffolding of protein complexes for the control of specific endothelial cell functions including the regulation of actin dynamics (Figure 1). A patient mutation in the CCM2 phosphotyrosine-binding (PTB) domain (L198R) that disrupts the binding of target proteins including CCM1 is consistent with in vitro data describing the PTB domain interactions of CCM2 being required for the normal maintenance of endothelial cell physiology (Denier et al., 2004; Zawistowski et al., 2005). Many CCM protein mutations found in the lesions are at splice junctions, suggesting that the protein may not be expressed because of non-sense-mediated mRNA decay (Stahl et al., 2008).</p><p>Of the three CCM proteins, CCM1 has the largest number of defined binding partners, including several that regulate the endothelial cell adherens junctions and vascular permeability (Figure 1) (Zawistowski et al., 2005; Glading et al., 2007). CCM3 has recently been shown by several groups to bind the STE20-like Mst4/STK25 serine/threonine kinases, which can phosphorylate the ezrin-radixin-moesin (ERM) cytoskeleton-associated proteins that function to link the actin filaments to the plasma membrane (Zheng et al., 2010; Ceccarelli et al., 2011; Fidalgo et al., 2012). CCM2 binds CCM3 outside of the PTB domain in what has been named the Karet domain (Zawistowski et al., 2005; Hilder et al., 2007; Li et al., 2010; Costa et al., 2012). CCM2 also binds the E3 ubiquitin ligase SMAD ubiquitin regulatory factor (Smurf1) (Crose et al., 2009). Given that the loss of CCM1, -2, or -3 protein results in similar pathological lesions, the common dysregulated biochemical pathway in endothelial cells is increased expression and activation of the small GTPase RhoA (Whitehead et al., 2009; Borikova et al., 2010; Stockton et al., 2010; Zheng et al., 2010). This dysregulated RhoA expression is consistent with the initial proteomic studies of Hilder et al. predicting that the CCM protein complex binds proteins that are involved in the regulation of the cytoskeleton (Hilder et al., 2007). The question becomes how do CCM1, -2, and -3 integrate their individual scaffolding functions to regulate the dynamic RhoA-mediated control of the endothelial cell actin cytoskeleton, and is RhoA signaling an important target to arrest CCM pathology?</p><!><p>The initial evidence for dysregulated RhoA signaling in CCM pathology came from experiments where RNAi-mediated loss of CCM1 or -2 protein expression led to an increased stress fiber formation (Glading et al., 2007; Whitehead et al., 2009; Stockton et al., 2010). Later studies also showed that a loss of CCM3 resulted in an increased stress fiber formation; however, one study challenged this finding and proposed that CCM3 loss did not increase stress fiber formation (Zheng et al., 2010; Chan et al., 2011). It is well chronicled in many cell types, including endothelial cells, that activated RhoA stimulates stress fiber formation (Ridley and Hall, 1992). Like other GTPases, RhoA is active when GTP is bound and inactive when GDP is bound to the guanine nucleotide-binding site of the GTPase (Figure 1). Specific guanine nucleotide exchange factors (GEFs) activate GTPases, such as RhoA, by stimulating the exchange of GDP for GTP, and GTPase-activating proteins (GAPs) inactivate the GTPase by promoting the intrinsic GTPase activity. The Rho GTPases also bind the guanine nucleotide dissociation inhibitors (GDIs), which block GDP dissociation, thereby keeping the GTPase in an inactive state (Jaffe and Hall, 2005).</p><p>Measurement of activated RhoA (GTP bound) levels in CCM1, -2, or -3-deficient endothelial cell lysates demonstrated an increased active RhoA compared to the wild-type control endothelial cells (Whitehead et al., 2009; Stockton et al., 2010; Zheng et al., 2010). This finding is consistent with the increased formation of stress fibers in the endothelial cells lacking CCM1, -2, or -3. Two primary RhoA effectors cooperate to control actin polymerization and stress fiber formation: Rho-associated coiled coil-forming kinase (ROCK) and mammalian homolog of Drosophila diaphanous (mDia) (Watanabe et al., 1999). mDIA is a formin protein that catalyzes the formation of long straight actin filaments (Figure 2A). The ROCK, in contrast, is a serine-threonine kinase whose substrates include myosin light chain (MLC), MLC phosphatase, and LIM kinase (LIMK) (Riento and Ridley, 2003) (Figure 2B). The phosphorylation of MLC phosphatase inhibits its catalytic activity, and the resulting increase in the phosphorylated MLC increases myosin cross-linking with actin, which results in actomyosin contractility. LIMK is activated when phosphorylated by the ROCK. LIMK-catalyzed phosphorylation of cofilin inhibits the actin depolymerizing and severing activity of cofilin. The treatment of cells with a ROCK inhibitor, such as Fasudil or Y-27632, results in the disruption of actin filaments leaving diffuse, disrupted actin filaments in the cell and abolishes increased stress fibers due to the activation of RhoA.</p><p>The increased RhoA activity in CCM1, -2, or -3-deficient endothelial cells results in the increased phosphorylation of the MLC, consistent with the increased stress fiber formation. Functionally, CCM1, -2, or -3-deficient endothelial cells display loss of migration, invasiveness, ability to form three-dimensional tubes, and form a stable permeability barrier as a monolayer (Whitehead et al.. 2009; Borikova et al., 2010; Stockton et al., 2010). Each of these functions was rescued by the treatment of the cells with the ROCK inhibitor or RNAi knockdown of the ROCK, consistent with the activated RhoA and increased actin-based stress fibers driving the CCM phenotype (Whitehead et al., 2009; Borikova et al., 2010; Stockton et al., 2010). Thus, the CCM proteins must be regulating RhoA activity.</p><!><p>In addition to the widely appreciated role of specific GEFs, GAPs, and GDIs in the regulation of RhoA activity, it is now realized that ubiquitin-mediated proteasomal degradation of RhoA is important for the control of localized RhoA signaling and the control of RhoA protein levels (Ding et al., 2011). Ubiquitination resulting in proteasomal degradation of RhoA is catalyzed by two different E3 ubiquitin ligases: Smurf1 and Cullin3. Smurf1 and Cullin3 are members of the HECT domain and the RING/U-box families of the E3 ubiquitin ligases, respectively.</p><p>Smurf1 has a C2-WW-HECT domain architecture with the E3 ligase activity encoded in the HECT domain. It is a member of the NEDD4 subfamily of HECT domain E3 ligases. The C2 domain targets Smurf1 to the membranes, and the WW domain generally binds the substrate proteins for ubiquitination by the HECT domain. Among its target substrates, Smurf1 ubiquitinates RhoA, leading to its degradation and, therefore, regulates cell shape, polarity, cell-cell contact, and motility (Wang et al., 2003). Smurf1 is thought to inhibit RhoA signaling through targeted degradation of active RhoA at the sites of active membrane protrusion during the dynamic regulation of cell polarity and migration that involves an integrated response of additional GTPases Rac1 and Cdc42 and the PAR6-PKCζ polarity complex (Wang et al., 2003). This localized RhoA degradation prevents reactivation of the targeted RhoA protein by a Rho GEF, which inhibits RhoA signaling in the cellular locations having active Smurf1. Interestingly, the overall levels of RhoA do not generally change significantly when Smurf1 is either overexpressed or knocked down using RNAi in the fibroblasts or cells of epithelial origin such as HEK293 cells. This finding has been used to support the notion that Smurf1 mediates the degradation of the localized active RhoA at sights of active membrane protrusion at the leading edge of migrating cells and not a generalized degradation of RhoA (Wang et al., 2003). The Smurf1 knockdown phenotype has not been defined in the vascular endothelial cells.</p><p>Cullin3 is a member of the Cullin-RING-Ligases (CRLs) that have been shown to target RhoA for ubiquitination and proteasomal degradation (Chen et al., 2009). RhoA was identified indirectly as a target for Cullin3 by the phenotype observed in the cells where Cullin3 had been knocked down by RNAi (Chen et al., 2009). The Cullin3 knockdown cells display a remarkable network of actin stress fibers, inhibited migration, and altered cell morphology. Unlike the Smurf1 knockdown cells, the Cullin3 knockdown cells had a markedly increased RhoA expression due to the loss of Cullin3-mediated RhoA degradation. The expression of other GTPases was not affected by the Cullin3 knockdown, indicating that RhoA is a selective GTPase for ubiquitination by Cullin3.</p><!><p>A hallmark of RNAi-mediated knockdown of CCM1, -2, or -3 in endothelial cells in culture is increased stress fiber formation due to the aberrant RhoA activity (Glading et al., 2007; Whitehead et al., 2009; Stockton et al., 2010; Zheng et al., 2010). This increased RhoA activity has also been seen with the RNAi knockdown of STK25, a serine-threonine protein kinase that binds CCM3 (Zheng et al., 2010). The knockdown of ezrin and moesin, members of the ERM cytoskeleton-associated proteins that are phosphorylation substrates for STK25, also activated RhoA (Zheng et al., 2010). This finding suggests that membrane-localized actin-associated complexes regulated by STK25 and the ERM proteins control RhoA activation. Studies using a well-characterized Förster Resonance Energy Transfer (FRET) biosensor for RhoA showed that the loss of CCM1, -2, or -3 did, indeed, result in an increase in RhoA activation in the endothelial cells (Borikova et al., 2010). The knockdown of CCM1 gave a pronounced activation of RhoA, but the loss of any of the three CCM proteins increased RhoA activity both at the sites near the plasma membrane and in the cell body.</p><p>Crose et al. demonstrated that CCM2 bound Smurf1 through a CCM2 PTB domain-Smurf1 HECT domain interaction that localized Smurf1 at the plasma membrane (Crose et al., 2009). In these studies, it was shown that the CCM2 interaction with Smurf1 regulated RhoA degradation. Interestingly, CCM2 was first cloned as a scaffold-like protein that was originally named Osmosensing Scaffold for MEKK3 (abbreviated OSM) (Uhlik et al., 2003). In this study, OSM was highly expressed in the primary macrophages and localized to the sites of newly polymerized actin in the membrane ruffles. This localization is consistent with a targeted role of CCM2/OSM in controlling RhoA function.</p><p>The Smurf1 knockdown in other cell types has indicated that there is a redistribution of F-actin to cortical actin and not a general increase in stress fiber formation throughout the cell body (Wang et al., 2003). Smurf1 is proposed to be localized to the membrane protrusions at the leading edge of the migrating cells and membrane ruffles, which is similar to the localization of CCM2 in the macrophages, where RhoA is ubiquitinated and degraded. Several laboratories have reported the knockdown of CCM1, -2, or -3 that resulted in stress fiber formation in human and mouse endothelial cells, suggesting that mechanisms, in addition to the dysregulated Smurf1 ubiquitination of RhoA, might contribute to the CCM phenotype if Smurf1 has similar regulatory functions in the cells of endothelial and epithelial origin (Whitehead et al., 2009; Stockton et al., 2010; Zheng et al., 2010).</p><p>Our studies using the stable shRNA knockdown of CCM1, -2, or -3 have demonstrated an increase in the RhoA protein levels in addition to the increased RhoA activity (Borikova et al., 2010). This experiment has been done in multiple endothelial cells of human and mouse origin. Other laboratories studying the phenotype of CCM knockdown in endothelial cells have generally used siRNA strategies with a significantly shorter time between the introduction of the siRNAs and analysis (~5–7 days) vs. the drug selection after lentiviral infection for the expression of shRNAs to stably knockdown the CCM proteins (~14 days). In general, the siRNA strategies have not given a significant increase in the RhoA expression that we have observed with the stable shRNA knockdown. The reason for this discrepancy is unclear because increased stress libers are apparent in endothelial cells for more than one study where siRNAs were used for CCM protein knockdown (Whitehead et al., 2009; Stockton et al., 2010; Zheng et al., 2010). This increase in stress fibers was not found in one study using the CCM3 siRNA knockdown in HUVECs, even though a loss of tube formation was observed (Chan et al., 2011). The CCM1, -2, and -3 shRNA knockdown phenotype, characterized by an activated RhoA, increased RhoA expression, and stress fiber formation, does not appear to be an off target effect of the shRNAs. Multiple shRNAs for targeting CCM1, -2, or -3 give similar phenotypes, and the loss of migration, invasion, and tube formation are reversed with the inhibition of the ROCK, demonstrating that the phenotype is driven by RhoA activation (Borikova et al., 2010). The difficulty in comparing the siRNA and shRNA studies is the turnover of two proteins, CCM1, -2, or -3, and RhoA is potentially being altered. The turnover of these proteins appears to be relatively slow, making the timing of the assays of RhoA activation and protein expression after knockdown critical. If both Smurf1 and Cullin3 are involved in the CCM phenotype, then the influence of their dysregulation may have different temporal as well as spatial consequences on RhoA function.</p><p>Our current understanding of the roles of Smurf1 and Cullin3 in regulating RhoA ubiquitination and degradation, in cell types other than vascular endothelial cells, suggests the possibility of distinct regulatory functions for the two E3 ligases in endothelial cells that could be dysregulated in CCM. Smurf1 has been shown to be involved in cell polarity and directed migration that is required for normal endothelial cell physiology. Cullin3 regulates RhoA protein levels, ROCK-dependent formation of stress fibers, and cell migration. The loss of Cullin3 expression using RNAi was sufficient to increase the basal activation of RhoA sufficiently to induce stress fibers (Chen et al., 2009). Functionally, CCM2 binds Smurf1, but it is presently unclear if the CCM proteins interact with the Cullin3 protein complex and regulate Cullin3-dependent RhoA degradation.</p><p>Mechanistically, the loss of CCM1, -2, or -3 could result in an increased RhoA GEF or decreased RhoA GAP activity. The functional regulation of RhoA by GDIs could also be deregulated. Each loss of the CCM protein-dependent change could result in increased basal RhoA activity and stress fiber formation. It is difficult to understand how the RhoA protein is increased with no change in transcription without decreased degradation of RhoA. Studies with the RhoA biosensor readily measured the spatial localization of the activated RhoA in cells (Borikova et al., 2010). The activated RhoA was located not just at the cell edge and membrane protrusions, but pronounced activity was also seen in the cell body. The increased RhoA activity was very pronounced in the cell body and the nucleus of the CCM1 knockdown endothelial cells. It will be of interest to determine what the effect of Smurf1 and Cullin3 knockdown has on the spatiotemporal activity of RhoA. Clearly, more studies are required to define how the CCM proteins regulate RhoA activity and if the control of RhoA degradation is a major mechanism for the CCM control of RhoA-dependent endothelial cell physiology.</p><!><p>Inhibition of the ROCK is able to rescue in vitro and in vivo the CCM phenotypes suggesting that the ROCK small molecule inhibitors could provide a pharmacological intervention for CCM (Whitehead et al., 2009; Borikova et al., 2010; Stockton et al., 2010). The ROCK inhibition or RNAi knockdown of ROCK2 rescues the endothelial cell actin cystoskeletal dynamics, in vitro tube formation, and permeability barrier function (Whitehead et al., 2009; Borikova et al., 2010; Stockton et al., 2010). In vivo, the ROCK inhibition decreased the vascular leak stimulated by LPS in CCM1- and CCM2-deficient mice (Stockton et al., 2010). Two structurally distinct ROCK inhibitors, H-1152 and Fasudil, rescue in vitro and in vivo the CCM phenotypes, with the RNAi knockdown of the ROCK rescuing in vitro the CCM1, -2, or -3-deficit phenotypes. Simvastatin inhibits HMG-CoA reductase and interferes with the production of geranylgeranyl pyrophosphate, which is required for RhoA lipidation, targeting RhoA to the membrane where it functions to control the actin cytoskeleton (Takemoto and Liao, 2001). Simvastatin, by inhibiting the geranylgeranylation of RhoA, is capable of rescuing the CCM phenotype (Whitehead et al., 2009). Cumulatively, the findings indicate that the activated RhoA seen with the loss of CCM1, -2, or -3 is responsible for the CCM phenotype and that inhibition of RhoA signaling or the ROCK activity can prevent and/or reverse specific endothelial cell defects seen in CCM. Simvastatin is an FDA-approved drug for lowering cholesterol, and Fasudil has been used in Japan for cerebral vasospasm after subarachnoid hemorrhage since 1995 and displays positive outcomes with a few adverse effects (Suzuki et al., 2007). Other small molecule ROCK inhibitors in early stage development for the treatment of cardiovascular disease have been well-tolerated in mice, suggesting that suppression of RhoA geranylgeranylation and/or ROCK inhibition provides viable molecular targets to prevent or treat the CCM lesions (Surma et al., 2011). Thus, despite our lack of complete understanding of how the loss of CCM1, -2, or -3 protein expression alters RhoA activation and RhoA protein levels, it is probable that a therapeutic strategy for patients can evolve from the discovery of the dysregulated RhoA signaling as a primary pathway responsible for the CCM endothelial cell pathophysiology.</p>

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One-pot fabrication of Ag @Ag2O core–shell nanostructures for biosafe antimicrobial and antibiofilm applications

Microbial contamination is one of the major dreadful problems that raises hospitalization, morbidity and mortality rates globally, which subsequently obstructs socio-economic progress. The continuous misuse and overutilization of antibiotics participate mainly in the emergence of microbial resistance. To circumvent such a multidrug-resistance phenomenon, well-defined nanocomposite structures have recently been employed. In the current study, a facile, novel and cost-effective approach was applied to synthesize Ag@Ag 2 O core-shell nanocomposites (NCs) via chemical method. Several techniques were used to determine the structural, morphological, and optical characteristics of the as-prepared NCs. XRD, Raman, FTIR, XPS and SAED analysis revealed a crystalline hybrid structure of Ag core and Ag 2 O shell. Besides, SEM and HRTEM micrographs depicted spherical nanoparticles with size range of 19-60 nm. Additionally, zeta potential and fluorescence spectra illustrated aggregated nature of Ag@Ag 2 O NCs by − 5.34 mV with fluorescence emission peak at 498 nm. Ag@Ag 2 O NCs exhibited higher antimicrobial, antibiofilm, and algicidal activity in dose-dependent behavior. Interestingly, a remarkable mycocidal potency by 50 μg of Ag@Ag 2 O NCs against Candida albican; implying promising activity against COVID-19 white fungal post-infections. Through assessing cytotoxicity, Ag@Ag 2 O NCs exhibited higher safety against Vero cells than bulk silver nitrate by more than 100-fold.Earth's biosphere is occupied by a plethora of microorganisms which encompass several categories, including bacteria, archaea, yeast, molds, algae, viruses and protozoa. Several benefits are provided by them in maintaining a balanced ecosystem, such as oxygen generation, nutrient supplementation, organic material decomposing and bioactive compounds production. Nonetheless, their pathogenicity represents a serious problem for public health and the entire ecosystem. The microbes causing infectious diseases are ubiquitous through several routes such as food manufacturing machines, water purification systems and polluted medical devices 1 . As a result, various antimicrobial agents, particularly antibiotics, were developed to combat the spread of pathogen-causing infections. However, the intense and widespread abuse of such biocides led to emergence of multi-drug resistant microbes (MDR). Recently, nanotechnology with its related products opens different avenues to face and solve the MDR threat. The metal and metal oxide nanoparticles either sole or in nanocomposite structures increased the antimicrobial activity by the virtue of expanding spectrum of enhanced features 2 .Silver nanoparticles (Ag Nps), are among the most attractive nanomaterials have been widely used in a range of biomedical applications, including diagnosis treatment, drug delivery, medical device coating, and for personal health care 3 . For years, knowledge about silver's ability to kill harmful bacteria has made its nanoparticles popular for creating various products. Silver has many advantages, including the fact that it is non-toxic to humans at very low doses and the use of silver was a common expedient for cooking procedures and for preserving water from contamination. Previous studies have presented improved bactericidal activity for lower nanoparticle sizes associated with higher surface area of the nanomaterials 4 . Silver ions are known to specifically react with the

one-pot_fabrication_of_ag_@ag2o_core–shell_nanostructures_for_biosafe_antimicrobial_and_antibiofilm_

<!>Materials and methods<!>Results and discussions<!>Morphological analysis. Scanning electron microscope (SEM)<!>The chemical mechanism.<!>OR<!>Cytotoxicity assessment.<!>Conclusion

<p>metabolic enzymes inside the bacteria; causing growth suppression. An oxide shell has also been demonstrated to boost biocidal action 5 . The preparation process of good quality nanoparticles (NPs) is vital to ensure their multidirectional effectiveness 6 . Several chemical and physical means were employed to synthesize NPs, the physical one involving laser ablation of a solid target in water 7 , condensation or evaporation and the thermal treatment of Ag NPs in an organic solvent at temperatures up to 360 °C in the gas atmosphere 8 . In the laser ablation procedure, the lack of any chemical reagents provides a unique benefit, but it is an expensive method. Chemical methods are alternatively applied, in which metal nanoparticles sizes are reduced leading to the formation of minute metal clusters. The synthesis of the nanoparticles in solution has important advantages as the ease with which the design, shape and size of the nanoparticles can be precisely controlled 9 .</p><p>In the present study, Ag@Ag 2 O core-shell nanocomposites (NCs) were synthesized via a simple chemical method. The as-prepared NCs were characterized structurally, morphologically and optically. Thereafter, the antimicrobial and antibiofilm efficiency of Ag@Ag 2 O nanocomposite against planktonic and biofilm-forming pathogens were evaluated. Additionally, the biocompatibility of Ag@Ag 2 O nanocomposites was assessed.</p><!><p>Methodology. The formation process of nano-sized silver composites (Ag@Ag 2 O) powder is a simple and safe system using alkali chemical techniques. 0.1 N of silver nitrate (AgNO 3 , 99.97%, Sigma Aldrich) aqueous solution which is used as a precursor of the silver element. It's added drop-wise to an alkali mixture solution, which contains [2.5 Wt% of Potassium Hydroxide (KOH, 99.97%, Sigma Aldrich), 10 Vol% of n-propanol (NPA) and deionized water, with stirring]. The reaction temperature is kept constant at 70-80 °C for 2 h. The solution was constantly stirred with a magnetic stir bar, until the solution turned into a grey colloidal suspension; indicating the fulfillment of the chemical reaction. Then, the precipitate powder is filtered, and dried at 60 °C overnight, as shown in Fig. 1a.</p><p>Characterization methods. The structural, composition and morphological properties of the (Ag@Ag 2 O) NCs composite powder were investigated using X-ray diffraction (XRD), Raman spectroscopy, Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), zeta potential and Fluorescence spectra. X-ray diffraction measurement was performed using Shimadzu 7000 XRD, with CuKα radiation (λ = 1.54 Å) generated at 30 mA and 30 kV with a scanning rate of 4° min −1 and 2θ values ranged between 25° and 80°. Raman spectrum was obtained at an excitation wavelength of 532 nm using Raman spectroscopy (Senterra, Germany). For the determination of the chemical bonds formed during the preparation process, Fourier Transform Infrared Spectrophotometer (FTIR, Bruker Corporation, Ettlingen, Germany) is used. The powder product morphology was investigated using Scanning Electron Microscopy [SEM, JEOL (JSM 5300)]. However, high resolution transmission electron microscope TEM (HR-TEM, JEOL-2100, Japan) was employed to examine morphology, high resolution d-spacing of the different structures, electron diffraction and mapping of silver and oxygen elements. X-Ray photospectroscopy (XPS) measurement was carried out using PHI 5000 Versa Probe III Scanning XPS Microprobe with Monochromatic Al source ranged from 0-1486.6 eV. Electrostatic potential was determined by the DLS technique using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) and the data were analyzed by Zetasizer software 6. Finally, the fluorescence spectrum was recorded at an excitation wavelength of 250 nm on fluorescence spectrophotometer (Agilent, G9800A, USA). Evaluation of the as-prepared Ag@Ag 2 ONCs as anti-bio-film agent. The inhibitory effect of Ag@ Ag 2 ONCs and AgNO 3 (5, 10, 25, 50 and100 µg/mL) against P. aeruginosa and S. aureus biofilms were assessed using tissue culture plate method. A sterile polystyrene 96-well microplate was seeded by 100 μL of tryptone soy broth (TSB) containing 10 8 CFU/mL of each tested strain. Simultaneously, two controls were run in parallel; positive control wells (medium containing a bacterial culture) and negative control wells (sterile TSB only). After 24 h of static incubation at 37 °C, washing, fixation and staining of the remained biofilm were carried out by 95% ethanol and 0.25% crystal violet, respectively. The absorbencies of adhered cells were measured spectrophotometrically at 595 nm. All the experiments were carried out in triplicate and the results are expressed as mean ± SD 11 . The following equation was employed to calculate inhibition percentage of biofilm formation where A represents the absorbance of the positive control wells and A 0 reveals the absorbance of the treated wells containing an antimicrobial agent.</p><p>Biofilm disintegrating assay. The potential of Ag@Ag 2 ONCs to degrade the already formed biofilms by P. aeruginosa and S. aureus were examined in comparison to AgNO 3 . Firstly, the bacterial lawn (10 8 CFU/mL) was inoculated into 96-well microplates and incubated statically at 37 °C for 24 h to permit biofilm formation. Secondly, the well contents were discarded aseptically. The diluted Ag@Ag 2 ONCs and AgNO 3 to concentrations (5, 10, 25, 50 and100 µg/mL) were added to each well. The incubation, processing, quantification and disintegration percentage of the biofilms were performed as previously described. All the experiments were carried out in triplicate and the results are expressed as mean ± SD. As stated by Cremonini et al. 12 the biofilm was deemed strong, medium and low at optical density (OD) ˃ 2, 1 ˂ OD ˂ 2 and 0.5 ˂ OD ˂1, respectively.</p><p>Antagonistic effect of Ag@/Ag 2 O NCs on the algal growth. The inhibitory effect of Ag@Ag 2 O NCs was evaluated against Chlorella vulgaris by adding (5, 10, 25, 50 and100 µg/mL) in parallel to exact concentrations of AgNO 3 . The algae were propagated in sterilized Bold's basal media (BBM) medium; incubated at 25 °C under illumination with daily cycles of 12-h light and 12-h night for 7 days 13 . The cell count was assessed with a hemocytometer under a light microscope (Olympus BH-2, Japan). The inhibition percentage was calculated as mentioned in Eq. (1), and the results are expressed as means ± SD.</p><p>Investigation of the cytotoxicity of Ag@Ag 2 ONCs comparing with silver nitrate against normal cells. Normal mammalian kidney epithelial cells (Vero) were used to detect cytotoxicity of the studied compounds. Vero cell line was cultured in DMEM medium-contained 10% fetal bovine serum (FBS), seeded as 4 × 10 3 cells per well in 96-well cell culture plate and incubated at 37ºC in 5% CO 2 incubator. After 24 h for cell attachment, serial concentrations of Ag@Ag 2 O NCsand silver nitrate were incubated with Vero cells for 72 h. Cell viability was assayed by MTT method 14 . Twenty microliters of 5 mg/mL MTT (Sigma, USA) was added to each well and the plate was incubated at 37 °C for 3 h. After removing the MTT solution, 100 µL DMSO was added and the absorbance of each well was measured at 570 nm using a microplate reader (BMG LabTech, Germany). The effective safe concentration (EC 100 ) value (at 100% cell viability) of the tested compounds was estimated by the Graphpad Instat software.</p><!><p>Structural analysis and chemical bonds formation. X-ray diffraction (XRD). The X-ray diffraction (XRD) spectrum of nano-composite (Ag@Ag 2 O)NCsis given in Fig. 2a 16 .On the other hand, the close overlap between Ag and Ag 2 O diffraction peaks and the difficulty to distinguish between the Ag + and Ag 0 peaks at the diffraction angle of 38.1° inferred a formation of a hybrid structure 17,18 . Sajjad Ullah et al. 18 ) in the samples. The structure could possibly have an Ag 2 O shell with Ag as the core with a decreasing gradient of oxygen from the surface to the core 6 . Despite the simplicity of preparation method, the silver element needs a special medium during its preparation. The individual crystallite size (t) was calculated using Scherrer's formula 19 given by Eq. ( 2).</p><p>where k is the Scherrer's constant (0.89-0.9), λ is the X-ray wavelength, β is the full width at half maximum (FWHM) and θ is the Bragg angle 19 . According to Eq. ( 2), the sample crystallite size for the plan (111) is calculated and is found to be approximately 18.6 nm.</p><p>Raman analysis. The molecular structure and phase identification of the Ag@Ag 2 Ocore shell are explored using Raman spectra. Figure 2b shows the Raman spectrum for the prepared nanopowder in a range from 0 to 4500 cm −1 . Two major peaks are clearly detected; the first one at 74.1 cm −1 with an intensity of 7900 cps for Ag 0 (Ag lattice vibrational mode) and the other one at 1046.7 cm −1 with an intensity of 583 cps for Ag 2 O (Ag-O stretching/bending modes) 20 .</p><p>Fourier-transform infrared spectroscopy (FTIR):. FTIR analysis reveals the functional groups of the Ag@/Ag 2 O nanocomposite synthesized using alkali chemical treatment (Fig. 2c).The broad band at3400 cm -1 indicates the O-H stretching vibrations of the hydroxyl groups 21 corresponding to H-bonded alcohols and also to intramolecular H bonds which are most probably from water molecules 22 . The peaks at 2357 cm −1 and 1655 cm −1 prove the existence of O-H carboxylic acids 23 and OH bending, respectively 24 . The band at 1387 cm −1 assigned to O-H bend of carboxylate 25 . The absorption band on 675 cm −1 is due to Ag-O stretching mode, which corresponds to Ag-O vibration in Ag 2 O.Furthermore, the appearance of follower peak at 868.68 cm −1 corresponds to the metaloxygen vibrations for the formation of (Ag@Ag 2 O) NCs 15 ; thus, synchronizing with the aforementioned XRD results; confirming the formation of (Ag@Ag 2 O) NCs.</p><p>(2) t = k. /β. cos θ www.nature.com/scientificreports/ X-ray photoelectron spectroscopy (XPS). The XPS data for the chemically prepared Ag@Ag 2 O NCs is illustrated in Fig. 3. Figure 3a shows the general survey analysis of the nanopowder, which exhibits a major detected peak of the Ag3d at a binding energy of 368.34 eV with an atomic ratio of 48.5%. Also, O1s peak is detected at a binding energy of 530.81 eV with an atomic ratio of 29.75% and K2p peak is observed at a binding energy of 293.5 eV with an atomic ratio of 2.18%. Finally, C1s peak is measured at a binding energy of 285.21 eV with an atomic ratio of 19.56%. In Fig. 3b, the high resolution of the Ag3d spectrum displays two main strong bands. Such two bands can be further de-convoluted into two pairs of sub-peaks. The peaks at 367.98 eV with an atomic ratio of 44.01% and 373.96 eV with an atomic ratio of 28.75% are respectively assigned to Ag 0 (3d5/2 and 3d3/2). The other set of bands is detected at 367.38 eV with an atomic ratio of 10.48% and 373.6 eV with an atomic ratio of 10.36% are attributed to Ag + (3d5/2 and 3d3/2, respectively) in the nanocomposite. Figure 3c confirms the oxidation of the silver nanoparticles through the existence of the O1s spectrum at 529.39 eV with an atomic ratio of 47.64%, at 530.77 eV with an atomic ratio of 25.32% and at 531.4 eV with an atomic ratio of 23.19%. Finally, the results confirm that there are two different configurations of silver species, namely Ag 2 O and Ag, which is consistent with many published reports [26][27][28] . The detected elemental carbon in the main survey analysis may have originated from the ambient atmosphere itself. The adsorption of hydrocarbons from the surrounding atmosphere, which results in the creation of a thin carbon layer on surfaces, is most likely the source of the carbon contamination 29 . result is nearly consistent with the result from XRD patterns in Fig. 2a. The silver oxide may have formed in as a solution mixture containing potassium hydroxide and n-propanol which have high oxidation potential 30 . Also, the time spent since the beginning of the reaction, i.e. when adding silver nitrate to the oxidized mixture and until the end of the reaction is not enough to produce the silver oxide in its final form. Thus, it is an incomplete reaction that results in the precipitation of the silver nanopowders. This step entails the formation of a layer of silver oxide on the surface of the silver powder nanoparticles as a result of remaining in the oxidizing solution for a longer time 31 . Thus, it is logical to form an Ag/Ag 2 O core shell compound of a spherical nature as a result of the lattice mismatch between silver metal and silver oxide 6 . However, the aggregation is more likely to occur due to too small size as shown in Fig. 4a and b. Generally, the smaller particle size is usually more beneficial for www.nature.com/scientificreports/ the antibacterial activity. Because the particle size is smaller, many more particles will be easily adsorbed on the surface of the bacterial cell membrane, and then successfully attack the cell, ultimately destroying the physiological functional groups of the cell 32 .</p><!><p>Transmission electron microscopy (TEM). TEM has been employed to characterize the size, shape, morphology and crystallinity of the synthesized Ag@Ag 2 O NCs. Zeta potential. The surface charge of Ag@Ag 2 O core shell was determined from Zeta potential by applying voltage across a pair of electrodes at either end of a cell containing the particle dispersed. The charged particles are attracted to the oppositely charged electrode and assessing the Zeta-potential value by − 5.34 mV (Fig. 5a). The Ag@Ag 2 O NCs show slightly low surface charges which tend to form agglomerates 33 . Moreover, the low surface charges of Ag@Ag 2 O NCs reflect the urgent requirement of a capping agent to prevent such agglomeration and keep nanocomposites stable for a long time 34 . However, upon antimicrobial application and cytotoxicity evaluation, the examined NCs were freshly prepared and examined after a short time of preparation (within 48 h of preparation). Subsequently, the prepared NCs, within such time, didn't exhibit aggregation and were still stable. Additionally, several reports 35,36 synthesized AgNPs and other metal-NPs in the same range of zeta and also exhibited antimicrobial activity.</p><p>Fluorescence spectra. The fluorescence emission peak of Ag@Ag 2 O NCs was detected using an excitation wavelength of 250 nm and appeared at about 498 nm in the visible range as shown in Fig. 5b. This fluorescence emission peak may be attributed to the relaxation of the electronic motion of surface plasmons 37 . The sharpening behavior in the peak may be due to the core shell structure and coverage of Ag by Ag 2 O, which prevents the nanopowder from combining with any water molecules as well as continuing the oxidation process 38 .</p><!><p>Based on the preceding experimental data, it is worth mentioning to explain the chemical mechanism of the nanocomposite (Ag@Ag 2 O) formation as demonstrated in Eq. ( 3). The reaction of silver nitrate with potassium hydroxide produces silver hydroxide via the following mechanism 24 :</p><p>(3) 4)- (7). Briefly, a part of AgOH may be reacting with the n-propanol, which acts as a wetting agent that decreases the recombination rate and the generation of silver propanoate (Ag-O 2 CCH 2 CH 3 ), as shown in Eq. ( 4), which is inferred from FTIR spectra as a sharp peak at 1655 cm −1 and 3400 cm −1 as shown in Fig. 2c 39 . Meanwhile, Ag-O 2 CCH 2 CH 3 is reacted with the hydroxyl group of KOH producing silver ions (Ag + ) in a continuous oxidation process [Eq. ( 5)]. The silver ion reacts with water and n-propanol in an alkaline medium via the presence of OHgroup to produce silver element (core); as shown in [Eq. ( 6a)]. Additionally, some of the silver ions re-interact with water and n-propanol for producing silver hydroxide as in [Eq. (6b)]. Therefore, the unstable silver hydroxide product (AgOH) is reduced to silver oxide (Ag 2 O shell) as shown in [Eq. ( 7)].</p><!><p>Antimicrobial efficiency of Ag@/Ag 2 O NCs against planktonic pathogens. Considering the health problems associated with microbial contamination, it is vital to find out effective antimicrobial agents that are able to control their outbreak. Thus, the current study is concerned with the antimicrobial activity of Ag@ Ag 2 O NCs against some prokaryotic and eukaryotic pathogens. The sensitivity of the examined pathogens to different concentrations of Ag@Ag 2 O NCs is shown through agar diffusion assay. Figure 6a and b illustrates the comparative results of antimicrobial activities of the Ag@Ag 2 O NCs and their precursor. Also, it demonstrated a dose-dependent manner in which the antimicrobial activity of different concentrations of Ag@Ag 2 O NCs against E. coli, B. cereus and C. albicans as representative models of pathogenic Gram-negative bacteria, Grampositive bacteria and Fungi, as well as C. vulgaris control before treatment with Ag@Ag 2 O NCs and C.vulgaris after treatment with 50 μg/mL of Ag@Ag 2 O NCs are shown in Fig. 7A-E respectively. Generally, Ag NPs displayed considerable effectiveness indicated by halo zones which exceeded 1 mm, where any antimicrobial agent was evaluated as "good" atan inhibition zone greater than 1 mm 40 . For all the examined pathogens, inhibition halos were directly proportional to the concentration of AgNPs. In addition, Gram-positive strains seemed to be more resistant than Gram-negative strains. That could be attributed to the lipophilicity of Ag NPs according to different cell wall polarity and compositional variations 41 .</p><p>As revealed by Pazos-Ortiz et al. 42 the thickness of the cell wall increases the resistance of bacteria to the exposed NPs. The thick peptidoglycan layer of the Gram-positive bacteria's wall, which is composed of teicoic acids and lipoteicoic acids, restricts the diffusion of NPs. Moreover, the tolerance response of each microbe depends on its metabolic properties. However, the cell wall of the Gram-negative bacteria is composed of thinner peptidoglycan layer together with lipoprotein and lipopolysaccharide, which together represent 25% of its mass. It is noteworthy to mention that the nosocomial infections and enteric fever are associated with P. aeruginosa, E. coli and S. typhi, respectively. Therefore, their inhibition is a pivotal issue. In agreement with our results 42,43 reported low reduction in S. aureus count (CFU/mL) and also halo zone in comparison to Gram-negative bacteria upon treatment by Ag@Ag 2 O NPCs. Besides, Ag@Ag 2 O NPCs biosynthesized by aqueous leaf extract of Eupatorium odoratum (EO) exhibited antagonistic performance coincident with the obtained results of current study 41 . In the www.nature.com/scientificreports/ same sense, D'Lima et al. 6 reported that Ag/Ag 2 O hybrid nanoparticles showed a considerable zone of inhibition against P. aeruginosa; declaring the enhancement of antibacterial activity upon combination with carbenicillin. In contrast, other studies reported higher susceptibility of Gram-positive bacteria for NPs treatment than Gram-negative one 11,44 . Remarkably, a considerable halo of mycostasis was noticed against C. albicans. Despite the oligodynamic nature of silver ions, which is due to their higher activity at minute concentrations, a potent antifungal efficiency of 50 μg of Ag@Ag 2 ONCs exhibited upon comparing with its precursor (Fig. 6); implying effectiveness in the treatment of COVID-19 post infections. Such fungal infections appeared recently in the second wave in India, in particular in patients who were put on mechanical ventilation in intensive-care units.</p><p>The fungicidal property of Ag@Ag 2 ONCs could be assigned to the damage of the glycoprotein-glucan-chitin cross-linkage of fungi cell wall followed by sever alterations in cellular biochemistry 11,45 . In addition, it has been suggested that Ag nanoparticles interact with the proteins of the plasma membrane, which is responsible for keeping trans-membrane electrochemical potential gradient such as H + ATPase protein. Such interaction leads to alterations of normal protein conformations and malfunctioning by blocking the regulation of H + transport across the membrane, which ultimately hindering growth, restraining respiration and ending with death [46][47][48] .</p><p>In coincidence with our results, Mallmann et al. 49 highlighted similar results with inhibitory influence of Ag@ Ag 2 O NCs against several species of Candida. Otherwise, Elemike et al. 41 demonstrated the dominant biocidal effectiveness ofAg@Ag 2 O NCs in bacteria than fungi.</p><p>Evaluation of the as-prepared Ag@Ag 2 ONCs against biofilm formation, biofilm disintegration and algal growth. Biofilms are multicellular sessile microbial communities embedded in a self-produced extracellular polymeric matrix (EPS) (e.g. DNA, proteins and polysaccharides) and attached toa living or inert substratum or interface. Actually, the viscoelastic nature of the EPS represents a serious concern, especially in water pipes, water purification systems and also in medical devices. Where, the biofilms have the capability to withstand different stress factors by the virtue of such feature. Hence, nanotechnology invasion has provided a significant tool to eradicate such problem at both environmental and medical levels 50 . The inhibitory effect of different concentrations of as-synthesized Ag@Ag 2 ONCs and their precursor salt on biofilm formation/ disintegration of both Gram-positive and Gram-negative bacteria was illustrated in Table 1. As noticed, P. aeruginosa biofilm was less susceptible for both treatments and under formation/ disintegration conditions, in comparison to S. aureus biofilm. As revealed by Hoseini -Alfatemi et al. 51 , P. aeruginosa and S. aureus biofilms were inhibited by 10 and 1 mg/mL of AgNPs, respectively; which makes our study characteristic. Where, 100 µg/mL suppressed (98.7% and 87.5%) and (93.1 and 74.8%) of S. aureus and P. aeruginosa biofilm synthesis and disintegration, respectively. Interestingly, Gram-negative biofilms were comparatively more resistant to antibiofilm treatments than Gram-positive as reported in several studies 42,51,52 . Generally, Ag@Ag 2 O NCs exhibited antibiofilm activity via several routes including, destruction of initial planktonic phase, damage of aggregated/sessile phase, disruption of EPS matrix, increasing of hydrophobicity of EPS and inhibition of quorum sensing system 53 . What is more, the inhibitory effect of Ag@Ag 2 ONCs against algal growth of C. vulgaris was studied. C. vulgaris is involved among other algal genera which are responsible for various environmental issues such as eutrophication and biofouling, especially in the availability of high concentrations of contaminants and in association with direct sunlight 53 . As illustrated in Table 1, Ag@Ag 2 O NCs exhibited a drastic algicidal effect on the proliferation and viability of algae with 98.4% growth inhibition. Severe damage of chloroplasts could be proposed due to yellowish to pale green color of algal growth in the presence of Ag@Ag 2 ONCs. Meanwhile, the control culture (without Ag@Ag 2 ONCs) appeared green and flourished during 7 daysof incubation as shown in Fig. 7D and E. Disintegration of algal cell organelles, thylakoid disorder and plasmolysis are common features associated with the destructive effect of Ag@Ag 2 ONCs on algal cell as stated by Duong et al. 54 . Therefore, the employment of Ag@ Ag 2 ONCs in restriction the algal blooms could result in constraining of their environmentally adverse influence.</p><p>As general observations, Ag@Ag 2 ONCs exhibited greater inhibitory activity than its precursor against all examined microbial forms. That could be assigned to the small size of nanoparticles and in relation to surface area. As pointed out by 55 , the antagonistic activity of NPs derived from their penetration ability which depends on www.nature.com/scientificreports/ sizes that are less than 100 nm. In addition, the biocide activity of Ag@Ag 2 ONCs uplifted linearly with increasing in Ag@Ag 2 O NCs concentration, which implies dose-dependent manner. However, NPs type, concentration, size, aggregation state, surface charge, synthesis conditions and tested microbe consider being governing parameters influencing of the effective doses 51 . Broadly, several strategies could be ascribed for NPs to display their toxicity against different microbial forms. The first strategy begins from puncturing and perforating the first protective barrier of the cell, which is cell wall, by interacting with its anionic components such as neuraminic acid, N-acetylmuramic acid, and sialic acid. However, as long as the NPs are smaller than 80 nm, their passage to cell membrane and later inside the cell is facile; causing phospholipid peroxidation, polysaccharides depolymerization and subsequently membrane detachment and integrity destruction 10,56 . At this stage, cell permeability increases followed by intracellular components leakage and proton motive force dissipation. Once NPs occupies intracellularly, more destructive features were exerted concerning metabolism and biochemical activities 10 . AgNPs showed higher affinity for binding with thiol group of amino acids; forming extra -S-S-bonds. By such way, deformation of protein configuration occurs, leading to proteins denaturation and ribosomes inactivation 56,57 . Further, NPs bind with nucleic acids such genomic and plasmid DNA; causing blockage of DNA replication and repair processes. With continuous release of Ag + ions and their oxide from Ag@Ag 2 O NCs, set of reactions (e.g., Fenton and Haber-Weiss reactions) are continuously and intensively generating Reactive Oxygen Species (ROS) such as hydroxyl radicals (OH − ), superoxide radicals (O 2 − ) and singlet oxygen ( 1 O 2 ). Under such oxidative stress, massive damage to the cell takes place and eventually lead to cell death. Tee et al. 58 and Pazos-Ortiz et al. 42 referred to the complexity of the mechanisms by which NPs exhibit their antagonistic influence. Figure 1b represents schematic illustration on the destructive effect of Ag@Ag 2 O NCs against different microbial forms.</p><!><p>After 72 h of incubation of the Ag/Ag 2 O NPs and silver nitrate precursor with normal renal epithelial Vero cells, it was found that their estimated safe doses on cell viability were 13.43 ± 1.63 µg/mL and 0.075 ± 0.001 µg/mL, respectively. This indicates that Ag/Ag 2 O NPs ismore safe than silver nitrate source. However, at 100 µg/mL of Ag/Ag 2 O NPs or silver nitrate caused death in Vero cells by 79.69% and 91.09%, respectively, as it is shown in Fig. 8a. Moreover, severe collapse in the normal spindle shape of silver nitrate-treated cells, at 25 µg/mL, confirmed its cytotoxicity in comparison to the normal morphology of Ag/Ag 2 O NPs-treated cells and untreated control healthy cells (Fig. 8b) 14 . The lower cytotoxicity of the prepared NPs, at < 13 µg/mL, may be related to their particle size (≥ 40 nm), negatively particle charge and high agglomeration potential (Fig. 4a,b) which results in increasing their size thus decreasing their cellular uptake and diminishes ROS generation 59 .</p><p>In support of this issue, Liu et al. 60 found that Ag NPs with size of 55 nm generated less ROS than 15 nm Ag NPs. Moreover, silver NPs' tendency to agglomeration increases in culture medium 61 . Besides, based on the previous finding, corona formation which is mediated by adsorption of fetal bovine serum (FBS), from culture medium, on silver NPs, mainly limits their cytotoxicity via reducing their cellular uptake 59,62 . All these factors contribute to minimize the cytotoxicity effect of Ag/Ag 2 O NCs on normal cells. This higher safety of Ag/Ag 2 O NPs on human normal cells (Fig. 1c) lends credibility to their biomedical applications compared to bulk silver nitrate.</p><!><p>Due to the globally identified antibiotic resistance among clinical pathogens, novel antimicrobial materials are needed to circumvent drug resistance. In this study, we have demonstrated a facile chemical method to fabricate Ag@Ag 2 O core-shell nanocomposites and their antibacterial, antifungal and antibiofilm activities against a wide range of microbial pathogens were examined. Structural, morphological and optical properties were studied using different techniques. XRD, Raman, FTIR, XPS and SAED indicated the formation of a hybrid NC structure with a crystalline nature. SEM and HRTEM showed the evidence of Ag@Ag 2 O with a spherical core-shell structure and its particle size ranging from 19 to 60 nm.</p><p>Furthermore, the antagonistic properties of Ag@Ag 2 O core-shell and its precursor AgNO 3 were compared in the range of 5-100 μg/mL. The image data declared the sensitivity order of pathogens versus examined Ag@ Ag 2 O as follows: S. typhi ˃ P. aeruginosa ˃ E. coli ˃ B. cereus ˃ S. aureus. Besides, a noticeable antifungal potency of Ag@Ag 2 O was observed at 50 μg/mL. Additionally, its antibiofilm activity and disintegration capability were increased with elevation of concentration. Generally, a dose-dependent behavior could describe the inhibition of examined pathogens by Ag@Ag 2 O. Eventually, the cytotoxicity of the NC was analyzed by Vero cells and its effective safe concentration value was estimated to be about 36.31 ± 1.53 µg/ml. The promising structural features and biocidal activity of Ag@Ag 2 O opens up employment in various technological sectors.</p>

Scientific Reports - Nature

Concentration-dependent <i>rhombitrihexagonal tiling</i> patterns at the liquid/solid interface

We report STM investigations on a linear oligophenyleneethylene (OPE)-based self-assembling Pd(II) complex 1 that forms highly-ordered concentration dependent patterns on HOPG. At high concentration, 2D lamellar structures are observed whereas the dilution of the system below a critical concentration leads to the formation of visually attractive rhombitrihexagonal Archimedean tiling arrangements featuring three different kinds of polygons: triangles, hexagons and rhombi. The key participation of the Cl ligands attached to the Pd(II) centre in multiple C-H/Cl interactions was demonstrated by comparing the patterns of 1 with those of an analogous non-metallic system 2.

concentration-dependent_<i>rhombitrihexagonal_tiling</i>_patterns_at_the_liquid/solid_interface

<!>Conclusions

<p>From bee honeycombs to ancient Roman mosaics and Moorish wall tilings, periodic polygonal patterns are ubiquitous in arts and science both for ornamental and technological purposes. 1 Tessellations of surfaces, i.e., the tiling of planes using one or various types of regular polygons, were rst classied by Kepler in 1619. 2 Regular tilings feature a single type of polygon (triangle, square or hexagon) repeated innitely whereas less frequent semiregular Archimedean tilings (AT) combine at least two different polygons placed edge-to-edge around a vertex. Scanning tunnelling microscopy (STM) provides a perfect tool to observe such patterns at the molecular level 3 and furthermore constitutes a link to nanodevice technology. 4 For instance, and besides various 1D patterns and/or 2D networks based on a wide variety of self-assembled systems, 5,6 AT arrangements have been visualized by STM. These systems, however, have been limited to trihexagonal (Kagomé) tilings, 6 in which two hexagons and two triangles are alternated on each vertex. Very recently, lanthanidebased polyphenyl systems have been demonstrated to form exciting snub square tilings combining two different (trigonal and square) polygons on Ag(111) through STM. 7 In this article, we move a step further towards complex tessellations at the liquid/solid interface by realizing a special type of rhombitrihexagonal AT patterns that feature up to three different polygons (triangle, rhombus and hexagon). Our system not only represents one of the most complex patterns ever visualized by STM but it can also be transformed into lamellar structures above a critical concentration. 8 To achieve this goal, we have taken advantage of recent ndings from our group on a self-aggregating oligophenyleneethylene (OPE) 9 -based Pd(II) derivative 1 (Fig. 1). 10 Complex 1 exists in a monomeric state in nonpolar solvents below z1 Â 10 À4 M. Above this critical concentration, micrometre-sized elongated supramolecular structures are formed, in which the OPE-based ligands of 1 adopt a more rigid conformation around the Pd(II) ion to maximize p-p and Pd/Pd interactions with neighbouring units in the stack.</p><p>Encouraged by these results, we questioned whether the distinct conformation of the units of 1 in solution depending on the concentration would also be reected in a different packing mode at the liquid/solid interface, as observed for other metallosupramolecular p-stacks. 11 To that end, a drop of a concentrated solution of 1 (c ¼ 5 Â 10 À4 M) in 1-phenyloctane was placed onto HOPG and investigated by STM. The images show the formation of highly ordered lamellar structures consisting of alternated bright and dark fringes (Fig. 1 and S1 †). In contrast, the pyridine-based ligand (precursor of 1) alone does not form any organized structures on HOPG even at millimolar concentration, thus revealing the importance of an extended aromatic surface and the presence of a Cl-Pd(II)-Cl fragment to produce organized ad-layers. Within the lamellar structures, the spots with higher local density of states can be assigned to individual aromatic rings belonging to the OPE segments of 1 whereas the darker striations attached to the edges of the aromatic system correspond to parallel-aligned dodecyl chains (Fig. 1). The unit cell of this pattern is highlighted in yellow in Fig. 1 and the corresponding parameters are: a ¼ 0.76 AE 0.02 nm, b ¼ 6.81 AE 0.06 nm, and a ¼ 75.0 AE 3.0 . On the basis of these dimensions, only four out of six dodecyl chains from each molecule (two outer and two inner) are adsorbed on the substrate whereas the remaining two chains are most likely embedded in the supernatant. 12 The density of the lamellar pattern was calculated to be 0.20 molecules per m 2 (plane group p1). 13 Remarkably, the parallel orientation of the OPE units at the HOPG/1-phenyloctane interface closely resembles that observed in the associates in nonpolar solutions, although one has to note that in the former assemblies the aromatic rings lie on the HOPG surface whereas in solution these are stacked on top of each other. These observations infer that monolayer formation is largely driven by adsorbate-substrate (epitaxial) and adsorbate-solvent (solubility) interactions. 14 Similarly to the solution behaviour, we questioned whether dilution of the system below a given concentration (1 Â 10 À4 M) would lead to a distinct molecular arrangement at the liquid/ solid interface. Fig. 2 shows the STM images of 1 obtained from a 100-fold more diluted solution (c ¼ 1 Â 10 À6 M) of 1 at the HOPG/1-phenyloctane interface. Interestingly, no sign of lamellar structures was observed at this concentration. However and to our surprise, a highly-ordered periodic pattern comprising three types of polygons (hexagons, rhombi and triangles) can be visualized (Fig. 2, S3 and S4 †). 15 These results bring to light that the structural phase transition in solution and at the solid/liquid interface occurs in a similar concentration range (below 1 Â 10 À4 M). On closer scrutiny, we noticed that the geometric shapes are separated from one another by bright segments that correspond to the aromatic rings of the molecules of 1 (Fig. 2a and b). Within this arrangement, each hexagon is sharing its edges with 6 rhombi and its vertices with 6 triangles leaving no gaps and overlaps, as shown in Fig. 2b and c. This exotic surface tessellation (plane group p6) resembles one of the AT of the Euclidean plane, the 3.4.6.4. rhombitrihexagonal tiling. 2 The unique difference from the regular rhombitrihexagonal tiling is the presence of rhombi instead of squares. The density of the AT patterns corresponds to 0.16 molecules per m 2 . By careful analysis of the STM images, we found out that the edges of all polygons are nearly equivalent in length (1.7 AE 0.2 nm) (Fig. 2b). This distance matches that of the aromatic backbone of a pyridine-substituted OPE ligand obtained by theoretical calculations (Fig. S10a †). Indeed, some individual aromatic rings can be distinguished in the magnication shown in Fig. 2b. According to our STM investigations, the Pd(II) centres are located at the vertices of the polygons, as all edges are occupied by the aromatic segments (see model in Fig. 2c). The repeat unit is represented by an equilateral triangular motif consisting of three molecules, whose edges are successively oriented towards the Cl-Pd(II)-Cl centre of a neighbouring unit within the triangle, as shown in Fig. 2c. Six such triangular subunits further pack into a hexagonal motif, thereby delineating an inner hexagonal cavity surrounded by six triangles and six rhombi in an alternated fashion (Fig. 2b and c). The dimensions of the unit cell are a ¼ 6.5 AE 0.1 nm, b ¼ 6.5 AE 0.1 nm, and a ¼ 60 AE 3 whereas the distance between the Pd centres within each triangular motif extracted from STM measurements was found to be 2.5 AE 0.2 Å. According to this dimension, the edge of one molecule and the Cl-Pd(II)-Cl fragment of a neighbouring unit are distant enough to enable the interaction between the central OCH 2 group of one molecule and the Cl ligand of the other one by C-H/Cl interactions (see proposed model in Fig. 2c and S5, S6 †).</p><p>As shown by us 16 and others, 17 metal-bound chlorine atoms have a strong propensity to interact with polarized C-H groups through hydrogen bonding interactions both in the crystalline state 18 and in solution. 16 In our system, only the methylene groups attached to the electronegative oxygen heteroatoms of the side chains are polarized enough to interact with such hydrogen-bonding Cl acceptors. Thus, on the basis of these considerations, STM analysis and theoretical calculations, two C-H/Cl interactions on either side of every molecule of 1 represent, along with the interaction of aromatic and aliphatic segments with the HOPG lattice, 19 the driving force for AT formation. It is worth noting that due to their lower tunnelling efficiency, a clear visualization of the dodecyl chains has not been possible. 20 We hypothesize that the alkyl chains will be concentrated in all polygonal cavities to maximize their interaction with the HOPG surface, which ultimately facilitates the AT formation. This situation is clearly possible in the hexagonal cavities, in which up to 12 dodecyl chains can be accommodated, two per monomeric unit (Fig. 2c). The cavities of the rhombi are slightly smaller and we postulate that four chains (see Fig. 2c and S5 †) can occupy these areas. Finally, the relatively high electron density observed in the triangular voids suggests that these areas are also considerably lled with alkyl chains.</p><p>However, due to their smaller size compared to the rhomboidal and hexagonal cavities only partial adsorption of the chains is possible, whereas other parts protrude above the adsorbate into the phenyloctane layer. 21 Our proposed model (See Fig. 2c, S5 and S6 †) clearly shows that a maximum of 6-7 carbon atoms from each dodecyl chain t in the triangular voids without inducing severe steric effects or distortions in the AT arrangement.</p><p>As particularly apparent in Fig. 2a and b, the majority of the molecules feature a nearly perfect linear geometry, indicating that the pyridine-based ligands are arranged with a 180 angle around the Pd(II) ion. There are, however, some areas in which some slightly bent molecules can be observed. This is evident in Fig. 2d and S4 † top, in which a small distortion of the ideal 180 angle is observed in few molecules resulting in a ring-like appearance. We also observed that this bending is not periodic but rather randomly distributed over the whole HOPG surface. The phenomenon of molecular curvature of systems exhibiting an extended p-conjugated surface has been previously observed for different classes of molecules. 22 In a particularly relevant example, Beton, Anderson and co-workers have recently reported on a novel 2D supramolecular organization of cyclic porphyrin systems by STM. 23 They describe the encapsulation of one cyclic polymer in a folded state into another unfolded polymer. The folded polymer undergoes bending where the subsequent strain is adequately made up by stacking stability. Accordingly, the bending energy of the complex in the AT patterns was calculated using the following equation:</p><p>where K is the bending coefficient, l is the length of the molecule and R is radius of curvature in the molecular arrangement. We applied this equation to calculate the bending energy of our 1 taking into account that the bending coefficient corresponds to 0.03 nN nm 2 (for monolayer systems), the molecular length is 3.8 nm and R can be approximated to 4 nm. The relatively small estimated energy (3.56 meV) required for the bending around the metal ion is well compensated by the high stability of the multipolygonal tiling that is attributed to C-H/Cl interactions and alkyl chain packing.</p><p>In order to nd out to what extent the existence of a Cl-Pd(II)-Cl fragment and thus, the participation of C-H/Cl forces is inuencing the AT formation, we have investigated a non-metallic OPE-based analogue 2 through STM. OPE 2 (Fig. 3) 10 is equivalent in size to complex 1 (see geometry-optimized structures in Fig. S11 †). However, the Cl-Pd(II)-Cl fragment has now been replaced by an alkyne functionality. This slight modication is expected to prevent C-H/Cl interactions and, consequently, the formation of multipolygonal patterns. Similarly to 1, OPE 2 forms one-dimensional associates in nonpolar solvents above 1 Â 10 À4 M, although the propensity of this system to aggregate is considerably reduced compared to 1. 10 In fact, when a 5 Â 10 À4 M solution of 2 in phenyloctane was used for STM under equivalent conditions to those of 1, no lamellar patterns were observed, highlighting again that the solution and interface behaviour are comparable. Moreover, dilution of the sample up to 10 À6 M did not lead to any changes in the molecular packing on HOPG, as shown in Fig. 3 and S8. † Regardless of the concentration, a highly regular grid-like pattern consisting of bright segments of 4.0 AE 0.2 nm in length is observed (Fig. 3a and b). The good agreement between this length and that extracted from molecular modelling (3.83 nm, Fig. 3c and S11 †) supports that these fragments correspond to the aromatic OPE core of 2.</p><p>In contrast to 1, the absence of a relatively exible Pd(II) centre increases the rigidity of the system to the point that bending of the molecules cannot be realized. Similarly to 1, the alkyl chains have not been visualized and are most likely adsorbed onto HOPG occupying the empty spaces between the OPE segments, as depicted in the model shown in Fig. 3c and S9. † The repeat unit comprises four molecules that delimit a cavity with a quadrilateral shape (Fig. 3b), yielding a unit cell whose parameters are a ¼ 5.2 AE 0.2 nm, b ¼ 5.2 AE 0.2 nm, and a ¼ 74.0 AE 3.0 (plane group p4). However and in contrast to 1, no rhombitrihexagonal structures are formed. This is inuenced by the absence of chlorine ligands that can participate in weak C-H/Cl hydrogen bonding interactions with polarized CH 2 groups. As a result, the grid-like pattern formed by 2 should be stabilized by other weak interactions. According to the proposed model shown in Fig. 3c, the patterns are maintained by weak CH/O forces between the hydrogens of the aromatic rings connected to the central triple bond and the oxygen atoms of the dodecyloxy chains. On this basis, each molecule interacts with four neighboring molecules through a total number of eight C-H/O contacts: four of them involving four of the oxygen atoms of the peripheral chains and the remaining four involving the two central aromatic rings, two on each side, thus creating a uniform structure exhibiting a network density of 0.15 molecules per nm 2 .</p><!><p>In summary, we have observed distinct patterns through STM by exploiting the self-assembly behaviour of an OPE-based Pd(II) complex 1. Above a critical concentration, the units of 1 are arranged in a parallel fashion into lamellar patterns. In more diluted solutions, however, the involvement of the Cl-Pd(II)-Cl fragment of 1 in C-H/Cl interactions with oxygen-polarized CH 2 groups of the side chains along with surface effects of the HOPG lattice lead to one of the most complex tessellations ever visualized by STM: a special type of semiregular rhombitrihexagonal tiling. The key inuence of the Cl-Pd(II)-Cl center is demonstrated by investigating a related non-metallic compound 2. Our ndings bring to light that unconventional non-covalent forces such as C-H/X interactions may become relevant enough to strongly inuence pattern formation. Such surface tessellations with uniform porosity may be exploited for the encapsulation of guest molecules on surfaces, providing access to surface-active 2D or 3D assemblies, as recently shown by Tait, Flood and co-workers.</p>

Royal Society of Chemistry (RSC)

Use of Boundary-Driven Nonequilibrium Molecular Dynamics for Determining Transport Diffusivities of Multicomponent Mixtures in Nanoporous Materials

The boundary-driven molecular modeling strategy to evaluate mass transport coefficients of fluids in nanoconfined media is revisited and expanded to multicomponent mixtures. The method requires setting up a simulation with bulk fluid reservoirs upstream and downstream of a porous media. A fluid flow is induced by applying an external force at the periodic boundary between the upstream and downstream reservoirs. The relationship between the resulting flow and the density gradient of the adsorbed fluid at the entrance/exit of the porous media provides for a direct path for the calculation of the transport diffusivities. It is shown how the transport diffusivities found this way relate to the collective, Onsager, and self-diffusion coefficients, typically used in other contexts to describe fluid transport in porous media. Examples are provided by calculating the diffusion coefficients of a Lennard-Jones (LJ) fluid and mixtures of differently sized LJ particles in slit pores, a realistic model of methane in carbon-based slit pores, and binary mixtures of methane with hypothetical counterparts having different attractions to the solid. The method is seen to be robust and particularly suited for the study of study of transport of dense fluids and liquids in nanoconfined media.

use_of_boundary-driven_nonequilibrium_molecular_dynamics_for_determining_transport_diffusivities_of_

Introduction<!>Methodology<!>Transport under Equilibrium: Benchmark Case<!>Transport under Non-Equilibrium<!>Boundary Driven Non-Equilibrium Molecular Dynamics<!><!>Boundary Driven Non-Equilibrium Molecular Dynamics<!>Molecular Interactions<!>Modeling Adsorption<!>Adsorption of Multicomponent Mixtures<!>Systems Studied<!>Simulation Details<!>Case I: Pure LJ Fluid<!>Adsorption Isotherms<!><!>Adsorption Isotherms<!><!>EqMD<!><!>EqMD<!>NEMD<!><!>NEMD<!><!>Comparison between EqMD and BD-NEMD<!><!>Comparison between EqMD and BD-NEMD<!><!>Case II: Methane<!><!>Case II: Methane<!>Case III: Transport of a Binary Mixture of Two Methane-Like Fluids with Different Adsorption<!><!>Case III: Transport of a Binary Mixture of Two Methane-Like Fluids with Different Adsorption<!><!>Case III: Transport of a Binary Mixture of Two Methane-Like Fluids with Different Adsorption<!>Case IV: Binary Mixture of LJ Fluid with Size Difference<!><!>Case IV: Binary Mixture of LJ Fluid with Size Difference<!><!>Case IV: Binary Mixture of LJ Fluid with Size Difference<!>Conclusions<!><!>Author Present Address<!>

<p>Understanding and modeling of mass transport in porous media is essential in nearly all branches of natural sciences and engineering given the ubiquity of fluid flow in natural and anthropogenic solids. Particularly in engineering applications, a significant number of processes exploit the relative differences in mass transport to separate different components of a fluid mixture. By employing porous matrices, such as membranes, the optimal design of separation units can lead to significant reductions in cost and energy consumption. An early example of separation of fluids using porous media is water desalination using reverse osmosis membranes, where salt is removed from water without the need for energy-intensive distillation units.1 Since the turn of the century, advances in technology and a perennially increasing control over nanostructure design has led to the production of porous materials with remarkable adsorption and transport properties, sometimes counterintuitive or unexpected. Fluids flowing within carbon nanotubes (CNTs)2−5 exhibit extremely large fluxes. Structured solids such as zeolites6,7 and metal–organic frameworks (MOFs),8−10 with very large surface area to volume ratios, have the potential of being selective to certain molecules due to a combination of steric and energetic effects, which makes them ideal for separation and catalysis processes. Extremely thin yet strong polymer membranes have been designed to be used in nanofiltration11,12 and more recently polymer membranes have been used as an alternative to distillation in the fractionation of crude oil.13 The issues associated with the production of oil through unconventional tight nanoporous shale rocks is another example of the unforeseen behavior drawn by the flow of fluids through ultraconfined media.14</p><p>Optimal design in separation processes has an enormous impact on overcoming current scientific and environmental challenges.15 In many instances, given the nanoscale porosities of these materials, understanding the precise mechanism of mass transport is challenging. It is not known to what extent traditional kinetic and phenomenological models can be used to predict transport in these materials. Generally speaking, there are three theories used to characterize transport in porous materials. The first is based on Fick's law, which employs an empirical constant to relate the transport (mass flux) to the macroscopic density (or pressure) gradient that drives it.16 The strategy is conceptually straightforward and commonly implemented in the analysis of experimental results. The resulting coefficients suffer from many drawbacks, namely, a lack of transferability, and a nontrivial pressure, temperature, and concentration dependence. The Onsager formulation, based on irreversible thermodynamics,17 recognizes that the driving force for transport is actually a gradient of chemical potential. The transport coefficients thus generated are fundamentally robust and better behaved than the Fickian counterparts, but they are conceptually challenging as chemical potentials cannot be directly measured. A final formulation, defined as the generalized Maxwell–Stefan theory,18 suggests the description in terms of so-called corrected diffusities. There is a formal link between all these formulations.19,20 However, in all of these descriptions, there is a need to experimentally determine the transport coefficients, i.e., there are no currently available fully predictive methods.</p><p>Experimental determination of mass transport and diffusion in nanoporous materials is challenging.21,22 A fundamental predictive theory behind transport in nanoconfined spaces is lagging behind the bulk–fluid counterpart, mainly due to the complexities of incorporating the surface–fluid effects, which are, in many cases, dominant.23 Thus, as a complementary tool to experiment and theoretical modeling, molecular simulations can provide useful insight into the mechanism of transport and separation in confined media.23</p><p>Molecular dynamics (MD), Monte Carlo (MC), and a combination of both are commonly used in literature to study transport of pure fluids and multicomponent mixtures.24−26 Calculations can be based on the analysis of systems in or away from equilibrium.27 In the equilibrium molecular dynamics (EqMD) method, the mean squared displacement, or velocities, of individual particles and their centers of mass are used to measure the motion of the fluid. To calculate transport diffusivities, additional information, namely, adsorption isotherms, are included. Otherwise, the tracking of particle positions or velocities is employed to predict transport diffusivities of multicomponent mixtures.18,28−32 A particularly relevant scenario to this manuscript is the calculation of transport diffusivities in nanopores.33−36</p><p>Calculating transport diffusivities using equilibrium molecular dynamics requires very long simulation runs,37 and additional simulations (typically Grand Canonical MC) to calculate the Darken correction factors from adsorption isotherms (∂μ/∂(ln ρ)) are required. As an alternative, nonequilibrium molecular dynamics (NEMD) techniques have been developed measuring transport by inducing a flux by external fields or by artificial gradients.27,38 One of the first reported nonequilibrium methods is the external-field NEMD (EF-NEMD) method of Evans and Morris,39 where an external field exerts a force on all fluid particles, generating a steady-state nonequilibrium flux. The ratio of the flux to the force is identified as the inherent transport coefficient or Onsager coefficient. This method has been successfully used to study pure systems27,38 and binary mixtures.40−42 A similar method of using walls to push fluid particles through pores in a pistonlike fashion has also been proposed by Wang et al.43,44 To report transport diffusivities using these techniques, adsorption isotherms are required to calculate the Darken factors (see the "Methodology" section).</p><p>Other NEMD methods have been developed that do not require adsorption isotherms to measure transport diffusivities. These methods rely on having density gradients across the pore, with the ratio of the induced flux to the density gradient being the transport diffusivity. One of the earliest of such direct measurement methods is the gradient relaxation molecular dynamics (GRMD)38 method where a system is initially set up with a density gradient and is then allowed to relax until fully equilibrated. The evolution of the fluid motion to an equilibrated state is then modeled by the diffusion partial different equation to measure the transport diffusivity. Given that the system never reaches steady state until fully equilibrated, it can be difficult to measure the transport diffusivity using ever changing density gradients and fluxes, leading to poor statistics. More efficient algorithms have been introduced by ensuring a steady-state gradient, and thus a steady-state flux, reducing the error in measured variables. One of the most common nonequilibrium steady-state methods is the dual control volume grand canonical molecular dynamics (DCV-GCMD) that has been used to measure transport diffusivities of pure fluids27,45−48 and mixtures.49,50 DCV-GCMD combines insertion and deletion methods of grand canonical MC with MD so that particles can be deleted downstream of the pore and inserted upstream to create a steady-state chemical potential and density gradient across the pore. The combination of MC with MD has clear advantages, as a steady-state gradient can be achieved and the pressures upstream and downstream can be clearly defined. However, the use of both MC and MD moves can be cumbersome with particle insertions being particularly difficult for high pressures and dense fluids. A more recent method fixing chemical potentials across a pore is that of Ozcan et al.51</p><p>Other NEMD techniques exist that do not rely on chemical potentials, and subsequently on insertions and deletions. One relevant approach is that of Li et al.,52 where a pressure gradient is achieved using a partially reflecting membrane. A membrane is positioned in the simulation box where particles crossing the membrane in a certain direction cross it without hindrance, yet particles attempting to cross the membrane in the opposite direction have a probability of being reflected back. This leads to a pressure gradient and a steady-state flow.</p><p>Finally, the boundary-driven NEMD (BD-NEMD) method of Frentrup et al.37 is a unique method of measuring transport diffusivities where MD is solely used to induce steady-state density gradients across nanopores. This is done by applying an external field only to a small region in the simulation box, positioned far from the pore. This tool has been extensively used in literature for pure components37,53−57 and mixtures.58,59</p><p>Although the BD-NEMD method has been extensively used in literature, it is not known to what extent transport diffusivities measured in this technique agree with other proven methods, such as the EqMD method. Moreover, there are significant assumptions that need to be addressed to ensure that transport diffusivities measured from the BD-NEMD and EqMD methods agree under all conditions. Moreover, although this method has been used to study multicomponent mixtures,58 only the self-transport coefficients have been measured, and the effect of the cross-species transport coefficients have not been studied. The values of the cross-species transport coefficients have not been previously measured using the BD-NEMD method, and it is not known to what extent transport diffusivities of multicomponent mixtures measured using this method agree with those from EqMD simulation. This work aims to address these issues.</p><!><p>Several excellent reviews discuss the relationship between the different transport coefficients which may be directly or indirectly measured in molecular simulations. The reader is referred to them for extension on this topic.19,60 For completeness, we will briefly discuss the most relevant expressions and relationships.</p><!><p>In molecular simulations, a common practice is to measure the motion of molecules using the self-diffusivity of individually tagged particles of type l, which can be calculated using the Einstein relation:611where rl,i is the vector describing the position of the ith particle of molecular type l, Nl is the total number of molecules of type l, and d is the dimension of the vector rl,i. In bulk, d has a value of 3 as particles can move in all three dimensions. However, inside porous materials, the value of d can range from 1 to 3, depending on the dimensions where particles can freely move. The angle brackets refer to an ensemble average.</p><p>Although self-diffusivity is informative in describing the motion of individual particles, it does not describe the collective mobility of molecules, i.e., how the collective (center of mass) motion of molecules of type l is related to the collective motion of all molecules of type m. Given that flow in porous materials is a consequence of the collective motion of molecules, self-diffusivity cannot be used to calculate transport of fluids apart from extremely dilute systems where particles do not experience strong intermolecular forces and thus move independently.62 A more general equation is thus used to describe the transport of particles of type l, influenced by the collective motion of particles of type m, denoted by Λlm:35,38,632</p><p>For a pure component system, Λ11 corresponds to the Maxwell–Stefan (MS) diffusivity, D̵MS, also known as the collective, or Darken corrected, diffusivity, Dc. Thus, eq 2 can be simplified for a pure component system:3</p><p>For multicomponent mixtures, elements of the matrix [Λ] are related, but they are not identical to the exchange coefficients of the MS diffusivity. Λij provides an indirect route to calculating transport diffusivities of multicomponent mixtures (eq 11). In addition to the Einstein relation, Λij can also be measured using Green–Kubo relations which use velocities instead of displacements.29 Although very commonly used, calculating collective diffusivities from equilibrium molecular dynamics simulations requires many independent simulations (or one very long simulation) to get acceptable statistics.33,62,64 This renders this method laborious and computationally inefficient.</p><p>Moreover, in order to compute transport diffusivities from equilibrium simulations, additional information is required in the form of the adsorption isotherms for the fluid in the porous material. While the equilibrium methods are accepted as the de facto "gold standard",65 they are computationally inefficient. It is in this space that nonequilibrium models can be an alternative to calculate transport diffusivities.</p><p>In the next section, we will discuss an implementation of a boundary-driven nonequilibrium method to binary mixtures and the relationship between the transport coefficients obtained as compared to those derived from other routes.</p><!><p>Transport diffusivity, Dt, is the mass transport coefficient used in the continuity and Fick's equations, relating the flux of a given species, i, in a porous medium to its concentration gradient:36,40,664Here, J is the n × 1 vector of fluxes of n components inside the pores, [Dt] is the n × n Fick or transport diffusion matrix, and ∇ρ is the n × 1 vector of density gradients. It is important to note that fluxes need to be measured relative to a specified frame of reference which is the porous solid and is assumed to be stationary. Moreover, it is also important to mention that ∇ρ corresponds to the density gradient of the fluid inside the porous media, i.e., the density of the adsorbate.</p><p>For a binary mixture where the flux is measured in one direction, z, the above equation can be written as675where Diit is the self-transport diffusivity, i.e., the contribution to the flux of species i due to its own concentration gradient, and Dijt is the mutual diffusivity, corresponding to the contribution to the flux of species i due to the concentration gradient of species j. For a binary mixture, none of the elements of the transport diffusivity matrix are necessarily similar to each other, i.e., Dijt ≠ Djit.</p><p>Crucially, a concentration gradient is not the only source of mass flux, e.g., the Soret effect68 describes the flux of mass driven by a thermal gradient which has been studied in bulk68,69 and under confinement70−72 using simulations. Similarly, consider a system in vapor–liquid equilibrium where there is a clear gradient in density; however, there is no net mass flux across the interface. As an alternative to the incongruities of the Fickian formulation, Onsager's treatment provides a fundamental starting point relating the fluxes to the underlying mass transport driving force. In an isothermal case, one could relate the fluxes to the gradients of the chemical potential difference of each species, ∇μ:176Here, [L] is the symmetric matrix of Onsager coefficients (phenomenological coefficients). An interesting inference of the Onsager treatment is that the flux of species i becomes dependent on the chemical potential gradient of both components i and j. There is an explicit recognition within this formulation that there are cross-component effects, i.e., that the flux of one component may have an impact on the flow characteristics of the other. In particular, the cross coefficient terms become important because the density of the system becomes liquidlike and correlation effects are strong.73 For a binary mixture7where [L] is symmetric8which is not true for mutual transport diffusivities. The matrix [Λ] with elements Λij (eq 2) is directly related to the Onsager matrix [L]. One can redefine the transport coefficients for a binary fluid system flowing through a porous media as follows:739where β is 1/RT where R is the gas constant and T is the temperature. Commensurate with eq 5, ρi is the density of the adsorbed fluid. The Onsager coefficients, Lij, do not have the units of length2/time customary to describing transport, while the modified term Λij has the same units as both transport and self-diffusivities. Going a step further, and using the Jacobian matrix for a chain of variables, eq 9 can be modified so that the driving force is given in terms of the adsorbed density gradients:10</p><p>Comparing eq 5 with eqs 7 and 10, the relationship between the diffusion coefficients may be expressed as11and12where #Comps is the number of components. The term βρi∂μi/∂ρj is commonly known as the Darken factor, Γij, and for a pure system, it is the proportionality constant relating transport to the collective diffusivity:13The Darken factor can be calculated from adsorption isotherms obtained from simulations or experimental data.</p><p>A critical review of some of the alternative techniques for calculating diffusion coefficients can be found in literature.17,27 Of particular note is the Maxwell–Stefan formalism.18,74−76 These models are all "equivalent" and the relationship between them has been presented elsewhere.16,77</p><!><p>Given a density gradient inside a pore and an appreciable flux, it is possible to directly assess the transport diffusivity of a fluid flowing inside a pore. To induce such gradient, we revisit the proposal of Frentrup et al.37 as applied to pure fluids, modifying key assumptions, and extending it to multicomponent mixtures. First, a simulation box composed of two bulk reservoirs in contact with the pore is set up. Fluid particles are added until a target global density is reached and the system is left to equilibrate. An external field is then applied in a small section of the simulation box, which in this work is 2 nm wide, far enough from the pore. This region is also the boundary of the two reservoirs. This external field exerts a directional force on all particles within the small region, pushing particles at the boundary of the two reservoirs to one side, thus increasing the concentration of the fluid in one reservoir, and depleting the amount of fluid in the other. The reason the external force is applied far away from the pore is to minimize the effects of the applied force on the flow across the pore, to ensure that the flow is being induced by the density gradient across the pore. The system eventually reaches steady state, and a steady-state concentration gradient occurs within the pore. This is illustrated in Figure 1 for a binary mixture. In terms of implementation, the directional force is the result of applying an acceleration to the particles. In this work, the same acceleration is applied to all species. Interestingly, in this method there are two bulk regions on each side of the pore. For small applied forces, the density of each bulk region remains fairly constant, and the observed density gradient is only within the pore. Applying different external forces to the boundary leads to different bulk compositions and different density gradients across pores, as can be seen in Figure 2, for a pure fluid flowing through a slit pore.</p><!><p>BD-NEMD method employed, as applied to a binary mixture. Top: Snapshot from the simulation, indicating the direction of flow, with different fluid species colored red or blue and the fixed pore colored gray. Arrows denote the regions where an external acceleration is imposed. Periodic boundary conditions is employed between left and right reservoirs. Bottom: Concentration profile of each species is color matched with the simulation snapshot. Dashed lines correspond to systems with no external forces, and solid lines are the steady-state result after applying external forces resulting in concentration gradients across the pore. Pore boundary regions are assumed to be in local equilibrium with neighboring bulk regions.</p><p>LJ fluid at T = 1.5 ε/kB, flowing through a FCC pore with pore height H = 20/3 σ, with the solid particles being the same as the fluid LJ particles. (left) ρ vs z using 20 different forces. All cases are plotted gray except for three: no force (black solid), highest force (red dashed and dotted), and medium force (blue dashed). To calculate density gradients inside the pores, the local equilibrium assumption is invoked, and the adsorbed densities at the boundaries (highlighted) are estimated using bulk densities from equilibrium simulations. (right) Adsorbed vs bulk densities of the same system used to estimate the adsorbed densities at the boundaries of the pore. Errors are within the size of the symbols.</p><!><p>In Figure 2 (left), different forces are applied to the fluid and a density gradient develops within the pore. It can be clearly observed that the density in the adsorbed phase and in the bulk are very different. This implies that density gradients across the pore, and consequently the transport diffusivities, are different dependent on which density (bulk or adsorbed) is used. It is important to mention that commonly in literature using BD-NEMD transport coefficients are calculated using reservoir densities37,54,56,78,79 which is only rigorous if the adsorbed densities are the same as bulk densities, i.e., for nonadsorbing systems. As will be shown later, an incorrect choice leads to significant discrepancy between measurements of transport diffusivities using the EqMD and NEMD methods.</p><p>Therefore, a key modification of the BD-NEMD method implemented in this work is to use density gradients inside the pore instead of calculating the density gradients using bulk (reservoir) concentrations. This can be particularly challenging, as the statistics inside the pore can be poor. In order to estimate the density gradients more robustly, an assumption is made that inside the pore (pore entrance and exit), the boundaries are at local equilibrium with the adjacent bulk reservoirs; thus, the amount adsorbed at the boundaries can be calculated from equilibrium simulations of a bulk region. Knowing the bulk densities on each side, the adsorbed density at the boundary inside the pore can be estimated, and the density gradient is calculated. An example of the such relationship between bulk and adsorbed density can be seen in Figure 2 (right).</p><p>Once the density gradient is calculated, the flux of each component, Ji, is calculated in the middle of the pore:14where Axy is the area of the plane in the pore perpendicular to the direction of the flow, trun is the total simulation running time, and Ni+ and Ni– are the total number of particles of species i that have passed the middle of the pore in the same and opposite direction to the direction of the external force, respectively. The choice of the middle of the pore is arbitrary if the cross-sectional area of the pore does not change.</p><p>By running simulations with different forces, one could obtain different density gradients and fluxes which can be used to improve the statistics. Transport diffusivities can be assessed using the following equation:15</p><p>For an m component mixture, Dt is the transport diffusivity matrix defined in eq 5, and ∂ρ/∂z and J are n × m matrices of concentration gradients and fluxes, respectively. n is the number of in silico experiments carried out, each with a different boundary force, and the superscript "T" denotes a matrix transpose.</p><p>One of the benefits of using the BD-NEMD technique over other nonequilibrium approaches is the fact that the force itself is not used to evaluate transport coefficients. It is applied in a region so far away from the pore that it does not affect the transport inside the pores. However, what the force does is to help in building up fluid on one side of the pore. Transport is a thus a consequence of the concentration gradient across the pore, and not the applied force.</p><p>Thus, it does not strictly matter what force is applied to either species, as long as there are measurable concentration gradients and fluxes of all species across the pore. If fluxes are only functions of concentration gradients, and not applied forces, then transport coefficients are independent of those concentration gradients and thus different forces can be applied to any species.</p><p>This is in contrast with other NEMD techniques, such as the external force NEMD (EF-NEMD) method, where a force is applied to all species. In that case, the applied force is directly responsible for the transport of both species and transport coefficients are calculated by relating fluxes to applied forces. In that case, if molecules have different masses, then one might observe buoyancy effects and artifacts that might affect the measurement of transport coefficients. To overcome that particles having different masses, accelerations should be modified to ensure the same force applied to different species.</p><!><p>All fluid molecules are modeled as single spheres, with the Mie potential describing intermolecular interactions between particles. Cross interactions are resolved by using the Lafitte et al. combination rules.80 Details are provided in the Supporting Information.</p><!><p>As highlighted previously in Figure 2 (right), in the BD-NEMD method it is important to relate bulk densities to the adsorbed densities. Moreover, if one wants to calculate transport diffusivities from the EqMD method (eqs 2 and 13), then adsorption isotherms relating adsorbed densities to chemical potentials or fugacities are essential.</p><p>To relate adsorbed densities to bulk densities, in this work both grand canonical Monte Carlo (GCMC)61,81 and EqMD simulations have been used. The GCMC method models the pore and the bulk separately using a defined chemical potential, whereas the EqMD method simultaneously models a bulk in equilibrium with the pore. For each GCMC simulation, 40 000 cycles were used, where each cycle consists of a displacement move, an insertion, and a deletion. For particle displacement moves, the probability of success was set to 0.25.</p><p>At higher densities, the GCMC method can become less efficient, as insertion and deletion of particles is more difficult. A particular advantage of the BD-NEMD method proposed is that the same system set up can be used to measure adsorbed densities by turning the external forces to zero, removing additional burden of setting up new simulations.</p><p>To calculate chemical potentials, for this particular force field one may use directly a molecular based equation of state (EoS), SAFT-γ Mie.82 The inputs of this EoS are the same as the Mie potential:16where ASAFT is the molar Helmholtz free energy of the fluid. The correspondence between the results of the equation of state and those from molecular simulations has been discussed elsewhere.83−90</p><p>An additional advantage of employing the SAFT-γ Mie EoS alongside the BD-NEMD method is that it allows accessing Λij (eq 2), given the condition that the boundaries of the pore are in equilibrium with the bulk regions:17where ∂μ/∂z is either calculated using SAFT-γ Mie EoS or from GCMC.</p><!><p>Although GCMC and EqMD could be used to calculate adsorbed densities of binary mixtures, given the additional degree of freedom stemming from considering the compositions, the pure component adsorption isotherms of each component are used as input to predict the adsorption of multicomponent mixtures through the ideal adsorption solution theory (IAST).91,92 In this paper, the IAST method is implemented using the pyIAST package, where the pure component adsorption isotherms (pressure vs adsorbed concentration) are used as inputs.93 Details are provided in the Supporting Information.</p><!><p>Four case studies are chosen in this study: (i) Case I: A pure LJ fluid within a slit pore; as a validation of the proposed BD-NEMD method by comparison to transport diffusivities calculated using EqMD. (ii) Case II: Pure methane within a slit pore; as an example of application to realistic fluids. (iii) Case III: A binary mixture of two methane-like fluids, with one having realistic parameters (as in Case II) and the other having augmented interactions with the pore; as an example application on the effect of the solid–fluid interaction on selectivity. (iv) Case IV: A binary LJ mixture, where one species is the same as Case I, and the other has a radius 30% larger; as an example application on the effects of size differences on self-and mutual diffusivities in binary mixtures</p><p>All parameters for the four systems studied are presented in Table 1. All walls are modeled as FCC lattices, and for all cases, the lattice constant is .</p><!><p>In all simulations, a system is set up with a solid pore of Lz = 12 nm positioned in the middle of a 36 nm simulation box, thus having two bulk regions on each side, each being 12 nm in length. The pore region is an FCC smooth slit pore. Details of its structure and the definition of the pore height are given in the Supporting Information. Lengths of the box (and the pore) in the x and y dimensions are the same, being greater than 10 σ. The large values of pore dimensions and simulation box sizes are chosen to minimize finite size effects. In particular, the ratio of particle size to pore length (σ/L) is always less than 0.05, and the ratio of pore height to pore length (H/L) is always less than 1. From previous studies,94 it is known that these dimensions are far from those resulting in significant finite size effects.</p><p>The total void volume of the simulation box is known and fixed, and particles are added to a target global fluid density. Equilibrium molecular dynamics simulation are then run in the NVT ensemble. Thus, the system equilibrates, and the adsorption isotherms can be assessed.</p><p>With the system equilibrated, the final configuration is used for two purposes. First, the same system is used as the initial configuration for the BD-NEMD simulations. Second, the pore section of the final configuration is isolated (the bulk regions removed and periodic boundary conditions imposed in the x- and z-directions) and used in EqMD simulations to calculate Λij in an essentially infinite pore setup. The EqMD and NEMD simulations are then used to calculate transport diffusivities which were compared.</p><p>Each simulation is run for 10 million time steps of 2 fs. The first 4 ns are used for equilibration, or reaching a steady state, and the remaining 16 ns were analyzed as the main production run.</p><p>For both the BD-NEMD and EqMD methods, following previous studies,37,57 simulations were run in the NVTW ensemble, where TW refers to the temperature coupling of the solid particles only. This means that no temperature coupling is used for the fluid as to avoid influencing its dynamics, and instead energy was added or removed using wall particles as a thermostat, i.e., the excess energy input of the external force is removed by the walls. To implement this, the pore solid particles are allowed to vibrate about their equilibrium position using position restraints of the harmonic form, with a bonding potential of 10 000 kJ mol–1 nm–2 and an equilibrium bond distance of σwall. By only applying temperature coupling to the solid particles, the temperature of both solid and fluid are kept constant about the equilibrium temperature, which can be found in the Supporting Information. Moreover, to ensure the positions of the fluid were not modified, no center of mass motion removal was used.</p><p>The BD-NEMD simulations are run using a modified version of GROMACS/5.1.295 and for each simulation at each state point, 20 different simulations were run using applied accelerations in the range of 0–0.004 nm ps–2 to keep the perturbations of the system within the "linear regime". EqMD simulations are run using GROMACS/201896 to ensure results are not subject to software bias. All visualizations of simulations have been rendered using the VMD package.97</p><!><p>For the pure LJ system in a slit pore of height 2 nm (6.67 σ), simulations are run from the dilute limit to highly concentrated systems, i.e., ρσ3 = 0.02–0.85 at T = 1.5 ε/kB, which is above the critical temperature of the LJ fluid (Tc = 1.31 ε/kB).98 The coexisting fluid density at the supercritical fluid–solid transition for a bulk LJ fluid is at ρσ3 = 1.01599 which set the upper limit of densities to 0.85, ensuring the fluid inside the pore did not undergo a phase transition to a solid phase.</p><p>Figure 2 (right) shows the measured adsorbed concentrations for all bulk concentrations studied. It could be clearly seen that the pore is very selective at lower densities, while at higher densities the concentration is only around five percent less than bulk concentrations. The latter underestimation is presumably an artifact of the particular definition of the pore height used in this work.</p><!><p>The BD-NEMD method can be used to measure both transport diffusivities (eq 15) and collective diffusivities, Λij, (eq 17). While the first calculation is direct, for the latter, one needs to find the chemical potential gradients across the pore. Otherwise, Λij can also be directly calculated using EqMD (eq 2). As a test of robustness of the BD-NEMD method developed, Λij is compared with both methods.</p><p>Furthermore, chemical potentials are required to evaluate the Darken factors, so Λij could be used to calculate Dt (eq 13). Chemical potentials are assessed using both GCMC and the SAFT-γ Mie EoS. While using the EoS, the chemical potential is calculated using the bulk density at equilibrium with the adsorbed density, whereas in GCMC (μVT ensemble) chemical potential is an input from which adsorbed densities can be determined. Although the two methods are fundamentally different to each other, a quantitative agreement between them can be seen in Figure 3. The disagreement at higher densities is attributed to sampling inefficiencies in GCMC. The equation of state is computationally much more efficient; thus, the SAFT-γ Mie EoS is chosen as the preferred method of measuring chemical potentials and Darken factors.</p><!><p>Adsorption isotherm of Case I. The SAFT-γ Mie approach uses direct MD simulations where the pore and the adsorbed fluid are in equilibrium with the bulk, and chemical potentials are estimated using the equation of state with the bulk densities as input. Errors are within the size of the symbols.</p><!><p>The relationship between chemical potential and adsorbed densities can be used to calculate chemical potential gradients across the pore from the BD-NEMD simulations, which can then be used to determine Λij using eq 17. Additionally, the Darken correction factor is also calculated from the same relationship:18</p><p>Γii is calculated as a function of the adsorbed concentration using SAFT-γ Mie EoS, and the results can be seen in Figure 4 (right). The Darken factor approaches the value of 1 at infinite dilution, corresponding to the expected value for an ideal gas. As more particles are added at lower densities, the presence of attractive forces lead to a decrease in the value of the Darken factor. The Darken factor increases as further insertion of fluid particles becomes less favorable, culminating in very large increases in the chemical potential. The density profile inside the pore can be seen in the Supporting Information. At low densities, there is only a small layer of adsorption nearest to the surface. At high density, the pore is saturated, and the fluid is highly ordered. The onset of saturation occurs at ρσ3 = 0.4, after which particle insertions become less favorable and the chemical potential increases.</p><!><p>Darken factor, Γ, vs adsorbed density, ρads calculated from the SAFT-γ Mie EoS.</p><!><p>For each value of the density of fluid inside the slit pore, 60 independent equilibrium simulations, or 300 million time steps in total, were run. Self-diffusivities were calculated using eq 1. Then, the mean square displacement (MSD) of the center of mass of the fluid in the two dimensions (plane) parallel to the pore surface is measured as a function of time. As can be seen in eq 2, in the limit of an infinite time, collective diffusivity, Λii is given by the slope of the linear line describing MSD as a function of time. Figure 5 shows the results for one density (ρσ3 = 0.5), and although the average value of all 60 simulations is linear, each simulation has a drastically different MSD profile relative to the average. Thus, the errors associated with measuring collective diffusivities can be particularly large. This is found to be true across all concentrations.</p><!><p>Centre of mass mean squared displacement of 60 different EqMD simulations of the same system (ρσ3 = 0.5). Each dotted line represents results of one simulation. The average of the 60 simulations is highlighted as the thick red line which is related to the collective diffusivity, Λ.</p><!><p>With the collective diffusivities measured from EqMD and the Darken factors from adsorption isotherms, it is possible to calculate transport diffusivities, Dt = ΓΛ (eq 11).</p><!><p>For different densities, 20 different forces are applied at the boundary and fluxes and concentration gradients are evaluated. As can be seen in Figure 6a,b, there is a linear relationship between the applied force and the flux as well as the density gradient, i.e., the system is kept within the linear response regime. Frentrup et al.37 observed a nonlinear response when employing forces an order of magnitude higher than that applied in this work.</p><!><p>Flux, J, and density gradient, ∂ρ/∂z, for fluid densities and external forces applied for the LJ fluid. (a) J vs force and (b) ∂ρ/∂z vs force, where different colors correspond to different fluid densities at equilibrium. (c and d) Contour maps of fluxes and density gradients respectively, showing maxima at intermediate loadings. Errors are within the size of the symbols.</p><!><p>In Figure 6a,b, the fluxes and density gradients are shown for different applied forces, respectively. The symbols are colored based on the equilibrium adsorbed densities, i.e., where the force is null. Figure 6c,d shows the contour maps of fluxes and density gradients as functions of the force and the fluid density. Although one would expect that increasing the external force leads to greater flux and density gradients, for a given applied force the value of flux (or density gradient) peaks at intermediate loadings. This could be explained by noting that at higher densities applying a force to particles at the boundary pushes them into a dense fluid region and hinders the flow. However, this does not mean that the transport diffusivity is highest at intermediate concentrations. The value of flux should not be taken as an indication of faster transport. It is only by relating fluxes to density gradients where a true measure of transport is given, which can be seen in Figure 7. The aforementioned figure clearly highlights that increasing pore loading leads to higher transport diffusivities.</p><!><p>Flux, J, vs density gradient, ∂ρ/∂z for the LJ fluid. Colors indicate adsorbed density at equilibrium. The slope of each line corresponds to the transport diffusivity for the given loading (eq 15). Errors are within the size of the symbols.</p><!><p>A summary of all transport coefficients measured using the EqMD and BD-NEMD methods is presented in Figure 8. From the EqMD method, self-diffusivity (Dself) and collective diffusivity (ΛEqMD) are calculated using eqs 1 and 2, respectively, and transport diffusivity (DEqMDt) is calculated using eq 13, i.e., by multiplying ΛEqMD by the Darken factor, Γ. From the BD-NEMD method, ΛBD-NEMD and DBD-NEMDt were calculated using eqs 17 and 15, respectively.</p><!><p>Summary of the different transport coefficients measured for the LJ system studied.</p><!><p>As can be seen, there is quantitative agreement between the BD-NEMD method and the benchmark equilibrium simulations across all densities. The BD-NEMD method captures the same trend as the EqMD simulations; however, both transport and collective diffusivities are underestimated in the nonequilibrium simulation. This underestimation is about 20% in the value of Λ and 15% in the value of Dt. The error is greatest at lower densities, which is presumably caused by uncertainties in the equilibrium simulations, as the error bars are very large and the values obtained using the NEMD method all lie within the error associated with EqMD.</p><p>It is important to note that while the coefficients approach the same value at infinite dilution, Dρ→0s = Λρ→0 = Dρ→0t ≈ 3.5 σ(ε/m)2, upon increasing densities, these coefficients show different trends. Self-diffusivity decreases with increasing densities and is roughly 20 times less at the highest density than at the infinite dilution limit, as particles in denser phases have smaller velocities and less free paths to diffuse uninterruptedly. This trend is not observed in the collective diffusivities, as the value of Λ peaks at intermediate densities of ρAdsσ3 = 0.45 and then slightly decreases at higher density (see Figure 6). Given that the Darken factor substantially increases at higher densities, transport diffusivities show the opposite trend to the self-diffusivities being up to 15 times larger at the highest density relative to the lowest. The fact that transport diffusivities can be orders of magnitude larger than self-diffusivities stems from the very significant collective motion of the fluids as a consequence of smoothness of the surface of the slit pore, and any individual movement of adsorbed molecules correlates with the movement of all other molecules in the system.</p><p>The key difference between this work and previous BD-NEMD approaches in literature is in the way density gradients are defined in the calculations of the transport diffusivities. As mentioned previously, a common practice is to use the unambiguous bulk reservoir densities. However, as can be seen in Figure 9, using bulk densities as the driving force for calculating transport coefficients inside the porous regions leads to drastically different profile for transport diffusivity as a function of density. Bulk density gradients do not take into account the effect of pore adsorption on transport and/or any pore entrance effects that may be present. Using bulk density gradients leads to significant overestimation of transport diffusivity at low loadings and underestimations at intermediate loadings, with a clear minimum at around ρAdsσ3 = 0.4. This minimum is neither observed for the BD-NEMD method developed in this work nor the EqMD benchmark (see Figure 9).</p><!><p>Comparison between transport diffusivities with the BD-NEMD method assuming the concentration gradient is given by the bulk compositions across the pore (open squares), as commonly used in literature, or if adsorbed densities are used (black squares), implemented in this work. Red squares correspond to benchmark EqMD results. Errors are within the size of the symbols.</p><!><p>To exemplify how this methodology could be employed to investigate transport of real fluids, the BD-NEMD method is further tested on a system consisting of supercritical methane in a slit pore at T = 300 K. Essentially, the system resembles that of Case I but the fluid force field can be traced back to a realistic model. The Mie parameters describing the intermolecular interactions have been previously optimized to reproduce vapor–liquid equilibrium100 (see the Supporting Information). The quantitative agreement between results obtained from MD with those obtained from EoS is a unique trait of the SAFT implementation used describing the macroscopic properties of the Mie intermolecular potential, allowing for fast and accurate description of the free energy, and thus chemical potentials of the system.</p><p>The top-down coarse-graining technique used to parametrize the force field is effective at producing robust models with transferability and representability, and can be used with confidence to describe transport and adsorption properties.101−104 The methane model presented in this work correctly predicts self-diffusivities of supercritical methane at 303 and 333 K at a range of different pressures (see the Supporting Information). The accuracy of the EoS in measuring the self-diffusivities of methane justifies the use of the model in measuring transport coefficients of methane in nanopores.</p><p>The slit pore is composed of particles explicitly modeled in an FCC lattice, with self-interaction parameters described using an ad hoc LJ potential, resembling an organic substrate. The height of the pore is 2.6 nm.</p><p>The relationship between adsorbed and bulk densities and Darken factors is seen in Figure 10 (left) and in the Supporting Information, where it is seen that at low bulk densities the adsorbed densities are higher than the bulk densities. The pore is slightly wider making surface adsorption less pronounced than the adsorption for Case I. Furthermore, relative to the solid particles used in Case I, the solid particles here are larger with a smaller ε, thus leading to weaker attractive energy density, and thus adsorption capacity.</p><!><p>(left) ρBulk vs ρAds and the Darken factors for methane in 2.6 nm wide slit pore specified in Case II. (right) Summary of diffusivities of pure methane in the pore in Case II. Squares are the collective diffusivities, and diamonds are transport diffusivities. The striped white symbols are from BD-NEMD simulations and blue and red are measured using EqMD.</p><!><p>The comparison between the BD-NEMD and EqMD techniques is presented in Figure 10 (right), showing the remarkable agreement between the two methods, with agreements from the dilute limit to very dense fluids spanning orders of magnitude.</p><!><p>Case II was extended by introducing an additional fluid component with similar characteristics to the methane model, henceforth described as MA. This new fluid, MB, has the same intermolecular interactions except that its interaction with the solid pore is less favorable. The cross species energy, εij, was 20% less than that of the MA–solid interaction (Table 1). In the bulk, there is no difference between the two fluids, and the mixture can be seen as having the same bulk properties as pure MA at the same total density. However, the reduction in the value of the cross-species interaction results in slightly different adsorption isotherms, and different pore selectivities.</p><p>In order to observe the adsorption behavior of the binary mixture, pure component adsorption isotherms are evaluated and can be seen in the Supporting Information. Ostensibly, the adsorption isotherms of the pure fluids look very similar, but given the different interactions with the pore, in a binary mixture it is expected that the pore would be more selective toward species MA. IAST is used to correlate the adsorption isotherms of the mixture using the pure component adsorption isotherm, estimating pore loadings using bulk pressures and compositions.91,92 The predictions of the IAST were then validated against EqMD simulations of binary mixtures, results that can be found in the Supporting Information. Generally, higher partial pressure of each species leads to higher adsorption of said species.</p><p>Pore selectivity toward species MA is defined as19where xi is the mole fraction of species i and the superscripts "Ads" and "Bulk" refer to compositions inside the pore and in bulk. A value greater than one refers to a pore that is more selective toward species MA.</p><p>Figure 11 (left) showcases pore selectivity, with selectivities of up to 16% at the lowest pressures (or bulk densities). With increasing pressure the pore become less selective. The main difference between the adsorption behavior of the two species is the amount adsorbed nearest to the pore surface, where species MA is adsorbed considerably more strongly than species MB, given the less favorable interaction between species MB and the solid. Free energy calculations found in the Supporting Information further showcase the stronger adsorption of species MA. With increasing densities, the first few monolayers near the pore surface become filled, and adsorption occurs in the middle of the pore, which is weakly affected by the solid surface, thus impartially allowing both species to adsorb in the middle resulting in a lower overall selectivity.</p><!><p>(Left) Pore selectivity as a function of partial pressures of MA and MB, with the red line (eq 20) being the region where transport diffusivities have been measured in this work. (right) Adsorbed densities of MA (solid black line) and MB (dashed red line) measured against pore height, for a system at equilibrium with a bulk equimolar mixture where ρMABulk σMA3 = ρMBBulk σMB3 = 0.25.</p><!><p>The region outlined by the red line in Figure 11 (left) was chosen as the subspace to measure the transport diffusivity matrix of the mixture. This region follows the following constraint:20where σ = σMA = σMB.</p><p>In this subspace, the total bulk pressure fluctuates between 46.5 and 48.0 MPa. As the species adsorb differently inside the pore, by keeping the global composition constant, there are different amounts of MA and MB in the bulk at different compositions, leading to a slight difference in pressure. The composition defined using the mole fraction of species MA, xMA, is the independent variable. The pore selectivity remains constant independent of fluid composition, having a value of SMA = 1.145.</p><p>To measure transport diffusivities from the EqMD method as benchmark cases, Darken factors were calculated using ∂ ln f/∂ρ as described in eq 13 (Figure 12). Generally, the values of the self-species Darken factor, Γii, increases with increasing concentrations of species i. Moreover, Γii is much larger that the cross-species Darken factor, Γij, indicating that the fugacity of one species is not as strongly correlated with the changes of composition of the other species as it is with changes of compositions of itself. The transport diffusivity matrix is evaluated using both the EqMD and BD-NEMD methods. For the binary mixture, there are four Λij and Dijt values to be evaluated with eq 2 used to measure collective diffusivity matrix, [Λ], from the EqMD method. The matrix, along with Γij, is used to calculate the transport diffusivity matrix, [Dt] using eq 13. The values of Λij at different compositions can be found in the Supporting Information. In general, for this particular system, given the identical interactions between all fluid particles independent of species, it is found that ΛMA,MA ≈ ΛMA,MB and ΛMB,MB ≈ ΛMB,MA, i.e., the flux of each species is equally influenced by chemical potential gradients of either species. This trend is also true for the transport diffusivities measured, and these can be seen in Figure 13, where filled symbols are transport diffusivities measured using the BD-NEMD method, and the empty symbols are those measured from the EqMD method. The first column describes the self-transport diffusivities, and the second column shows the cross coefficients, i.e., the contribution to the flux of species i due to concentrations gradient of species j.</p><!><p>Darken factors of the binary methane-like fluids, where "1" refers to species MA and "2" refers to species MB.</p><p>Transport diffusivities evaluated for the binary methane-like mixture at total reduced density of ∑iρiσi3 = 0.5. Empty symbols are from the EqMD method, and filled symbols are from the BD-NEMD method.</p><!><p>Diit and Dijt demonstrate positive linear correlations with molar composition of species i, with the diffusivity of each species approaching zero in the limit of zero concentration. For species MA, in the limit of pure component, i.e., xMA → 1, DMA,MAt approaches the value for the pure component system at ρσ3 = 0.5, presented in Figure 10. For Case II, DBD-NEMDt ≈ 3800 × 10–5 cm2 s–1 which is in quantitative agreement in the limit of pure component for DMA,MAt. This is not the same for species MB, and the slope describing DMB,MBt as a function of composition is steeper than that describing DMA,MAt. In the limit of pure component of each species, DMB,MBt is 10% larger than DMA,MAt. This does not necessarily mean that species MB travels faster than species MA inside the pore, as a concentration gradient in each species causes significant fluxes in the other species. Nevertheless, the high transport coefficients of MB can be explained by the lower selectivity of species MB, as MA particles adsorb more strongly to the surface and are slightly slowed down. If we were interested in evaluating transport selectivity, then transport diffusivities could be used in continuum models to assess changes in composition downstream of a pore.</p><p>Commensurate with previous cases, it is seen that transport diffusivities measured using equilibrium methods have large uncertainties. Moreover, the boundary driven method consistently overestimates the diffusivity, with an average of 18% for all elements at all compositions. However, the trends are qualitatively consistent with the values measured using the BD-NEMD method within the uncertainty of the EqMD measurements. This deviation is not significant given that values of transport diffusivities span orders of magnitude with changing concentrations, as previously discussed for Cases I and II.</p><!><p>To understand the effect of size heterogeneity on the elements of the transport diffusivity matrix for a binary mixture, the system studied in Case I was modified by adding another species which is 30% larger than the original LJ fluid, keeping all other self-interaction parameters the same. The pore height is kept the same and the wall particles are thermostated at a temperature of 1.5 ε, resulting in a supercritical fluid. Henceforth the original LJ species will be referred to LJF-1, and the larger species will be referred to as LJF-2. From Table 1, it can be seen that the cross interaction energy, εij, between LJF-2 and LJF-1 and between LJF-2 and the solid (LJW) is slightly less than 200 K. This is a consequence of the combining rules used, where the cross interaction energy scales down if the two species have large size differences.</p><p>For this system, transport diffusivities were measured for all compositions where ∑iρiAdsσi3 = 0.2–0.7. Compared with Case III, there are slight differences in the transport behavior in this system. To highlight the differences, the concentration constraint used to study Case III, i.e., ∑iρiAdsσi3 = 0.5, was also investigated. The results can be seen in Figure 14 (left), where it can be seen that the relationship between transport diffusivity and mole fractions is no longer linear. At intermediate mole fractions, xLJF-1 ≈ 0.5, the self- and cross-transport diffusivities of species LJF-1 are lower than a linear correlation (y = x), and the self- and cross-transport coefficients of LJF-2 are conversely higher than expected. This is in agreement with previous studies, where strong correlation effects lead to the slowing down of a more mobile species and less strongly adsorbed species by the less mobile species.31 Without a linear relationship between mole fractions and transport coefficients, pure component transport diffusivities cannot be used to approximate the self-transport coefficients in the binary diffusivity matrix as functions of composition.</p><!><p>Transport diffusivities, Dt, evaluated for Case IV LJ mixture at ∑iρiAdsσi3 = 0.5. Here "1" and "2" refer to species LJF-1 and LJF-2 respectively. For this plot, total reduced density was maintained at ∑iρiσi3=0.5. (left) Transport diffusivities measured against mole fraction of species LJF-1, xLJF-1. (right) Dt measured against volume fraction of said species, vLJF-1. The inset plots show the nonlinear relationship of Diit vs mole fraction and a linear relationship with volume fraction.</p><!><p>Interestingly, a linear relationship becomes apparent when transport is measured against volume fractions, v or by multiplying densities by σ3. When volume fraction of species i, vi, is zero, the self-transport diffusivity of species Diit is also zero. The self-transport coefficient linearly increases with increasing volume fractions until the point where vi approaches 1, when its value approaches the pure component transport diffusivity.</p><p>As with previous cases, in this binary mixture, self-transport diffusivities are orders of magnitude larger than self-diffusivities. For the systems presented where reduced density is kept constant, self-diffusivities are independent of composition, having a value of 13 × 10–5 cm2 s–1 for LJF-1 and 10 × 10–5 cm2 s–1 for LJF-2. Again, this is very different to the transport diffusivities seen in Figure 14, emphasizing the fact that self-diffusivities are not adequate parameters to be used in understanding transport in mesoporous materials.</p><p>Moreover, for this system the cross coefficients behave differently from the ones studied in Case III. The cross transport coefficient for each species is not similar to the self-term. For species LJF-1, the cross term DLJF-1,LJF-2t is larger than the self-term, DLJF-1,LJF-1t. For the other species, the opposite trend is observed. As previously described, the mutual diffusivity, Djit, quantifies the influence of concentration gradients of species i on the flux of species j. To understand how the concentration gradient of species i affects the flow of both species (i and j), the ratio Diit/Djit was compared with xi/xj at different compositions. If the values of the diffusivity ratio are the same as the composition ratio, then it can be concluded that the flow is fully mutualized and that there is no transport selectivity. This is because each column of the transport diffusivity matrix describes the resultant fluxes emerging from the same concentration gradient, and if the ratio of the elements of each column of the transport diffusivity matrix ratio is the same as the ratio of molar compositions in the pore, then it implies that the fluid is fully mixed, i.e., "toothpaste" or ideal piston flow. An exhaustive discussion is provided in the Supporting Information. Table 2 shows the comparison of ratios and a quantitative agreement is observed.</p><!><p>The results agree well with the ratio of the molar compositions at different volume fractions at ∑iρiAdsσi3 = 0.5. This agreement alludes to the fact that the flow is fully mutualized. The subscripts "1" and "2" refer to species LJF-1 and LJF-2 respectively.</p><!><p>Although the transport is fully mutualized, composition still affects the overall transport. Increasing the composition of the lighter species leads to a faster flow, which can be quantified by measuring the total fluxes and the total density gradient across the pore (refer to the Supporting Information). The smaller species acts as a diluting agent for the larger species, enhancing overall transport.</p><p>In this case, the self-transport coefficient of the smaller species is at least 20% larger than that of the larger one in the pure component limit, and the cross coefficient of the smaller species is 7 times larger. Although these metrics would ostensibly allude to a faster transport of species LJF-1, this is not the correct conclusion, as the fluid is fully mutualized, yet it could be seen that for high density fluids inside slit pores, if the adsorption is ideal and the fluid is well-mixed, then the self-transport diffusivity of each component can be estimated using the linear relationship with respect to the volume fractions. Moreover, if the transport is fully mutualized, then the cross terms can be estimated using the ratio of the molar compositions.</p><p>The complete picture of transport diffusivity matrix of this system as a function of the adsorbed concentration can be found in the Supporting Information.</p><!><p>This work was carried out to validate the assumptions currently used in the BD-NEMD methods in measuring transport diffusivity of pure components. By benchmarking values of transport diffusivity obtained using the BD-NEMD method against those from the EqMD method, it was highlighted that current implementations of this nonequilibrium method, where it is assumed that concentration gradients can be calculated using bulk reservoir concentrations on either side of the pore, lead to spurious values of transport diffusivity. By relating bulk concentrations on each side to their adsorbed concentrations at equilibrium and using adsorbed concentration gradients, it was shown that the BD-NEMD method can be used to compute accurately transport diffusivities with much lower uncertainty than the EqMD method.</p><p>To relate EqMD collective diffusivities to transport diffusivities, Darken correction factors are required. Commonly, these correction factors are assessed using GCMC methods. In this work, a novel method was introduced, whereby the Darken factors are evaluated for systems where intermolecular interactions are described using Mie potentials. Using the same system used for the BD-NEMD method at zero force, the equilibrium bulk compositions for each system are used to calculate chemical potentials from the molecular based equation of state, SAFT-γ Mie. A Python code for the evaluation of the SAFT-γ Mie EoS is available.105</p><p>For dense fluids and liquids, it is shown that transport coefficients are orders of magnitude larger than self-diffusivities. Although there is no formal expectation that these two quantities match except at the ideal gas limit, it is a frequent working assumption which is proven wrong.</p><p>In dense binary systems, the influence of the adsorption selectivity on transport is minimal, at least for modest differences in pore attraction. The influence of the size of the particles is, on the contrary, more pronounced. Larger particles dominate the transport, and it is seen that the transport cross coefficients become relevant. These significant values of cross coefficients result in mutualized flow in slit pores at high densities, i.e., no transport selectivity is observed. We do make the caveat that these heuristic observations are relevant only to smooth pores, and a significant difference is seen upon the consideration of transport in rugous nanopores, as we will describe in a future communication.</p><!><p>Full details of the molecular interactions; pore structure and pore height characterization; thermostating; accuracy of the methane model in describing thermodynamic and transport properties; ideal adsorption solution theory for binary mixtures; density profiles inside the pores; enhanced discussion on flow mutualization and transport inside the pores; binary transport coefficients for cases III and IV; free energy calculations of binary mixtures (PDF)</p><p>jp1c09159_si_001.pdf</p><!><p>∥ Princeton Plasma Physics Laboratory (PPPL), Princeton, NJ 08536, United States</p><!><p>The authors declare no competing financial interest.</p>

PubMed Open Access

Microstructural control of polymers achieved using controlled phase separation during 3D printing with oligomer libraries: dictating drug release for personalized subdermal implants

Controlling the microstructure of materials by means of phase separation is a versatile tool for optimizing material properties. In this study, we show that ink jet 3D printing of polymer blends gives rise to controllable phase separation that can be used to tailor the release of drugs. We predicted phase separation using high throughput screening combined with a model based on the Flory-Huggins interaction parameter, and were able to show that drug release from 3D printed structures can be predicted from observations based on single drops of mixtures. This new understanding gives us hierarchical compositional control, from droplet to device, allowing release to be 'dialed up' without any manipulation of geometry. This is an important advance for implants that need to be delivered by cannula, where the shape is

microstructural_control_of_polymers_achieved_using_controlled_phase_separation_during_3d_printing_wi

<!>Materials Library<!>Screening materials at the drop scale (10 picolitres)<!>Macro-scale cast samples (15 microliters)<!>Macro-scale 3D ink-jet printing of drug eluting samples<!>Conclusions<!>MA-A macromers end capping.<!>Materials library preparation<!>Microarray preparation.<!>Flory-Huggins interactions parameter.<!>Degree of conversion.

<p>highly constrained and thus the usual geometrical freedoms associated with 3D printing cannot be exploited, bringing a hitherto unseen level of understanding to emergent material properties of 3D printing.</p><p>Often it is not possible to exploit design freedoms due to limitations in the manufacture or the implementation of a device. A pertinent example is the long-term subdermal delivery implant. This is usually cylindrical with a size (maximum diameter 2 mm, length range 1 to 4 cm) defined by a combination of implantation method and anatomical positioning [1][2][3] . Currently, such devices are manufactured by a process which heats a blend of polymer and active pharmaceutical ingredient (API) to around 100°C, extrudes and then cuts them to size. [4,5] However, current systems are not personalizable, nor is it possible to combine multiple drugs into a single treatment. One route to achieving personalization is through 3D printing. Recent advances in 3D printing have shown it can be used for controlling drug elution, most commonly through variation in geometry, or variation in composition [6,7] . Whilst Fused Deposition Modelling and Binder Jetting are popular and show promise, for the manufacture of implants they are limited by their resolution. Ink jet based 3D printing, however, offers multiple benefits including its scalability, high resolution and importantly, drop by drop deposition that can provide both control over material properties at the microscale as well as the ability to co-deposit multiple materials (drugs). In this work, we exploit the latter to develop microstructural control at the sub droplet level which then permits the precise tailoring of the drug release. This microstructuring only emerges as a function of the drop by drop deposition inherent to ink jet printing.</p><p>In developing this concept, we report the creation of a library of multicomponent inks, whose diversity of physicochemical properties allows for the range of phase separation behaviour required for tailoring drug release. We show that, by understanding the 3 mechanisms that drive formation of this microstructure, we can predict microstructure that arises out of the ink jet printing process and reliably design and manufacture implants for tailored release.</p><!><p>To create the library of printable, functional materials, we first synthesized a range of low molecular weight (5 kDa) biodegradable oligomers with the following head-terminal group combinations, -OH, MA-OH and MA-A (Figure S2). The end functionalities were varied to control; (a) the drug release by influencing chain-end centered degradation, and (b) reactivity.</p><p>The oligomers (PCL, PLA and PTMC) were synthesized from three core monomers (caprolactone, DL-lactic acid and trimethylene carbonate), offering a range of degradation rate/mode, crystallinity and thermal/mechanical properties. These polymers are widespread in the biomedical industry, and selected for an easier pathway to adoption compared to completely new materials. [8] The oligomers were synthesized by ring opening polymerization using metal free organocatalysis, [9] chosen for low toxicological impact of any subsequent medical devices. [10] The library was created by combining nine hydrophobic oligomers in a 1:1 ratio with two relatively hydrophilic reactive solvents, n-vinyl pyrrolidone (NVP) and poly(ethylene glycol diacrylate) (PEGDA) 250 Mw, giving a total of 18 inks (Figure S3). After degradation studies, we formulated with a combination of NVP and PEGDA to further tune the degradation behaviour and enhance the structural integrity of the fast release formulations.</p><!><p>Microarrays of single drops of each of our 18 inks were rapidly deposited and cured on a glass slide using a high throughput (HTP) method, ready for characterization (see supplementary). [11] In this single drop screening (SDS) images were taken of single 200 um spots (Figure 1B), each representing the deposited drops within ink-jet processes. Drops' surface chemical and microstructure/phase separation properties were evaluated using optical microscopy, time-of-flight secondary ion mass spectrometry (ToF SIMS) and automated peak force quantitative nanomechanics (QNM) atomic force microscopy (AFM). This analytical combination provided the phase separation and oligomer distribution data (Figure 1B and Figure S4) required to create a phase separation 'taxonomy' which was then related to a Flory-Huggins interaction parameter (χ) prediction of the likelihood of phase separation (see supplementary, Table S1-2) (Figure 1C). Our observations indicated that the mixtures exhibit three different types of microstructures, (a) homogeneous, completely interspersed mixture (b) dispersed droplets indicating nucleation and growth of domains and (c) spinodal decomposition (core-shell) mixture (Figure 1B). Consequently, the materials were ordered according to their χ values and compared to the observed microstructure taxonomy (Figure 1C). Broadly, the material microstructure conformed to classification by χ and approximate χ values demarking boundaries between microstructure types were identified: (a) below 0.025 presented one single phase (b) with values between 0.025 and 0.06 phase separated into dispersed droplets and (c) above 0.06 exhibited a core-shell microstructure. These material combinations were further screened using a HTP methodology designed to assess "printability" to eliminate those materials that cannot be easily printed using ink-jet printing (Table S3-5). [12]</p><!><p>Degradation rate: To determine the influence of microstructure on the cured ink's macromaterial/degradation, cast cylinders (length 4 mm, radius 0.5 mm) of the 18 inks were prepared and subjected to degradation studies. The duration of degradation was sixteen weeks and the mass loss rates are shown in Figure 2A and Figure S5. As most of the structures printed with NVP didn't maintain their integrity following curing, we replaced the NVP only formulations with PEGDA/NVP solvent combinations. These were used in the biocompatibility and drug release studies to enable faster degradation than PEGDA alone, whilst maintaining some structural integrity.</p><p>Drug Release: A cardiovascular disease hypertension active, trandolapril, was used as a model drug in drug release screening from candidate formulations (see supplementary). Here, the χ parameter was used to predict which ink component trandolapril has more affinity with and would migrate toward (Table S6). With PEGDA alone, Figure 2C, we observed that the fastest release profiles were those from PCLMA/PEGDA and PTMC/PEGDA, each of which presented a core-shell microstructure in the SDS. PTMCMA/PEGDA exhibited the next fastest release and presented a dispersed droplets microstructure. In each of these cases, χ analysis predicted that trandolapril had more affinity for and would migrate toward the PEGDA (Table S6). In contrast PTMCMAA/PEGDA, PLAMA/PEGDA and PLA/PEGDA had significantly slower release, and χ forecasted greater active affinity for the oligomers over PEGDA, suggesting that the release is significantly slowed due to hydrophobicity of these materials. Therefore, we hypothesized that when using PEGDA alone, cast drug release rates are predominantly controlled by their affinity for PEGDA and that the microstructure provides a secondary tuning parameter. In contrast, all formulations with PEGDA/NVP, with the sole exception of PTMCMAA, exhibited very similar dissolutions rates which were seemingly unaffected by the microstructure, i.e. the drug release appeared to be dominated by the PVP behaviour. The AFM and optical images of the PTMCMAA/PEGDA/NVP formulation surfaces indicate segregation behavior quite unlike those observed in other formulations, suggesting a complex set of interactions that may be leading to exceptional drug release behavior.</p><!><p>The SDS and cast samples evaluation were used to guide the choice of materials for 3D inkjet printed drug releasing devices. Our screens indicated that two "levers" controlled deposition for an API, namely NVP inclusion and microstructure. Thus, we chose formulations predicted to show a range of release rates and therefore were guided by the release from cast materials (leading to the addition of NVP to PCLMA/PEGDA, and PTMCMA/PEGDA) and also by the SDS and degradation data (leading to use of PLAMA/PEGDA/NVP). We also incorporated a second active, the cholesterol lowering drug pitavastatin, into these formulations to demonstrate the predictions' effectiveness for multiple actives. Pitavastatin offers an effective contrast since it has a similar log P to trandolapril (3.97 and 3.45 respectively) but different aqueous solubility (0.426 mg/L and 2.5 mg/L respectively). The amount of drug released from 3D printed constructs was quantified on day 3, 15, 30 and 60, and 3D OrbiSIMS was used to unambiguously identify each compound [13] (Figure S11) and ToF-SIMS micro-scale resolved maps of bulk composition (via depth analysis) were used to understand the role of phase separation on drug distribution and release (Figure 3B).</p><p>Drug release and ToF-SIMS assessment showed that both PCLMA/PEGDA and PTMCMA/PEGDA constructs exhibited similar extended-release profiles and material distributions within the construct (Figure 3B). The latter were similar to those observed in the SDS microarray, indicating its reliability in predicting 3D printed material separation. ToF-SIMS (Figure 3B) also confirmed the affinity between the API and the PEGDA within a formulation, as we predicted from the χ values (Table S6). This indicated that microstructure dominated the drug release behavior in these printed samples, with the release likely via diffusion from exposed PEGDA, whilst also suggesting that the SDS, cast and printed samples have the same microstructure.</p><p>Using PCLMA, PTMCMA and PLAMA oligomers with NVP/PEGDA resulted in a range of drug release profiles that broadly followed the degradation rates of the core polymers. We noted that PCLMA/PEGDA/NVP's release was not statistically different to that from PCLMA/PEGDA, whilst using PTMCMA resulted in a substantial increase and PLAMA gave full release in < 20 days. This behavior, in combination with insights from ToF-SIMS 3D mapping (Figure S12, Figure 3B) leads us to propose that, whilst cast samples are not a reliable guide to release when using NVP, the similarity in microstructure in SDS and 3D printed samples indicates the reliability of SDS as a guide to performance when manufacturing via printing. In each case (with or without NVP) the microstructure is a determining factor, but via a different mechanism. When using NVP, ToF-SIMS confirmed that the drug and PVP are homogeneously distributed, as expected when using χ values to inform PVP:drug compatibility. Thus, the release is driven by PVP dissolution rather than diffusion of the drug within the PVP and will be controlled by the extent of the exposed PVP, a feature dependent on the microstructure. This degradation is also dependent on the oligomer degradation speed, i.e. rate of exposure of further PVP surface area, leading to the rapid release seen when using PLAMA (Figure 2A).</p><!><p>We have shown that the microstructure formed in our polymer blends was process dependent and arose as a function of the drop-by-drop deposition technique. As a result, we demonstrated that it is possible to functionally tailor the composition of 3D printed constructs to successfully control the release of drugs incorporated within them. We selected suitable 3D printing inks using complementary HTP methodologies that allowed us to screen for various desired properties and down select formulations from these screens. Screening of behavior in single drops, combined with the Flory-Huggins interaction parameter provided a prediction of phase separation, and thus drug release, in 3D printed structures. In summary, we demonstrated a reliable toolkit for the development of formulations suitable for 3D printing that can be used to tailor long term drug release on demand. Carbosynth. In all cases the vials were dried in an oven at 50 °C overnight prior to use, and the HEMA and DCM were stored over molecular sieves and under an inert atmosphere.</p><p>Benzyl alcohol (BA) and hydroxyethylmethacrylate (HEMA) initiated ROP of the oligomers. The oligomers were synthesized by ring opening polymerization using metal free organocatalysis, [1] chosen for low toxicological impact of any subsequent medical devices. [2] ROP experiments were performed adopting 'standard laboratory' conditions, i.e. ambient temperature and atmosphere. [3] -OH ended macromers were initiated using BA, -MA and MA-A macromers were initiated using HEMA. Macromers were synthesized following the procedure by Ruiz et al [1] . Briefly, 1000 mg of cyclic monomer (caprolactone, lactide or trimethylene carbonate) and BA or HEMA ([M]:[I] or DP0 ratios targeted to produce final molar masses of 5000 Da were weighed into a vial, which had been dried in an oven at 50 °C overnight and capped with a rubber septum. DCM (5 ml), was then added via syringe and the mixture was allowed to dissolve at room temperature (RT) for 5-10 minutes. Varying amounts of catalyst (1% mol/mol of TBD for lactide and trimethylene carbonate, 2 % mol/mol of TBD for caprolactone) were then added to trigger the ring-opening process.</p><p>Reactions were observed to occur in time-frames ranging from 15-120 minutes, according to the monomer:initiator :solvent :catalyst adopted ratios. The reaction was terminated by catalyst deactivation upon adding an acidic solution and the polymer purified by means of multiple precipitation steps and dried in a vacuum oven.</p><!><p>The MA-OH macromers were further functionalized with an acrylate end using a Stenglich coupling esterification following the same procedure by Taresco, et al [4] . Briefly, PCLMA, PLAMA or PTMCMA (0.2 mmol) and DMAP (0.04 mmol) were added to DCM (5 ml) at room temperature in a glass vial under magnetic stirring until complete dissolution. A second solution was prepared by dissolving 1 mmol of EDC and 1 mmol of acrylic acid in 2 ml of DCM. After dissolution, both solutions were mixed.</p><p>The reaction was allowed to stir for 48 hours. The modified macromers were purified under multiple precipitation steps and dried in a vacuum oven.</p><!><p>Our library of inks was composed of a new set of biodegradable, UV curable materials, which were screened using high throughput methodologies to identify key characteristics suitable for use as an implant, such as printability, biodegradability, cytotoxicity and drug elution (Figure S1).The library was created by combining the nine hydrophobic macromers with two different relative hydrophilic reactive solvents in a 1:1 ratio (w/v) resulting in a total of 18 inks. The solvents functioned as diluents and cross-linkers. Polyethylene glycol diacrylate (PEGDA) 250 Mw and n-vinyl pyrrolidone (NVP) were chosen as the reactive solvents. PEGDA is commonly used as a plasticizer to reduce the glass transition temperature of polymers such as PLA, [5] which helps reduce viscosity without the need of using high temperatures. It is also non-degradable so will not be depleted from the structure. Meanwhile, NVP was selected because once polymerized into poly(N-vinylpyrrolidone) (PVP) it has the ability to form a water-soluble composite structure with insoluble active substances and improve the release and solubility of drugs. [6,7] Additionally, NVP is also known to increase the reactivity of acrylate resins [8] and will degrade in hydrolytic conditions so be removed from any printed structure during use. All the formulations were a 50% w/v solution of the macromer (s) in the solvent (s). Whilst the reactive solvents both functioned as diluents, NVP is monofunctional so gives rise to linear polymer chains and PEGDA is difunctional so functions as a cross-linker/branching monomer so giving rise to a 3D network structure.</p><p>Additionally, both were chosen as the reactive solvents because they are commonly used in pharmaceutical formulations owing to their ability to interact with hydrophilic and hydrophobic solvents, polymers and drugs, [9,10] and they exhibit different degradation behavior. Formulations contained 1% Irgacure 2959 as photoinitiator.</p><!><p>DMF (75% w/v) was used as a non-reactive solvent for all the formulations in this experiment in order to study their properties in high throughput without the need to optimize viscosity in advance. The microarrays were prepared on polyHEMA coated glass slides using the using XYZ3200 dispensing station (Biodot) and quilled metal pins (946MP6B, Arrayit) under argon atmosphere (< 2000 ppm oxygen) maintaining between 40 and 50% relative humidity. Each spot had an average diameter of 200 µm. The spots were UV polymerized under argon atmosphere for 10 min after printing. To remove the solvent, glass slides were dried the in vacuum oven for a week. Atomic Force Microscopy. Height, Peak Force error, DMT modulus, logDMT modulus, adhesion, deformation and dissipation images were simultaneously acquired by Peak Force QNM-AFM measurements (Bruker Fast Scan). Images of 5x5 m per spot were recorded by using a programmable stage. AFM cantilevers with a nominal stiffness nominal k= 40 N/m (RTESPA 300) were used. A Poisson's ratio of 0.3 was used in all cases. Three images were acquired per polymer spot throughout the micro array. The spring constant of each cantilever was estimated by using the thermal tune. Sample standards of polystyrene (PS) were also used to validate tip characterization. Images were analyzed using the NanoScope Analysis software.</p><p>ToF SIMS: ToF-SIMS of microarray samples was carried out using a TOF.SIMS IV instrument from IONTOF GmbH (Muenster, Germany). ToF-SIMS analysis of positively charged secondary ions was carried out using a TOF.SIMS IV system from IONTOF GmbH (Münster, Germany) using 25 keV Bi3 + ion beam operated in the high current bunched mode delivering 0.3 pA with 100 µs cycle time, resulting in a mass range between 0 and 694 u.</p><p>Secondary ion maps were acquired using the stage raster mode. The whole area was scanned once with one shot per pixel, ensuring static conditions.</p><!><p>To investigate the phase separation in pin printed droplets, we used a combination of the Flory-Huggins theoretical model and experimental characterization methods. The Flory-Huggins parameter (χ) describes the excess free energy of mixing and governs phase behavior for polymer blends and block copolymers [11] . In order to calculate the χ value we first obtained the Hansen solubility parameter of the individual components of the formulation using the HSPiP program, were the δd, δp, δh and δTOT were obtained using the DYI tool of the software. We calculated the χ values following the procedure described by Imre et al. [12] by using equation 1,</p><p>where Vr is the molar volume of the repeating unit of the oligomer, R is the gas constant, T the absolute temperature and δ1 and δ2 are the total solubility parameter (δTOT) of the solvent and the oligomer respectively.</p><p>The phase separation taxonomy was created from observations from the printed spots and the χ values. The boundaries were chosen such that we included all the samples that exhibited the dispersed droplet phenomena. This resulted in two exceptions that showed either core-shell or homogeneous microstructure, which reflected the fact that the boundaries were not hard, and could be influenced by other physical properties such as viscosity and curing rate; we estimated the likely error in the boundary by calculating the average difference between χ at the boundary and at the exceptions, resulting in an approximate error of ±0.01.</p><p>Printability screening. To investigate the printability of the inks we used a HT method developed by Zuoxin et al [13] where the viscosity and surface tension are measured using a liquid handler and the printability calculated using the Ohnesorge number (Z=1/Oh). The Ohnesorge number has been identified as the appropriate grouping of constants to characterize drop formation [14] . Reis & Derby used numerical simulation of drop formation to propose 10 > Z > 1 for stable drop formation [15] . To identify printability at different temperatures, eighteen based inks formed by the combination of the nine different macromers mixed with PEGDA and NVP were selected and screened using ranges from 40 ºC to 70 ºC.</p><p>Degradation. This study was performed on the 18 primary inks. Cast cylindrical samples with dimensions of 4 mm length and 1 mm radius were used for this test.</p><!><p>Samples were analyzed with a Perkin Elmer Frontier FTIR-ATR spectrometer (Seer Green, UK) from 4000 cm −1 to 600 cm −1 with a scan resolution of 2 μm and step size of 0.5 cm −1 . Three scans were collected for each sample. Prior to sample spectrum collection, a background was collected on the clean ATR crystal. The degree of curing was calculated by quantifying the reduction of the C=C acrylate stretches (1636 cm -1 ) and the CH 2 acrylate twist 810 cm -1 when the macromers were combined with the reactive solvent PEGDA. The degree of conversion of on the samples mixed with NVP was calculated by looking at the reduction of the C=C vinyl groups of the NVP (1639 cm -1 ).</p><p>Cytotoxicity (Extract test). To test biocompatibility (Figure 2B), we performed an indirect cytotoxicity test for a period of 30 days to determine any evolving cytotoxicity of leached products, either through residual monomers or products emerging through polymer degradation. BJ6 fibroblasts were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) foetal calf serum, 1 % MEM non-essential amino acids solution (Sigma-Aldrich), and 1% antibiotics/antimycotics (100 units/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/ml amphotericin B; Life Technologies). Cells were cultured until they reached 80% confluency and subsequently detached from the culture surface using trypsin/EDTA (0.25%/0.02% w/v), centrifuged at 200 x g for 5 min and resuspended in culture medium. Cells were seeded in a 96 well plate at a density of 5,000 cells per well and allowed to attach for 24 hours before the cytotoxicity experiments. A new seeded well plate was used for each time point.</p><p>Triplicates of each formulation cast samples were sterilized under UV light (0.05 mW/cm 2 , 265nm) for 50 minutes and transferred into a 48-well plate. Each well containing a sample was filled with 1 ml of culture medium. Samples were incubated in the medium for a total of thirty days to allow for leaching of any cytotoxic components. After day 1, day 3 and day 30 of incubation, 200 µl of the supernatant were transferred in triplicate to the cells seeded in the 96-well plates. Cells cultured in in standard medium were used as negative control. Cells were incubated for 24 hours with the supernatant with cells cultured in fresh culture medium used as a negative control. Cytotoxicity was measured using Presto BlueTM (Invitrogen)</p><p>following the manufacturer's instructions. The fluorescent signal was measured with an automated microplate reader (Tecan) using an excitation wavelength of 560 nm and an emission wavelength of 590 nm. For the cytotoxicity percentage calculations, the fluorescent background control was first subtracted from all the samples. Then the percentage was calculated by multiplying the fluorescence of each sample by 100 and then dividing the total by the average fluorescence of the negative control.</p><p>Drug release study. The drug release profile was screened for a period of eight weeks on 16 of the formulations, with PCLMAA/PEGDA and PLAMAA/PEGDA being eliminated as they were not within the printable range. The drug trandolapril was selected for this screening. Formulations containing 0.65% w/v of trandolapril were casted in the same way than for the degradation study. Samples were transferred to individual vials containing 3 ml of phosphate buffered saline solution and placed in an incubator at 37˚C. 500 µl of the PBS solution were collected and each timepoint and filtered (0.45µm) for the HPLC analysis. The PBS solution was refreshed at each timepoint. For the drug release studies of the 3D printed samples the formulation were prepared in the exact same way than the cast ones.</p><p>HPLC. Samples were characterized with an Agilent (Santa Clara, USA) HPLC Series 1260 system, equipped with an auto sampler, degasser, UV lamp and multi-diode array detection.</p><p>A wavelength of 210 nm was used to quantify trandolapril and 280 nm for pitavastatin.</p><p>Method mobile phase compositions were 65% buffer and 35% acetonitrile (Fisher HPLC gradient grade). Phosphate buffer was composed of 6.8 g/L monobasic potassium phosphate (anhydrous, Sigma Aldrich) adjusted to pH 3.0 with phosphoric acid (85-90%, Fluka). An Ultimate LP-C18 column (5 μm, 25 cm × 4.6 mm diameter) was used to separate the samples at 40 °C. A flowrate of 1 mL/min using a 10 μL injection volume was implemented; runtime was 10 min. Trandolapril stock solutions were prepared by sonicating trandolapril/pitavastatin (nominally 1 mg, Carbosynth) in 10 mL methanol (Fisher HPLC grade) and diluting the volume with dissolution media in a 10 mL volumetric flask. Standards were prepared with the stock solution and dissolution media.</p><p>Printing. The formulations were printed using a Dimatix Materials printer (DMP-2830 Fujifilm). The printer was enclosed in a metallic environment box and filled with nitrogen gas. The oxygen level was kept between 0.25 ± 0.05% during the printing process to minimize the inhibition effect caused by oxygen during the free radical photo-polymerization curing procedure. A 10pL disposable printhead, Dimatix Materials Cartridge (DMC-11610, Fujifilm) was used for printing. In-line UV curing was applied at the cartridge height immediate after each swath of ink droplets are deposited, by using a LED UV unit (365nm, 800mW/cm 2 , Printed Electronics Limited, Tamworth, UK) attached and move with the printhead unit. The printing temperature was set to 28°C. The sample was printed at 30 µm for the first layer and reduced to 20 µm for all the following layers. The height of the printhead was set to 700 µm with an increment of 9 µm after each layer printed.</p><p>The samples were 3D printed using an inkjet printer (Dimatix DMP 2800) on the substrate polyethylene naphthalate. Samples dimensions were 5x5x 1 mm.</p><p>Individual sessile droplet size when deposited varied depending on the mixture being processed.</p><p>ToF-SIMS of printed samples was carried out using a 3D OrbiSIMS (hybrid SIMS) instrument from IONTOF GmbH (Muenster, Germany). Secondary ion mass spectra were acquired in negative ion polarity with delayed extraction mode using a 30 keV Bi3 + primary ion beam delivering 0.3 pA.The ToF analyser was set with 200 µs cycle time, resulting in a mass range between 0 and 3493 mass units .For the surface spectra, the primary ion beam was raster scanned over different areas with the total ion dose kept under the static limit of 10 13 ions/cm 2 . The 3D depth profiling data were acquired in dual-beam mode by raster scanning the primary ion beam over regions of up to 150 x 150 µm 2 at the centre of 300 x 300 µm 2 sputter craters formed using an argon gas cluster ion beam (GCIB). The GCIB was operated with 20 keV and 2000 atoms in the cluster delivering a pulsed 5 nA beam current.</p><p>The analysis was performed in the "non-interlaced" mode with a low-energy (20 eV) electron flood gun employed to neutralise charge build up. 3 sputter frames were performed per cycle with 15 analysis scans per cycle and a pause time in between cycles of 0.5 s. Optical profilometry was used to determine the crater depth after ToF-SIMS depth profiling experiments and calibrate the depth scale. Scans were obtained using a Zeta-20 optical microscope (Zeta Instruments, CA, USA). All maps were produced using SurfaceLab and 3D visualisations were produced using the simsMVA software [16] . Intensities were normalised by total ion counts to correct for topographic features. The final 3D representations were created by combining rendered isosurfaces ranging from 40% to 90% of the maximum normalised intensity for each ion.</p><p>orbiSIMS of a cross section of a multi-layer printed sample containing all compounds of interest was carried out using a 3D orbiSIMS (hybrid SIMS) instrument [17] A 20 keV Ar3000 + imaging GCIB of 5 µm diameter was used as primary ion beam, delivering 18 pA . We saw no significant reduction in cell viability on day 3 when compared to the control group (cells cultured in standard medium). However, there was a 10% viability reduction on day 30 when the cells were cultured with medium from the PLA based samples (Figure 2B), most likely due to the acidic degradation by products. [18] This images shows pH changed observed in the medium after been incubated with the samples for 30 days. The change in pH was detected due to the presence of phenol red in medium. The lighter the colour the more acidic the medium. Table S6. Flory-Huggins interaction parameter of the different components of the formulations with trandolapril and pitavastatin. When the formulations contained PEGDA/NVP (1:1) as the reactive solvent, there was an initial burst release of more than 20% in all cases, directly attributable to the dissolution of PVP when immersed in an aqueous environment.</p>

ChemRxiv

The Natural Product Butylcycloheptyl Prodiginine Binds Pre-miR-21,\nInhibits Dicer-Mediated Processing of Pre-miR-21, and Blocks Cellular\nProliferation

SUMMARY Identification of RNA-interacting pharmacophores could provide chemical probes and, potentially, small molecules for RNA-based therapeutics. Herein, using a high-throughput differential scanning fluorimetry assay, we identified small molecule natural products with the capacity to bind the discrete stem-looped structure of pre-miR-21. The most potent compound identified was a prodiginine-type compound, butylcylcoheptyl prodiginine (bPGN), with the ability to inhibit Dicer-mediated processing of pre-miRNA-21 in vitro and in cells. Time dependent RT-qPCR, western blot, and transcriptomic analyses showed modulation of miR-21 expression and its target genes such as PDCD4 and PTEN upon treatment with bPGN, supporting on-target inhibition. Consequently, inhibition of cellular proliferation in HCT-116 colorectal cancer cells was also observed when treated with bPGN. The discovery that bPGN can bind and modulate the expression of regulatory RNAs such as miR-21 helps set the stage for further development of this class of natural product as a molecular probe or therapeutic agents against miRNA-dependent diseases.

the_natural_product_butylcycloheptyl_prodiginine_binds_pre-mir-21,\ninhibits_dicer-mediated_processi

INTRODUCTION<!>Screening for Pre-miR-21 Thermal Modulators Identifies bPGN as a RNA Binding\nMolecule<!>bPGN Rapidly Modulates miR-21 but not Pre-miR-21 Expression in Cells<!>bPGN can Inhibit Dicer Activity in Vitro and in\nVivo<!>bPGN-Induced miR-21 Downregulation Causes Upregulation of Pdcd4 and\nPTEN<!>bPGN Inhibits Proliferation by Upregulating miR-21 Target Genes<!>DISCUSSION<!>STAR* METHODS<!>CONTACT FOR REAGENT AND RESOURCE SHARING<!>Cell culture<!>RNA and Biochemicals<!>Differential Scanning Fluorimetry (DSF)<!>Differential Scanning Calorimetry (DSC)<!>RNA/DNA Quenching and Direct Binding (Kd) Assays<!>Cell Culture, XTT cell viability assay, and bPGN treatment<!>RT-qPCR<!>Western Blot Analysis<!>Dicer Processing Assay<!>Live Cell Imaging<!>NanoString Analysis<!>QUANTIFICATION AND STATISTICAL ANALYSIS<!>DATA AND SOFTWARE AVAILABILITY

<p>MicroRNAs (miRNAs) play pivotal roles for maintaining cellular homeostasis and are involved in virtually all developmental, physiological, and disease processes such as proliferation, migration, cell cycle, and apoptosis (Asangani et al., 2008; Connolly et al., 2010; Krichevsky and Gabriely, 2009). The fundamental principles of the biogenesis of miRNAs are generally well understood (Lin and Gregory, 2015). First, primary-miRNA (pri-miR) transcripts are processed into short stem-looped precursor-miRNAs (pre-miRs) in the nucleus by Drosha-DGCR8 proteins. Pre-miRs are then transported into the cytoplasm by Exportin 5-RAN-GTP where they are further processed into mature ~22 nucleotide double stranded miRNAs by the Dicer-TRBP complex. Once formed, a double stranded miRNA is incorporated into the RNA-induced silencing complex (RISC) after which the passenger strand is degraded, forming the mature silencing complex (Gregory et al., 2006). Functionally, miRNAs mediate translational repression or transcript degradation through antisense interactions with target mRNAs and a single mRNA can be regulated by numerous miRNAs (Bartel, 2018; Bartel and Chen, 2004; Lin and Gregory, 2015). Studies have shown that miRNA-mediated post-transcriptional regulation requires high levels of specific miRNAs and thus, aberrant over- or under- expression of miRNAs has been linked to disease pathologies such as cancer (Denzler et al., 2016). A prime example, miRNA-21–5p (miR-21), is a small 22-nucleotide regulatory oncogenic miRNA that is overexpressed in most cancers including colorectal cancer (CRC). miR-21 regulates numerous driver genes, oncogenes, and tumor suppressor genes such as PDCD4, PTEN, STAT3, and MYC (Asangani et al., 2008; Krichevsky and Gabriely, 2009; Ma et al., 2013). In addition to cancer, dysregulation of miR-21 expression has been implicated in other diseases including obesity (Seeger et al., 2014), cardiovascular diseases (Bauersachs, 2012; Gryshkova et al., 2018), and diabetes (Jiang et al., 2017; Zhong et al., 2013). Thus, modulation of dysregulated miR-21 processing and function could provide a promising strategy for future drug discovery with broad potential therapeutic applications.</p><p>In recent years, there has been an increase in RNA-based therapeutic research that has focused on delivering short sequence-oligonucleotides (RNAi) to silence target mRNAs in cancer cells (Crooke et al., 2018; MacLeod and Crooke, 2017). Our efforts and others' have centered on the identification of chemical pharmacophores that bind specifically to discrete RNA structures as potential starting points for developing chemical probes and/or therapeutic agents. To that end, a growing body of evidence shows that miRNAs and pre-miRNAs are potential targets for small-molecule modulators (Connelly et al., 2017; Jiang et al., 2015; Lorenz et al., 2015; Naro et al., 2015). For example, Disney et al. developed a method called Inforna to identify small molecule binding partners for discrete RNA secondary structures (Disney et al., 2016). In addition, the small molecule AC1MMYR2 (2,4-diamino-1,3-diazinane-5-carbonitrile) has also been shown to specifically target miR-21 biogenesis in cells and in vivo despite non-selective inhibition of other miRNAs in vitro (Ren et al., 2015; Shi et al., 2013). Furthermore, Garner et al. developed a high-throughput assay to screen small-molecule ligands that may bind pre-miRNAs to inhibit Dicer processing (Lorenz et al., 2015). These studies have demonstrated that specific small-molecule inhibitors of miRNA processing are obtainable from suitably controlled assay systems and that structured RNAs can indeed be druggable targets.</p><p>Here, we use a RNA-based differential scanning fluorimetry (DSF) assay to identify modulators of miRNA biogenesis and processing. This effort resulted in the identification of the natural product butylcylcoheptyl prodiginine (bPGN, 1) (Figure 1A) which can regulate the expression level of miR-21 in HCT-116 colorectal cancer cells. 1 belongs to a family of bioactive tripyrrole natural product compounds called prodiginines produced by bacteria with diverse reported biological activities including antibacterial (Gondil et al., 2017), antimalarial (Castro, 1967), antifungal (Woodhams et al., 2018), and anticancer (Perez-Tomas and Vinas, 2010). Well-known derivatives from this family include obatoclax (Gariboldi et al., 2015) and prodigiosin (Perez-Tomas et al., 2003) (Figure 1A). Obatoclax has been tested in clinical trials and shows promise against hematological malignancies (Joudeh and Claxton, 2012; Schimmer et al., 2008; Schimmer et al., 2014) but has displayed limited efficacy in solid tumors (Arellano et al., 2014; Goard and Schimmer, 2013). Despite the clinical development of obatoclax, the reported molecular mechanism of prodiginine anti-cancer activity is still not fully understood (Chen et al., 2014; Xie et al., 2015), thus further elucidation of its molecular mechanism may provide important data toward improving its therapeutic applications. Here, we report for the first time that 1 can bind to pre-miR-21 and inhibit Dicer-mediated processing at non-cytotoxic concentration which results in cellular proliferation arrest by downregulating miR-21 expression. The previously unreported interaction of a prodiginine-type natural product with regulatory non-coding RNA to mediate downregulation of cancer-associated genes broadens the chemical classes identified as RNA-interacting pharmacophores for the development of molecular probes and provides avenues for potential therapeutic applications.</p><!><p>Compounds that modulated the thermal stability of pre-miR-21 in vitro were identified using a high-throughput differential scanning fluorimetry (DSF)-based screen. Briefly, we performed a screen of 3682 pure natural products against an annealed pre-miR-21 oligonucleotide and identified which compounds could modulate its thermal stability in vitro (Figure S1). This screen resulted in the identification of 32 compounds that either increased or decreased the thermal stability of pre-miR-21 (Table S1). In a single specificity screen using another stem-looped RNA structure, these compounds did not significantly affect the in vitro thermal stability of the 5'-UTR stem-loop A (SLA) RNA fragment from the Dengue-II virus genome (data not shown). Based on a literature search, 7 of the 32 active compounds have not been previously identified as RNA-binding molecules and thus were tested for their ability to inhibit growth in cells (Figure 1B, compounds 1, 4 - 9). Previous studies have shown the significance of miR-21 in growth and proliferation of HCT-116 cells (Chen et al., 2017), thus we chose to treat these cells with the compounds ranging from 0 – 25 μM and assessed for viability 24 hours post-treatment (Table 1). Among these compounds, aklavin (AKL, 4), trigilletine (TRG, 5), and an anthraquinone (ANT, 6) all showed high nanomolar half maximal growth inhibitory concentrations (GI50), close to the value of 5-fluorouracil (5-FU) positive control (0.72 μM). Lobinaline (LOB, 7), prumycin (PRU, 8), and dehydronuciferin (DHY, 9) all showed ≥ 10 μM GI50 (Table 1). The most active compound bPGN, 1, showed a GI50 as low as 0.035 μM in HCT-116 cells. Concomitantly, we also tested 2 other prodiginine compounds, obatoclax and prodigiosin, and determined their GI50 to be 0.10 μM and 0.24 μM, respectively against HCT-116 cancer cells. Significantly, the GI50 of 1, obatoclax, and prodigiosin in normal colon cells were all determined to be ≥ 10 μM, signifying a selective mechanism of these compounds against cancer cells.</p><p>In vitro, a dose dependent titration of 1, obatoclax, and prodigiosin (up to 20 μM) afforded a maximum melting temperature shift (ΔTm, max) of +3.1, +7.0, and +3.2°C, respectively against pre-miR-21 (Figure 1C). Using the intrinsic fluorescence of the highly conjugated tripyrrol backbone, we measured the direct binding affinity (Kd) of 1, obatoclax, and prodigiosin to pre-miR-21 and calculated an affinity of 0.41, 0.09, and 0.16 μM, respectively (Figure 1D). Navitoclax (structure shown in Figure S3) was used as a control for these experiments as navitoclax and obatoclax have been shown to share a common molecular mechanism as BH3-mimetics in cells (Chen et al., 2011; Koehler et al., 2014). Our results showed no significant interaction between pre-miR-21 and Navitoclax (Figure 1C). Using fluorescence displacement assay, Navitoclax did not show apparent binding affinity (Kdapp) to pre-miR-21 (data not shown), consistent with the lack of molecular interaction to pre-miR-21 in our DSF assay. Taken together, these results suggest that the tripyrrolic backbone is indeed responsible for RNA binding by the prodiginine family of compounds. Furthermore, we posit that the disconnect between the binding affinity (410 nM) and cellular growth inhibitory activity (35nM) of 1 may be due to an additive effect between bPGN inhibition of pre-miR-21 processing and other previously reported mechanisms for this class of compounds (i.e. as a BH3 mimetic) which would certainly enhance cellular activities.</p><p>Finally, to discern specificity of 1 in vitro, we tested the binding of 1 against other structured RNAs such as tRNA (Lys,3), HIV TAR, and pre-miR-638, and showed that 1 did not significantly change the Tm of these RNAs up to 5-fold molar excess of 1 compared to modulation of pre-miR-21 Tm (Figure 1E). Furthermore, results using differential scanning calorimetry (DSC), a dye-independent measurement of thermal stability, show similar stabilizing effects on pre-miR-21 structure in the presence of 1 consistent with DSF (Figure S4). These results directed us to focus our additional studies on elucidating the mechanism of action of 1 in modulating the expression and function of oncogenic miR-21.</p><!><p>In addition to its pro-apoptotic effect, obatoclax have been shown to inhibit proliferation of leukemia and colorectal cancer cells by inhibiting cell cycle progression, but the exact mechanism remained unclear and targets remain unidentified (Konopleva et al., 2008; Or et al., 2016). Here, a concentration dependent reduction in cell proliferation and viability was also observed when HCT-116 cells were treated with compound 1 at 0 (DMSO only), 0.005, 0.05, and 0.5 μM (Figure 2A). This static effect resulted in 55% reduction of cell number 24-hours post-treatment at 0.05 μM (p < 0.01), but complete cell death at concentration > 0.5 μM (p < 0.001). To determine if the proliferation arrest we observed is indeed caused by the inhibition of pre-miR-21 processing by 1, HCT-116 cells were treated with 50 nM of 1 and the expression levels of pre-miR-21 and miR-21 were evaluated via quantitative RT-PCR (RT-qPCR) at time points ranging from 0 to 24 hours post-treatment. GAPDH and U6 snRNA were used as reference control genes and miRs −1246, −203a-3p, −200b-3p, and −361 expression levels were used to assess selectivity. Figure 2B shows that 1 rapidly reduced miR-21 levels by ~80% (p < 0.001) within 2 hours post-treatment but was less effective in downregulating the expression level of the other tested miRs (up to ~30% reduction). miR-1246 is known to be only highly expressed in HCT-116 spheroid cells (Yamada et al., 2014), and thus remained at low levels up to 24 hours post-treatment. Interestingly, miR-21 expression level gradually recovered up to 24 hours post-treatment, suggesting involvement of a compensatory mechanism due to its pivotal role in cell survival (i.e. the miR-21:STAT3 feed-back loop (Krichevsky and Gabriely, 2009)). Notably, pre-miR-21 expression level did not significantly change up to 24 hours post-treatment indicating that Drosha-mediated processing of pri-miRNA to pre-miRNA was unaffected by 1 (Figure 2B). Furthermore, transfection of exogenous miR-21 mimic 4 hours before treatment of 1, afforded greater percentage of cells 24 and 48 hours post-treatment (Figure 2C), signifying that the proliferation arrest caused by 1 can be mitigated by the addition of exogenous miR-21.</p><!><p>To determine if 1 can inhibit Dicer-mediated pre-miR-21 processing, we performed two in vitro Dicer activity assays. First, we showed by gel shift assay that Dicer activity can be inhibited by 1 in a dose dependent manner (Figure 3A). Lanes 1, 2, and 3 are controls showing pure pre-miR-21, miR-21, and pre-miR-21 with DMSO only, respectively. Major bands, at ~55 and ~22 bp corresponding to pre-miR-21 and miR-21 respectively, are observed. Lane 4 shows pre-miR-21 with 100 μM (4-fold excess) of 1 without Dicer. Here, two bands were observed at ~55 and ~35 bp, suggesting that two major distinct structured species of pre-miR-21 exist in the presence of 1. We hypothesize that 1 induces a supercoiling structural change because of the lower bp band similar to previous reports shown for other compounds binding to RNA (Jain et al., 2013). Supercoiling of RNA can have a thermal stabilizing effect as more intramolecular interactions are made. This result is also consistent with our DSC results where we observe two thermal transitions (Tm1 = 42.3°C; Tm2 = 68.0°C) for pre-miR-21 in the presence of 1 (Figure S4), where presumably the higher melting temperature (trace 2) corresponds to the supercoiled species. Lanes 5–7 show the effect of increasing 1 concentration on pre-miR-21 processing by Dicer. In Lane 5, depletion of the ~55 bp band and the concomitant increase in ~22 bp band is observed in the absence of 1. In Lanes 6 and 7 a dose dependent decrease in the ~22 bp band is observed in the presence of 50, and 100 μM of 1, suggesting a dose dependent inhibition of Dicer processing of pre-miR-21 in vitro.</p><p>To further support these results, we also used DSF as a more sensitive method to determine if Dicer processing of pre-miR-21 was inhibited by 1. Here, under Dicer reaction conditions, the Tm of pre-miR-21 was 62.2°C and 59.6°C in the presence (beige) and absence (black) of 10 μM 1, respectively (Figure 3B). In the presence of Dicer and only DMSO (blue), we observe a shift in the Tm by −7.2°C, but a −2.8 and −0.6°C shift in the presence of 50 (aqua) and 100 (green) μM 1, respectively (Figure 3B). These results suggest that the presence of 1 can reduce the global residency of pre-miR-21 on Dicer in vitro. Pure miR-21 (red) was also tested as a control and had a Tm of 52.4°C, strongly correlating with the sample of pre-miR-21 incubated with Dicer with only DMSO (blue). As an assay control, we used the same method to evaluate known Dicer inhibitors: hexachlorophene (Lorenz et al., 2015) and regorafenib (Chen et al., 2017). Indeed, high concentration of hexachlorophene or regorafenib strongly inhibited Dicer activity consistent with our results with 1 (Figure S5). Taken together, these data strongly support that the binding of 1 to pre-miR-21 can lead to inhibition of Dicer-mediated processing of pre-miR-21 in vitro which may correspond to its activity in cells.</p><p>Finally, work by Su et al. showed that prodigiosin induces cytotoxicity in hepatocellular carcinoma cells via intercalation into DNA grooves in a copper-mediated manner (Su et al., 2015). To determine if this is true for 1, we assessed its cellular localization using fluorescence microscopy (max 545/580 nm) by treating cells with either DMSO or 50 nM of 1. Importantly, 1 is intrinsically fluorescent due to the highly conjugated tripyrrolic backbone as also previously reported for prodigiosin (Darshan and Manonmani, 2016; Han et al., 2014), thus no chemical modification was necessary. As an additional control, we also show that incubation of 1 with purified total RNA and DNA (gDNA or plasmid DNA) does not quench fluorescence of 1 up to 48 hours of incubation in vitro (Figure 3C). 1 hour prior to imaging, cells were treated with 0.5 μg/mL Hoechst nuclear stain. 3D laser scanning confocal imaging shows that 1 predominantly accumulated at the cytoplasmic space up to 24 hours post-treatment (Figure 3D). Analyses of the x-y plane as well, as the subsequent x-z and y-z orthogonal slices, reveal minimal presence of the 1 in the nucleus. Furthermore, quantification of fluorescence intensity shows minimal overlap between bPGN and Hoechst signals (Figure 3E). Taken together, our data indicate that at 50 nM, 1 does not induce cellular proliferation arrest via intercalating DNA. It also supports our hypothesis that 1 most likely inhibits Dicer-mediated processing of pre-miR-21 which occurs in the cytoplasm, but not the inhibition of Drosha-mediated processing which takes place in the nucleus.</p><!><p>Overexpression of miR-21 in colorectal cancer cells downregulates expression of tumor suppressor protein, programmed cell death 4 (Pdcd4) (Asangani et al., 2008), which is known to inhibit cell proliferation in other cancer cells (Wang et al., 2016a). In addition, it has been shown that aberrant PI3K-Akt pathway signaling plays an important role in tumorigenesis and is often associated with the absence (or mutation) of PTEN (Vivanco and Sawyers, 2002). PTEN, a highly regulated miR-21 target, negatively regulates the PI3K-Akt pathway via dephosphorylation of PIP3, ultimately inhibiting activation of Akt and consequently inhibiting cellular proliferation signaling. To determine if the reduction of miR-21 by treatment of 1 influences the expression of PDCD4 and PTEN in cells, we performed time dependent RT-qPCR and western blot analyses. RT-qPCR showed that the PDCD4 and PTEN transcripts were upregulated 2-fold by 8 hours post-treatment with 1 and continuously increased up to 2 – 4-fold by 36 hours post-treatment (Figure 4A). To see if Pdcd4 and PTEN protein expression could be modulated by miR-21, siRNA miR-21 mimic or inhibitor were transfected in HCT-116 cells. Results showed by western blot analysis that protein expression was decreased by ~40% when treated with the miR-21 mimic and increased by 22–58% when treated by the miR-21 inhibitor 48 hours post-transfections (Figure 4B). Consistent with the siRNA results, cells treated with 50 nM of 1 showed a time dependent increase in Pdcd4 and PTEN protein levels within 24 hours post-treatment (Figure 4C). Taken together, our data reveal that on-target inhibition of miR-21 expression in HCT-116 cells leads to the upregulation of the PDCD4 and PTEN transcripts and protein levels in a time dependent manner. Importantly, upregulation of these proteins is sufficient to induce the anti-proliferation effect observed upon treatment with 1.</p><!><p>To gain greater insight into the differential effects of 1 to miR-21 target genes, we performed a time course transcriptomic study using the NanoString Technologies platform to collectively identify which genes were modulated. TargetScan (targetscan.org, (Agarwal et al., 2015)) and miRDB (mirdb.org, (Wong and Wang, 2015)) both predict over 200 target genes for miR-21[−5p], many of which have been experimentally validated (Buscaglia and Li, 2011; Krichevsky and Gabriely, 2009). In our analysis, we found 28 of 41 predicted genes that were significantly differentiated from untreated control cells (p < 0.05) (Figure 5A, Table S2). Most notably, miR-21 target genes that are reported to play a role in suppressing cellular proliferation, such as PTEN, STAT3, and TGFB1/TGFBR2, were significantly upregulated. In addition, a differential pathway analysis using our NanoString data reveals multiple pathways important for cell proliferation as highly modulated upon treatment with 1 including modulation of the cell cycle, DNA replication, and PI3K-Akt pathways (Figure 5B). Taken together, our data indicate that the inhibition of cellular proliferation observed when cells are treated with 50 nM of 1 can be due to modulation of miR-21 expression and function which leads to the upregulation of target genes such as PTEN, which could then lead to the subsequent inhibition of cell proliferation. Figure 5C summarizes the connection between PTEN and other target genes and their effect on cellular proliferation.</p><!><p>As an increasing number of important cell functions are determined to be regulated, at least in part, by small regulatory RNAs, the identification of pharmacophores that interact with miRNAs are of more importance. Thus, development of methodologies to identify these pharmacophores will be very useful for the growth of the field. Here, for the first time, we employ a RNA-based high-throughput DSF-based screen and identified butylcycloheptyl prodiginine (bPGN, 1), a derivative from the class of tripyrrolic natural product called prodiginines, as an active RNA-binding small molecule. Currently, numerous evidences suggest that at clinical concentrations of prodiginines such as obatoclax or prodigiosin, proapoptotic Bcl-2 family proteins are activated in a p53-independent manner. This has been proposed to be the primary mechanism of tumor cytotoxicity for this class of compounds (Soto-Cerrato et al., 2004). Other observed mechanisms of anticancer activities for prodiginines include intracellular acidification, copper-dependent DNA cleavage, modulation of signal transduction and MAPKs, cell cycle arrest, and most recently, the inhibition of Wnt/β-catenin signaling pathway (Perez-Tomas and Vinas, 2010; Wang et al., 2016b). In this report, we add another previously unreported mechanism and show for the first time that 1, and derivatives thereof, can bind at nanomolar concentration and stabilize the discrete stem-looped structure of pre-miR-21. Importantly, we show that binding of 1 can interfere with Dicer-mediated processing of pre-miR-21, which leads to rapidly downregulates mature miR-21 expression in cells. We extended these findings by showing that 1 can consequently induce transcript and protein expression of Pdcd4 and PTEN, which can then lead to the observed reduced proliferation state of HCT-116 colorectal cancer cell. Indeed, the complexity of the mode of action(s) of prodiginines may account for the disconnect between our in vitro binding assay with pre-miR-21 (~410 nM) and cellular activity (~35nM) as we could be observing a more potent cellular activity by intrinsic additive effects. Nevertheless, the anti-proliferative, a static rather than cytotoxic effect of 1, by binding regulatory RNAs represents yet another apoptosis-independent mechanism by which prodiginines elicit anticancer activity.</p><p>Indeed, other studies have observed the same antiproliferative and reduced cell viability effect with other prodiginines, but the underlying mechanisms and/or targets in these studies remained unidentified. For example, Hsieh et al. showed that dephosphorylated Akt could lead to GSK-3β activation when treated with prodigiosin, but the molecular basis for how Akt was dephosphorylated was not elucidated (Hsieh et al., 2012). Here, our functional gene analysis shows significant upregulation of PTEN transcript (increased by 121 %). Consistently, studies have shown that PTEN is a direct target of miR-21 (Luo et al., 2017; Meng et al., 2007), but can also be indirectly downregulated by miR-21 through the suppression of SPRY genes (Chai et al., 2018; Feng et al., 2011). Thus, it was not surprising that inhibition of pre-miR-21 processing by 1 caused an increase in PTEN and, to a lesser degree, SPRY2 transcripts (Figure 5A), which could consequently lead to inhibition of PI3K-Akt signaling and ultimately inhibit cellular proliferation, as previously observed.</p><p>Prodigiosin have been shown to stabilize topoisomerase I/II-DNA complex in lymphocyte cells which may lead to DNA-damage-induced cytotoxicity in these cells (Montaner et al., 2005). It is hypothesized that the selective cytotoxicity of prodigiosin to cancer cells is due to the higher copper levels in cancer cells compared with normal cells. However, our live cell imaging studies revealed that 1 accumulates in the cytoplasmic space for up to 24 hours post-treatment, suggesting that the proliferation arrest we observed upon treatment of HCT-116 cells with 1 at sub-cytotoxic concentration is independent of DNA-damage and must be due to increased levels of miR-21 targets such as Pdcd4 and PTEN. Consequently, we posit that selective growth inhibitory effect towards cancer cells observed here is mediated by the inhibition of overexpressing miR-21 which increases its target genes, leading to reduced cell proliferation of HCT-116 cells. It is also important to note that Figure 2B shows downregulation of other miRNAs, albeit not as significant as miR-21, but in the same rapid manner. Previous reports have shown that the half-life of miRNAs is measured in days rather than hours (Gantier et al., 2011), but rapid degradation of stable miRNAs is not unusual and have been observed in many studies (Hwang et al., 2007; Krol et al., 2010; Ruegger and Grosshans, 2012). Unlike the biogenesis of miRNAs however, turnover and regulation thereof, are still poorly understood. Enzymes that play a role in miRNA turnover are slowly being identified (Chatterjee and Grosshans, 2009; Ramachandran and Chen, 2008) such as PNPT1 which have been shown to degrade mature miRNAs without affecting pri-/pre-miRs (Das et al., 2010). Here, future endeavors to identify factors responsible for rapid miRNA turnover and how 1 can activate rapid degradation of miRNAs is of great interests but requires further investigation.</p><p>Finally, recent work by Disney et al. has shown the importance of fully understanding the effects of clinical agents on regulatory RNAs or the transcriptome, beyond their initially-described protein targets (Velagapudi et al., 2018). Our studies here show that 1 can have substantial effect on pre-miR-21 stability through direct binding which leads to a reduction in Dicer processing and subsequent down-regulation of miR-21 expression. This data provides yet another example of a unstudied chemotype that can bind pre-miRNA. In contrast to pre-miR-21, we did not observe significant thermal modulation of other stem-looped structured RNAs (Figure 1E), thus we posit that 1 may confer selectivity to specific pre-miRNAs (e.g. via specific loop sequences or alternating nucleotide sequence as seen with prodigiosin binding to DNA). It is also possible that 1 may have additional direct effects on the actions of miR-21, which will require further structural and functional studies to deduce. To this end, further investigation of the global binding of 1 to other pre-miRNAs to identify vulnerable pre-miRNA targets are currently ongoing. In addition, structural insights of the molecular interactions of 1 to pre-miR-21 will also enhance our knowledge of the selectivity of this chemical pharmacophore.</p><!><p>Detailed methods are provided and include the following:</p><!><p>Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Barry O'Keefe (okeefeba@mail.nih.gov)</p><!><p>HCT-116 and was purchased from American Type Culture Collection (ATCC) and was established from an adult human colorectal cancer. HCT-116 was cultured in Roswell Park Memorial Institute (RPMI) media with 10% Fetal bovine serum (FBS). CCD 841 CoN was purchased from ATCC and was established from a normal colon epithelial cells of a 21 weeks gestation fetus. CCD 841 CoN cell line was cultured in Eagle's Minimum Essential Medium (EMEM) with 10% FBS. Both cell lines were subject to 1:5 passaging every 3 days and incubated in 37°C with 5% CO2. Cells were purchased directly from ATCC but were not authenticated in house.</p><!><p>Unmodified, desalted, and HPLC purified pre-miR-21 was purchased from Eurofins Scientific (Louisville, KY, USA). RNA was folded via resuspending with buffer (10 mM Tris-HCl pH 7.5 with 20 mM NaCl), and heated to 95°C for 90 sec, and allowed to cool to room temperature overnight. Folded RNA was aliquoted and stored in −20°C until assayed. The following sequence of pre-miR-21 was purchased:</p><p>5'-UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCU GUCUGACA-3'</p><p>Butylcycloheptyl prodiginine (bPGN) was supplied by Developmental Therapeutics Program (DTP), Division of Cancer Treatment and Diagnosis in the National Cancer Institute (NCI). Plates for high-throughput screens were provided by NCI, Molecular Targets Program (MTP). Compounds such as 5-FU, obatoclax, prodigiosin, navitoclax, spermidine, methoctramine, hexachlotophene, and regorafenib were purchased from Sigma Aldrich or selleckchem.com.</p><!><p>The high throughput DSF-screen was performed in a total reaction volume of 6 μL containing 1 μM pre-miR-21, 1× SYBR Green Dye (ThermoFisher, Cat#S7567), 2% DMSO, and buffer (0.25 mM Tris-HCl and 0.25 mM NaCl) in a 384-well white polypropylene reaction plate (Roche, Cat# 04729749001) and was treated with 3 μM concentration of compounds from the NCI Molecular Targets Program (MTP) pure compound library using an automated liquid handler. Spermidine and methoctramine were used as a stabilizing or destabilizing control, respectively. In a specificity screen, pre-miR-21, Lys3 tRNA, HIV TAR, and pre-miR-638 were treated with 5 μM bPGN to determine ΔTm. To obtain maximum change in Tm, DSF dose response was performed from a master mix containing 1.0 μM pre-miR-21, 1× SYBR Green, 2% DMSO in a reaction buffer (0.25 mM Tris-HCl and 0.25 mM NaCl). Then, bPGN, obatoclax, prodigiosin, or navitoclax was titrated ranging from 0 – 20 μM in each well at final volume of 6 μL. All DSF analysis was performed using Roche LightCycler 480 Instrument II using the following parameters: manual mode with integration time set at 0.75 seconds, continuous acquisition mode up to 99°C, ramp rate of 0.09°C/s and 5 acquisitions per °C. Raw data was imported into GraphPad Prism 7.03 for analysis and high-resolution figures. From the raw data, the temperature which gave half-denatured state (Log GI50) of pre-miR-21 was calculated (labeled as Tm) using the non-linear regression fit (Sigmoidal, 4PL) function, y = fluorescence, x = temperature. The exact Tm of premiR-21 with or without bPGN can be extrapolated using differential scanning calorimetry (below).</p><!><p>DSC experiments were performed using VP-DSC (Malvern Instruments). 30 μM Pre-miR-21, with or without 300 μM bPGN, was prepared in 1.5% DMSO adjusted buffer (0.25 mM Tris-HCl pH 7.5 with 0.25 mM NaCl), degassed, and added into the sample cell. Cell compartments were capped, adjusted for positive pressure (30 psi), and allowed to progress through a series of 8 alternating up and down scans, all scans running at a rate of 60°C/hr with a filter period of 16 seconds. The completed sample thermogram was first corrected for buffer-DMSO effects (subtracting the first up scan) followed by a normalized correction of the absolute baseline, the cubic approximation of the pre-transition (structured) and the post-transition (unstructured) regions of the thermogram. The baseline-corrected thermogram was fitted to a non-2-state unfolding model according to manufacturer's protocol, the fitting error evaluated for single versus multiple unfolding units. From this model-fitting, values for melting temperature (Tm) and enthalpy of unfolding (ΔHcal) were extrapolated for each transition. Analyzed data were imported into GraphPad Prism 7.03 to render figures.</p><!><p>Total RNA (50 ng/μL) and gDNA (25 ng/μL) from HCT-116 or 50 ng/μL plasmid were incubated with 0, 12.5, 25, 50, and 100 nM of bPGN in buffer (0.25 mM Tris-HCl and 0.25 mM NaCl). Fluorescence intensity was measured using SpectraMax i3x (Molecular Devices) at excitation 545 nm and emission 580 nm. Direct binding assay was performed in a 40 μL reaction volume containing 1.0 μM pre-miR-21 and compounds (bPGN, obatoclax, and prodigiosin) ranging from 0 – 10 μM in a reaction buffer (0.25 mM Tris-HCl and 0.25 mM NaCl) and the fluorescence intensity was using the same parameters. Raw fluorescence data were imported into GraphPad Prism 7.03 for analysis and high-resolution figures. Kd was calculated using the non-linear regression fit (Sigmoidal, 4PL) function.</p><!><p>HCT-116 cells (ATCC CCL-247) were cultured in RPMI with 10% FBS. Normal colon cells (CoN ATCC CRL-1790) were cultured in EMEM with 10% FBS. For dose response experiments, 2 × 103 cells were seeded in 96-well plates and incubated for 24 hours in a 5% CO2 / 37°C incubator. Cells were then treated with compounds ranging from 0 – 10 μM and incubated for another 24 hours. The XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) cell viability assay was performed according to manufacturer's protocol (ThermoFisher, Cat#X6493). Absorbance (450 nm) was read using Tecan-Magellan plate reader (Tecan Trading AG, Switzerland). Cells for RT-qPCR, NanoString mRNA expression, and protein analyses were cultured in 100 mm cell culture plates until 50–60% confluence before treatment. Cells were treated with 50 nM bPGN and collected for time point studies at 0, 1, 4, 8, 16, 24, 36, 48, and 72 hours post-treatment and subjected to protein and total RNA extraction following manufacturer's protocol for mirVana PARIS (ThermoFisher). For siRNA targeting miR-21, cells were treated with DMSO (control), 30 nM mimic (Ambion #4464066), or 30 nM inhibitor (Ambion #4464084) using Lipofectamine 2000 as the lipid carrier and incubated for 48 hours prior to mirVana PARIS extraction. All experiments were performed in experimental triplicate.</p><!><p>Materials for RT-qPCR for mRNA and miRNA expression profiling were EXIQON ExiLERATE (#303402) and miRCURY (#203351) LNA reagents and primers (Exiqon, Woburn MA, USA). Standard designed primers include hsa-miR-21–5p, hsa-miR-1246, hsa-miR-203a-3p, hsa-miR-200b-3p, hsa-miR-361, UniSp6 (loading control), U6 snRNA (reference gene), and GAPDH (reference gene). Pre-miR-21 and PDCD4 were ordered using the following primers:</p><p>pre-miR-21: forward – TGTCGGGTAGCTTATCAGAC, reverse – TTCAGACAGCCCATCGACTG</p><p>PDCD4: forward – TAGATTGTGTGCAGGCTA, reverse – GTTCAGCTTCAGATATGTCTC</p><p>RT-qPCR was performed according to manufacturer's protocol using the following conditions: Denature – 95°C for 10 min. Then Cycle 45 times of 10 sec at 95°C, 1 min at 60°C, and 10 sec at 95°C. End by extension – 55°C for 1 min and 4°C hold. PDCD4 fold change expression was determined using the following equation: 2−ΔΔCP, where ΔΔCP = ΔCPtreated – ΔCPuntreated and ΔCP is the difference in the CP values of PDCD4 and GAPDH (i.e. ΔCP = PDCD4 – GAPDH).</p><!><p>Cell lysates were subjected to standard trichloroacetic acid (TCA)/acetone precipitation. The isolated protein fraction pellet was dried, reconstituted in 8 M urea, and the total protein concentration was determined by UV spectrophotometry. For SDS-PAGE, a quantity of 20 μg protein was loaded per lane of NuPAGE 4–12% Bis-Tris gels (Invitrogen) and separated at 200 V constant voltage. Gels were briefly washed in 20 % ethanol and proteins were transferred to PVDF membranes using the iBlot 2 dry transfer device (Invitrogen) using the following method: 20 V for 1 minute, 23 V for 4 minutes, and 25 V for 3 minutes. Membranes were blocked in Odyssey Blocking Buffer in PBS (LiCor) and probed with mouse anti-PDCD4 or PTEN (Santa Cruz Biotechnology) and rabbit anti-GAPDH (Cell Signaling Technology) primary antibodies. Mulitplex detection was accomplished using goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD secondary antibodies (LI-COR Biotechnology).</p><!><p>Dicer reaction assays were set up according to manufacturer's protocol (Genlantis, San Diego CA, Cat#T510001). 10 μM of pre-miR-21 was incubated with 10U of DICER with or without bPGN (or other compounds) up to 36 hours at 37°C. The reaction was quenched, and RNA was purified by ethanol precipitation (or an RNA purification kit). 1 μM of the RNA is used for DSF assay (see above for protocol) and the rest was loaded into a pre-stained, pre-casted 4% agarose get for the gel-shift electrophoresis assay (Invitrogen, E-Gel Power Snap Electrophoresis System, Cat#G8342ST). Experiments were performed in experimental duplicates.</p><!><p>2 × 105 HCT-116 cells were cultured in a 35 mm glass bottom culture dish (Cellvis, CA, USA, #D35–141.5N) and treated with 50 nM bPGN and incubated for 24 hours. Cell were then treated with 0.5 μg/mL Hoechst stain 1 hours prior to imaging. A Nikon TI-Eclipse microscope outfitted with 20 X objective (0.8 NA) an Andor Zyla camera (Belfast, UK) and a Tokai Hit (Shizuoka-ken, Japan) stage-top incubator was used for wide field fluorescence imaging. A Leica SP8 laser scanning confocal microscope with a 40X (0.85 NA) air objective and a Tokai Hit stage-top incubator collected 3D images of the bPGN and Hoechst fluorescence using HyD detectors in photon counting mode. Cells were maintained at 37°C and 5% CO2 during the entire period of imaging, and bPGN fluorescence was measured using the tetramethylrhodamine channel. Experiments were performed in duplicates and on 3 separate days. Images were minimally processed (cropping) and analyzed with FIJI (intensity profiles; NIH) and Imaris (othogonal slicing; Bitplane, Concord, MA) software.</p><!><p>NanoString analysis was performed by the National Cancer Institute (NCI), Center for Cancer Research(CCR) Genomics Core Facility. The NanoString nCounter Human Pan-Cancer Pathway Panel assay kit (http://www.nanostring.com/) was employed to identify altered mRNAs with or without 50 nM treatment of bPGN at time points: 0, 1, and 24 hours post-treatment. 100 ng of total RNA was used for nCounter sample preparation according to manufacturer's instructions (NanoString Technologies). Raw data was normalized to 40 Housekeeping genes using the NanoString nSolver software. For our analysis, a 5 fM signal threshold was implemented as statistically significant, thus we excluded 227 genes because of low signals. Furthermore, in addition to the 40 housekeeping genes used as internal controls during analysis, all data were normalized to untreated control (time point 0). P-values were calculated using Pearson correlation as a distance metric and pairwise complete-linkage as a clustering method. nSolver Pathway Analysis was used to obtain differential pathway scores. Hierarchal clustering figure was made using GraphPad Prism from the normalized values.</p><!><p>All plots show means with error bars representing S.E.M., unless otherwise noted. Experiments were completed at least in triplicate except for nanoString mRNA analysis which are two replicates. Data were plotted in Graphpad Prism 7.03.</p><!><p>nSolver and Advanced Analysis software are available in nanoString Technologies website (https://www.nanostring.com/). FIJI and Imaris are available from NIH and Oxford Instruments, respectively.</p>

PubMed Author Manuscript

Synthesis and Characterization of the Arylomycin Lipoglycopeptide Antibiotics and the Crystallographic Analysis of their Complex with Signal Peptidase

Glycosylation of natural products, including antibiotics, often plays an important role in determining their physical properties and their biological activity, and thus their potential as drug candidates. The arylomycin class of antibiotics inhibits bacterial type I signal peptidase and is comprised of three related series of natural products with a lipopeptide tail attached to a core macrocycle. Previously, we reported the total synthesis of several A series derivatives, which have unmodified core macrocycles, as well as B series derivatives, which have a nitrated macrocycle. We now report the synthesis and biological evaluation of lipoglycopeptide arylomycin variants whose macrocycles are glycosylated with a deoxy-\xce\xb1-mannose substituent, and also in some cases hydroxylated. The synthesis of the derivatives bearing each possible deoxy-\xce\xb1-mannose enantiomer allowed us to assign the absolute stereochemistry of the sugar in the natural product and also to show that while glycosylation does not alter antibacterial activity, it does appear to improve solubility. Crystallographic structural studies of a lipoglycopeptide arylomycin bound to its signal peptidase target reveal the molecular interactions that underlie inhibition and also that the mannose is directed away from the binding site into solvent which suggests that other modifications may be made at the same position to further increase solubility and thus reduce protein binding and possibly optimize the pharmacokinetics of the scaffold.

synthesis_and_characterization_of_the_arylomycin_lipoglycopeptide_antibiotics_and_the_crystallograph

Introduction<!>Lipoglycopeptide synthesis<!>Structural analysis of a lipoglycopeptide arylomycin bound to SPase<!>Biological activity<!>Conclusions<!>General experimental procedures<!>Production and co-crystallization of SPase \xce\x942\xe2\x80\x9376 and lipoglycopeptide BAL4850C and data collection<!>Phasing, model building, and refinement<!>Determination of antimicrobial activity

<p>The clinical and agricultural use of antibiotics imposes a relentless selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens, and novel classes of antibiotics are needed.1,2 Bacteria produce a large assortment of antibiotics, possibly to gain advantage over competing microorganisms for limited resources,3–9 and these compounds have proven to be the richest source of antimicrobials for development into therapeutics. While most if not all of these natural products are produced as families of related compounds, the significance of this diversity is debated.10–14 The arylomycins, first isolated in 2002 from a strain of Streptomyces, consist of three related series of compounds, each of which has a conserved C-terminal tripeptide macrocycle attached to an N-terminal lipopeptide (Fig. 1).15–17 The macrocycle of the A series compounds is unmodified, while those of the B series and lipoglycopeptides are nitrated and glycosylated (and in some cases hydroxylated), respectively.15,16</p><p>The arylomycins inhibit type I signal peptidase (SPase, EC 3.4.21.89),16,18,19 which is an essential membrane-bound serine endopeptidase with a highly conserved active site that is required to remove the amino-terminal leader (signal) sequence during, or shortly after protein translocation across the cytoplasmic membrane. SPase acts via a unique Ser-Lys catalytic dyad with an unusual nucleophilic attack on the si-face of the substrate, as opposed to the re-face attack characteristic of the more common Ser-His-Asp catalytic triad serine proteases.20 Moreover, its position on the outer surface of the cytoplasmic membrane should make it relatively accessible to inhibitors, although penetration of the outer membrane could limit accessibility in Gram-negative bacteria. While their novel mechanism of action originally generated much enthusiasm, excitement for developing the arylomycins waned when it was concluded that their activity was limited to only a few Gram-positive bacteria.16,17 However, after reporting the first synthesis of an arylomycin, the A series member arylomycin A2 (Fig. 1), as well as several derivatives,21 we demonstrated that they actually have a remarkably broad spectrum of activity,19 including potent activity against both Gram-positive and Gram-negative bacteria, but that activity is limited in some cases by one of at least two resistance mechanisms: target mutation, specifically, the presence of a proline residue in SPase;19 or a second as yet undefined mechanism that confers Streptococcus agalactiae with resistance to the A series derivatives.22 Moreover, following the synthesis of a B series arylomycin, arylomycin B-C16, we demonstrated that the nitro group does not negatively impact activity against bacteria that are sensitive to arylomycin C16, and importantly, that it overcomes the resistance of S. agalactiae and imparts the scaffold with a reasonably potent minimum inhibitory concentration (MIC) of 8 μg/ml.22</p><p>Like the lipoglycopeptide arylomycins, many antibiotics are glycosylated, and in some cases the sugar substituents are required for activity.23,24 From a medicinal chemistry perspective, glycosylation can also impact an antibiotic's potential for development as a therapeutic by affecting its pharmacokinetic properties, including absorption, distribution, metabolism, and excretion, at least in part due to changes in solubility and serum binding.25 The most common sugar substituents are 6-deoxysugars, of which more than a hundred have been identified among different secondary metabolites,26 and indeed the sugar substituent of the lipoglycopeptide arylomycins was identified as deoxy-α-mannose,16 although its absolute stereochemistry was not determined.</p><p>The lipoglycopeptide arylomycins have been shown to have moderate activity against several bacteria, inhibiting a strain of Streptococcus pneumoniae with MICs ranging from 8 to >64 μM; a strain of Staphylococcus aureus, with MICs ranging from 32 to >64 μM; and a strain of Haemophilus influenzae, with an MIC of 64 μM.16 While they are not active against intact Escherichia coli, they were shown to have activity against permeabilized mutant strains, leading to the suggestion that their development as therapeutics would require the optimization of outer membrane penetration.16 However, the potential role of the resistance conferring proline has not been examined.</p><p>E. coli SPase is 324 amino acids in length, (molecular weight 35,960 Da and pI 6.9)27 and contains two amino-terminal transmembrane segments (residues 4–28 and 59–77), one small cytoplasmic region (residues 29–58), and a large carboxyl-terminal periplasmic catalytic domain (residues 78–324).28,29 Proteinase K digestion,29,30 gene-fusion,31 and disulfide cross-linking studies32,33 are all consistent with both the N- and C- termini of E. coli SPase facing the periplasmic space. The catalytically active periplasmic domain of E. coli SPase (SPase 2–75) has a molecular weight of 27,952 Da34 and pI of 5.6.35 It has been sub-cloned, purified,34 characterized35 and crystallized.36 To date, four crystal structures of E. coli SPase have been reported (all with the Δ2–76 enzyme), including the unbound protein,37 a binary complex with a β-lactam inhibitor,20 a binary complex with an A family arylomycin (arylomycin A2),18 and a ternary complex with arylomycin A2 and β-sultam.38 While these structures have helped elucidate the mechanisms of the molecular recognition underlying the inhibition of SPase by the arylomycins, the effects of macrocycle glycosylation remained unclear.</p><p>We now report the first synthesis of an arylomycin lipoglycopeptide and its biological characterization, as well as the structural analysis of the binary complex with a related glycosylated and hydroxylated derivative. Total synthesis allowed us to assign the absolute stereochemistry of the deoxy-α-mannose substituent and to determine that the spectrum of activity of the glycosylated derivative is limited by the same mechanisms of resistance as are the A series compounds. The structural analysis revealed that the inhibitor binds in a fashion similar to that previously reported for the A series derivative, and that the sugar is oriented away from the active site and into the aqueous environment. Consistent with these structural studies, in addition to finding that glycosylation does not interfere with SPase binding or activity, we find that it appears to increase aqueous solubility and reduce protein binding based on MIC values in the presence of serum proteins. In all, the data reveal that contrary to previous conclusions,16 glycosylation does not interfere with the antibacterial activity of the arylomycins, including activity against Gram-negative bacteria, and that similar types of modifications might be used to optimize the pharmacokinetics of this promising scaffold.</p><!><p>To synthesize the lipoglycopeptide arylomycins, we modified our previously reported syntheses of the arylomycin A21 and B22 series compounds to increase flexibility and material throughput. Because the absolute stereochemistry of the sugar was not known, we targeted the synthesis of both the deoxy-α-L-mannose and the deoxy-α-D-mannose variants. The required lipopeptide 2 was prepared using the procedure of Zhu and co-workers (Scheme 1).39,40 Briefly, the previously reported tripeptide 1 was acylated with isopalmitic acyl chloride, generated in situ from isopalmitic acid and oxalyl chloride in DCM, yielding the fully protected fatty tail, which was hydrolyzed under Nicolaou's conditions (Me3SnOH/DCE) to afford the lipopeptide 2 in 51% yield.</p><p>Macrocycle synthesis commenced with the preparation of the hydroxyphenylglycine-alanine iododipeptide 8 from commercially available 4-hydroxyphenylglycine 3 (Scheme 2). Briefly, Boc protection of 3, followed by mono iodization41 afforded 4 in 80% yield, which was subsequently converted into oxazolidinone 5. After reduction by triethylsilane in TFA, followed by reinstallation of the Boc group, the desired N-methyl amino acid 6 was obtained without racemization. Acid 6 was then coupled to L-Ala-OMe to afford dipeptide ester 7. Finally, hydrolysis of the methyl ester using Me3SnOH provided iododipeptide 8 in quantitative yield. This sequence produced 8 in seven steps with 40% overall yield, which required only one column purification for product 7.</p><p>The protected tyrosine pinacol boronic ester 11 was prepared from iodotyrosine in four steps (Scheme 3). Briefly, triply protected iodotyrosine 10 was prepared from commercially available iodotyrosine 9 in greater than 90% yield with only one column purification. The boronic ester of compound 11 was installed via a Miyama reaction,42 and then deprotection, followed by HOBt/EDC-mediated coupling to dipeptide 8 provided tripeptide 12. To optimize macrocyclization of 12, we first employed conditions that we found to be optimal for the cyclization of the bis-methyl phenol protected A or B series macrocycle cores (PdCl2(dppf)/NaHCO3, DMF). However, in this case the desired product was obtained in less than 25% yield, suggesting that while the free phenol may minimize the epimerization of 12,39 it also appears to adversely affect the reaction. Thus we re-optimized the cyclization conditions (Table 1) and found that Pd(tBu3P)2 and K2CO3 provided 13 in a satisfactory 48% yield.</p><p>The only remaining challenge was to glycosylate the free phenol of the macrocycle without O- to C-glycoside rearrangement under the required Lewis acidic reaction conditions (Table 2).43 Due to the low nucleophilicity of the phenol and the steric hindrance of the neighboring O-benzyl group, we first attempted to glycosylate 13 using glycosyl bromides 15 in the presence of AgOTf and 4 Å molecular sieves in DCM, but no glycosylated product was detected. We then investigated trichloroacetimidate 16a as a glycosyl donor. TMSOTf-promoted glycosylation, either in catalytic or super-stoichiometric quantities, yielded the desired macrocycle in less than 40% yield. However, 10 equivalents of BF3-Et2O44 resulted in simultaneous glycosylation and Boc deprotection, and yielded the desired glycosylated macrocycle 14a in 76% yield. Similarly, 14b was obtained in 70% yield using the same conditions.</p><p>With lipopeptide tail 2 and both glycosylated macrocycles 14a and 14b in hand, it only remained to couple and deprotect the two halves of the molecule. Compound 2 was coupled to 14a or 14b to provide the fully protected natural products 17a and 17b. Global deprotection was carried out in three mild reactions to avoid the possible elimination of the D-Ser hydroxyl group. Pd/C catalyzed hydrogenation, NaOMe induced deacetylation, and hydrolysis with Me3SnOH provided the candidate natural product candidates 18a and 18b in greater than 50% yield. Comparison of the 1H NMR spectra of the two candidate natural products with that of the authentic natural product (kindly provided by Dr. Sheng-Bing Peng, Eli Lilly) revealed that while almost all of the proton resonances of the 18a spectrum are nearly identical to the natural product, those of the sugar are significantly different (Fig. S1). In contrast, the 1H spectra of 18b and the natural product are virtually identical, as are the 13C spectra. Thus, we conclude that the lipopeptide arylomycins are glycosylated with deoxy-α-L-mannose. Furthermore, similarities in the NMR spectra in the region corresponding to the sugar of the different members of the lipoglycopeptides,16 suggest that they are all glycosylated with deoxy-α-L-mannose.</p><p>While the arylomycins, including the lipoglycopeptides, are naturally lipidated with different fatty acids ranging in length from 12 to 16 carbons, our analysis of the A and B series compounds were performed with a straight chain C16 tail.21 Thus, for systematic comparison of biological activity, we used the above protocol but with the straight chain C16 lipid, to synthesize the corresponding lipoglycopeptide derivative. As expected, this synthesis proceeded with indistinguishable yields. For simplicity, we refer to these derivatives as arylomycin A-C16, arylomycin B-C16, and arylomycin C-C16, corresponding to the A, B, and lipoglycopeptide compounds, respectively (Fig. 1).</p><!><p>Structural studies focused on analysis of the glycosylated and hydroxylated lipoglycopeptide antibiotic BAL4850C and SPase Δ2–76. BAL4850C contains the same sugar that is characteristic of the lipoglycopeptide class of arylomycins, but is differentiated from arylomycin C-C16 by macrocyclic hydroxylation and lipidation by an unsaturated C16 fatty acid (Fig. 1). All attempts to soak BAL4850C into preformed crystals of SPase Δ2–76 were unsuccessful, so we instead developed a co-crystallization method which yielded well ordered crystals that diffracted to beyond 2.4 Å resolution (Table 3). The initial Fo - Fc difference map revealed a well-defined rod-shape density corresponding to the N-terminal tripeptide, as well as ring-shape electron density corresponding to the three residue macrocycle core of the inhibitor with clear electron density for the mannose substituent (Fig. 2A). The modeled L-stereochemistry for the mannose substituent is consistent with the electron density, but the resolution is not high enough to independently confirm the assigned stereochemistry. The electron density of the C16 fatty acid tail of the lipoglycopeptide is weak at 1.0 σ, suggesting the fatty acid tail is disordered. Similarly, no density was observed for the fatty acid tail in the reported complex with arylomycin A2.18</p><p>The refined structure revealed that the arylomycin is positioned within the SPase binding site with its C-terminal macrocycle oriented towards the catalytic residues and both the peptide backbone and side chains tightly packed within the substrate binding groove (Fig. 2 and 3). Similar to the complex of SPase Δ2–76 and arylomycin A2,18 one C-terminal carboxylate oxygen (O45) interacts with three catalytically essential residues of the enzyme, including the nucleophile Ser91 Oγ, the general base Lys146 Nζ, and a component of the oxyanion hole, Ser89 Oγ. (Note that the numbering system used with previous SPase structures18,20,37,38 is different by one residue due an error in the originally reported sequence of the E. coli protein.30 The sequencing error occurred in the cytoplasmic region, between the two N-terminal transmembrane segments, which is not present in SPaseΔ2–76, and therefore does not affect the register of the residues within the crystal structures, only the residue numbering system. The numbering system used in the currently reported structure matches that in the Swiss-Prot sequence data base, accession number: P00803.) The other C-terminal carboxylate oxygen (O44) is hydrogen-bonded to Ile145 N and the general base Lys146 Nζ. The peptide backbone of the arylomycin forms eleven direct hydrogen-bonds with SPase, forming parallel β-strand interactions with the β-sheets that make up the substrate binding groove (SPase residues 143–146 and 82–86), seven of which are mediated by the macrocycle tripeptide and four by the N-terminal tripeptide. Macrocycle residues N33 and N28 form hydrogen-bonds with Asp143 O and Gln86 O, respectively, and there is a conserved water molecule (water 14 in Chain A and water 15 in Chain B) within hydrogen-bonding distance of O45. Finally, the C30 methyl group of Ala6 is directed toward the S3 substrate-specificity pocket and is in van der Waals contact with the side chain of SPase residue Ile145. Clearly, the macrocycle provides the majority of the interactions involved in SPase recognition. Interestingly, although the deoxy-α-L-mannose is in van der Waals contact with SPase residue Pro88, it is predominantly solvent exposed (Fig. 2 and 3).</p><p>A superposition of the structures of the three SPase-arylomycin complexes solved to date reveals that very little adjustment within the protein is needed to accommodate the sugar (Fig. 4). Moreover, the presence of the sugar does not appear to significantly alter the structure of the macrocycle, which the superposition reveals is similar, and engages the protein in a similar way, in each complex. However, the superposition also reveals that the binding mode for the three N-terminal residues (D-MeSer2–D-Ala3–Gly4) of the inhibitor is more variable, particularly at D-Ala3. These observations are consistent with the macrocycle mediating the majority of interactions between the inhibitor and the enzyme and suggest that the N-terminal peptidic tail may be more flexible.</p><!><p>The antibacterial activity of the lipoglycopeptide arylomycin C-C16 was characterized by determining the MIC required to inhibit the growth of several Gram-positive bacteria (Table 4). Against S. epidermidis, S. pyogenes, S. pneumoniae, C. glutamicum, and R. opacus arylomycin C-C16 has activity that is indistinguishable from the analogous A series compound. Moreover, like arylomycin A-C16, but unlike arylomycin B-C16, arylomycin C-C16 has no activity against S. agalactiae strain COH1, demonstrating that the ability of the nitro substituent to impart the B series scaffold with activity against this pathogen is unique.</p><p>We next determined the activity of arylomycin C-C16 against S. aureus, E. coli, and P. aeruginosa. As with both the A and B series compounds, arylomycin C-C16 has no activity against these pathogens. Also as with the A and B series compounds, arylomycin C-C16 does have significant activity against the mutant pathogens where the arylomycin resistance-conferring proline residue is mutated to a residue that does not confer resistance (P29S in the S. aureus protein, and P84L in the E. coli and P. aeruginosa proteins)19 (Table 5). Moreover, the addition of polymyxin B nonapeptide, which permeabilizes the outer membrane of Gram-negative bacteria, did not significantly affect the MICs. Thus, while the spectrum of arylomycin C-C16 is limited by the same resistance mechanisms as is the A series compound, in contrast to previous conclusions,16 the presence of the sugar does not impair the inhibitor's ability to penetrate the outer membrane of Gram-negative bacteria.</p><p>Although not unprecedented among therapeutics,45–48 from a drug development perspective, the fatty acid tails of the arylomycins might prove to be a liability, for example due to decreased solubility and increased serum binding. Thus, to begin to examine the effects of glycosylation on aqueous solubility and serum binding we redetermined the MICs of arylomycin A-C16 and arylomycin C-C16 against wild type S. epidermidis and sensitized E. coli in the presence of pooled human serum (25 – 100%) or bovine serum albumin (4 – 10%) in MHIIB. While the value of MICs varied under the different conditions tested, the MIC observed for arylomycin C-C16 was consistently 2- to 4-fold lower than that of arylomycin A-C16 under these conditions. These small but reproducible effects are consistent with glycosylation increasing the concentration of the free inhibitor available for SPase binding.</p><!><p>Our previous demonstration that the arylomycin class of antibiotics has a broader spectrum of antibacterial activity than previously appreciated, and that where resistance does exist, it results from the presence of a specific proline mutation in the target SPase protein,19 makes the arylomycin scaffold promising for development as a therapeutic. Toward the further exploration of this class of natural product antibiotics, we found that the lipoglycopeptide variants can be by synthesized in reasonable yield, with the key steps being a Pd(tBu3P)2/K2CO3-mediated macrocycle cyclization and a BF3-Et2O-mediated trichloroacetimidate glycosylation. Characterization of the synthetic product allowed us to unambiguously determine that the natural products are glycosylated with deoxy-α-L-mannose, and contrary to previous reports,16 we found that glycosylation does not appear to interfere significantly with activity. The structural analysis revealed that the lipoglycopeptides bind SPase in a manner analogous to the arylomycin A series compounds with the sugar moiety oriented away from the enzyme active site and largely solvent exposed. Nonetheless, the structural analysis also revealed that the hydrophobic portion of the sugar interacts with active site residues, and that glycosylation does affect the interactions between the peptidic position of the inhibitors tail and SPase. Thus, it remains possible that glycosylation affects activity against bacteria that were not examined in the current study. While the selection pressure, if any, that favors glycosylation of the arylomycin scaffold in nature remains unclear, it does appear that glycosylation increases the solubility of the scaffold, an important pharmacokinetic attribute for any candidate therapeutic. Thus, derivatization at the same position with other substituents, e.g. other sugars, phosphates, sulfates etc., might further improve the pharmacokinetic properties of the arylomycin scaffold and aid in its potential development as a therapeutic. Experiments to test this hypothesis are currently in progress.</p><!><p>Dry solvents were purchased from Acros. Commercially available amino acids were purchased from Bachem (Torrence, CA), Chem-Impex (Wood Dale, IL), or Novabiochem (EMD Chemicals, Gibbstown, NJ). Celite 545 filter aid (not acid washed) was purchased from Fisher. Anhydrous 1-hydroxybenzotriazole (HOBt) was purchased from Chem-Impex. All other chemicals were purchased from Fisher/Acros or Aldrich. Reactions were magnetically stirred and monitored by thin layer chromatography (TLC) with 0.25 mm Whatman pre-coated silica gel (with fluorescence indicator) plates. Flash chromatography was performed with silica gel (particle size 40–63 μm, EMD chemicals). 1H and 13C NMR spectra were recorded on Bruker DRX 500, or Bruker DRX 600 spectrometers. Chemical shifts are reported relative to either chloroform (δ 7.26) or methanol (δ 3.31) for 1H NMR, and either chloroform (δ 77.16) or methanol (δ 49.00) for 13C NMR. High resolution time-of-flight mass spectra were measured at the Scripps Center for Mass Spectrometry. ESI mass spectra were measured on either an HP Series 1100 MSD or a PESCIEX API/Plus. For all compounds exhibiting atropisomerism or isolated as semi-pure mixtures, NMR spectra are provided in Supporting Information. Yields refer to chromatographically and spectroscopically pure compounds unless otherwise stated.</p><p>All preparative reverse phase chromatography was performed using Dynamax SD-200 pumps connected to a Dynamax UV-D II detector (monitoring at 220 nm) on a Phenomenex Jupiter C18 column (10 μm, 2.12 × 25 cm, 300 Å pore size). All solvents contained 0.1% TFA; Solvent A, H2O; Solvent B, 90% acetonitrile/10% H2O. All samples were loaded onto the column at 0% B, and the column was allowed to equilibrate ~10 min before a linear gradient was started. Retention times are reported according to the linear gradient used and the % B at the time the sample eluted.</p><!><p>SPase Δ2–76 was expressed and purified as described previously.36 Prior to co-crystallization, SPase Δ2–76 (18.0 mg/ml in 20 mM Tris-HCl, pH 7.4, and 0.5% Triton-100) was combined with the lipoglycopeptide BAL4850C (10 mM in DMSO) in a 1:1 molar ratio and incubated on ice for one hour.</p><p>Co-crystallization trials for SPase Δ2–76 in complex with BAL4850C (provided by Basilea Pharmaceutica International Ltd., Grenzacherstrasse 487, CH-4058, Basel, Switzerland) were carried out by the sitting-drop vapor diffusion method. The final optimized reservoir condition that produced high quality crystals for data collection was 22% (w/v) PEG 4000 and 0.2 M KCl. The drop consisted of 2 μl of protein/inhibitor complex described above, 2 μl of reservoir solution, and 2 μl of 0.025 M n-dodecyl-β-D-maltoside (DDM). This 6 μl drop was equilibrated over 1 ml of reservoir solution at 20 °C. The crystals formed from a light precipitate after approximately two weeks and had an average size of ~0.2 × 0.1× 0.5 mm.</p><p>Before data collection, the crystal was transferred by a pipette from the growth drop to a cryoprotectant composed of 24% w/v PEG 4000, 0.2 M KCl, 0.008 M DDM, 20% glycerol) for 30 s. The crystal was mounted on a Hampton Research loop and flash-cryo-cooled by directly placing it into a gaseous nitrogen stream at 100K. The X-rays (wavelength 1.5418 Å) were generated from Cu Ka radiation via a Rigaku MicroMax-007 Microfocus X-ray rotating-anode generator running at 40 Kv and 20 mA and equipped with Osmic Confocal VariMax High Flux optics. The crystal-to-detector distance was set to 200 mm. All frames (280) were recorded on a R-AXIS IV++ imaging-plate detector with a 0.5° oscillation angle and an exposure time of 240 s per frame. The data revealed diffraction to beyond a resolution of 2.4 Å. Data were collected, indexed, and scaled using the program CrystalClear.49 The crystals belong to the tetragonal space group P43212. The unit cell dimensions were determined to be a = 72.0 Å, b = 72.0 Å, and c = 262.6 Å. The Matthews coefficient (Vm) is 3.03 Å3/Da for two molecules in the asymmetric unit. The fraction of the crystal volume occupied by solvent was 59.3%, calculated by the program Matthews in the CCP4i suite of programs.50,51 For crystal and data collection statistics see Table 3.</p><!><p>A molecular replacement solution was found using the program Phaser in the CCP4i suite of programs.51 The atomic coordinates used for the search model was taken from a 2.5 Å crystal structure of SPase Δ2–76 (PDB code, 1T7D; molecule A).18 The topology and parameter files for the inhibitor were generated using the program PRODRG.52 Coordinates for the inhibitor were manually docked into clear electron difference density (Fo - Fc) near the active site. In addition, the main chain trace and the side chain assignments for the dynamic regions corresponding to residues Phe197-Asn201 and Asp305-Leu315 in chain B were built in manually. Water molecules were added to well-defined peaks (2.0 σ and greater into the Fo – Fc maps). Model building and analysis was performed with the program Coot.53,54 Refinement of the structure was carried out using the program Refmac 5 in the CCP4i suite as well as CNS.51,55 The cycles of refinement were carried out for both protein model and inhibitor model using rigid body and restrained NCS refinement in the program Refmac 5, and simulated annealing, energy minimization, and B-factor refinement was performed in CNS. In addition, a cycle of TLS refinement was carried out using the TLS Motion Determination Sever and restrained TLS refinement protocol of Refmac 5 within the CCP4i suite.51 In all cycles of refinement, 5% of the reflections were set aside for cross-validation. Final refinement and analysis statistics of the complex are provided in Table 3. The stereochemistry of the structure model was analyzed with the program PROCHECK.56 No stereochemical outliers were observed in the Ramachandran plot, with 96.3% of the residues in the preferred regions. An all atom superposition of the arylomycin complexes was performed with the program Pymol57 using molecule B of the lipoglycopeptide arylomycin complex (PDB: 3S04), molecule A of the ternary complex with arylomycin A2 and a β-sultam (PDB: 3IIQ),38 and molecule A of the arylomycin A2 complex structure (PDB: 1T7D).18 Figures were prepared using the programs ISIS Draw version 2.5 (MDL Information Systems, Inc.), and PyMol.57 The atomic coordinates (accession code: 3S04) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org).</p><!><p>Antimicrobial activity was examined using thirteen bacterial strains, Staphylococcus epidermidis RP62A, Staphylococcus aureus NCTC 8325, Escherichia coli MG1655, Pseudomonas aeruginosa PAO1, Staphylococcus epidermidis RP62A SpsIB(S29P) (PAS9001), Staphylococcus aureus NCTC 8325 SpsB(P29S) (PAS8001),19 Escherichia coli MG1655 LepB(P84L) (PAS0260),19 Pseudomonas aeruginosa PAO1 LepB(P84L) (PAS2008),19 Rhodococcus opacus DSM 1069, Streptococcus agalactiae COH-1, Streptococcus pyogenes M1-5448, Streptococcus pneumoniae R800, and Corynebacterium glutamicum ATCC 44475. Minimum Inhibitory Concentrations (MICs) were determined from at least three independent experiments using the CLSI broth microdilution method. Briefly, inocula were prepared by suspending bacteria growing on solid media into the same type of broth used in the MIC experiment and diluting to a final concentration of 1 × 107 colony forming units/ml. 5 μl of this suspension were added to the wells of a 96-well plate containing 100 μl of media with the appropriate concentrations of compound. The MICs of E. coli, P. aeruginosa, S. aureus, S. epidermidis, R. equi, R. opacus, and C. glutamicum were determined in Cation-adjusted Mueller Hinton II broth. MICs of S. pyogenes and S. pneumoniae were determined in Todd Hewitt broth. The MICs of S. agalactiae were determined in Cation-adjusted Mueller Hinton II broth and in Todd Hewitt broth (MIC values differed by at most two fold between these two media). In all cases MICs were defined as the lowest concentration of compound to inhibit visible growth.</p>

PubMed Author Manuscript

Evolutionary Importance of the Intramolecular Pathways of Hydrolysis of Phosphate Ester Mixed Anhydrides with Amino Acids and Peptides

Aminoacyl adenylates (aa-AMPs) constitute essential intermediates of protein biosynthesis. Their polymerization in aqueous solution has often been claimed as a potential route to abiotic peptides in spite of a highly efficient CO 2 -promoted pathway of hydrolysis. Here we investigate the efficiency and relevance of this frequently overlooked pathway from model amino acid phosphate mixed anhydrides including aa-AMPs. Its predominance was demonstrated at CO 2 concentrations matching that of physiological fluids or that of the present-day ocean, making a direct polymerization pathway unlikely. By contrast, the occurrence of the CO 2 -promoted pathway was observed to increase the efficiency of peptide bond formation owing to the high reactivity of the N-carboxyanhydride (NCA) intermediate. Even considering CO 2 concentrations in early Earth liquid environments equivalent to present levels, mixed anhydrides would have polymerized predominantly through NCAs. The issue of a potential involvement of NCAs as biochemical metabolites could even be raised. The formation of peptide-phosphate mixed anhydrides from 5(4H)-oxazolones (transiently formed through prebiotically relevant peptide activation pathways) was also observed as well as the occurrence of the reverse cyclization process in the reactions of these mixed anhydrides. These processes constitute the core of a reaction network that could potentially have evolved towards the emergence of translation.T he biosynthesis of peptides involves aminoacyl adenylates (aa-AMPs), formed through the reaction of ATP with a-amino acids (aas) (Fig. 1), that are subsequently used to aminoacylate tRNA. Their standard free energy of hydrolysis value DGu9 5 ca. 270 kJ mol 21 , determined for Tyr-AMP 1 , ranks them among the energy-richest biochemicals. Aa-AMPs possess a phosphate group transfer potential much higher than ATP 1 and might then constitute adenylating agents as well as aminoacylating agents 2,3 . The otherwise unfavourable 1 reaction of ATP with a-amino acids (K 5 3.5 3 10 27 ) is driven towards completion by selective stabilization of aa-AMPs in the active sites of aminoacyl tRNA synthetases (aaRSs). They usually remain sequestrated by the enzyme and are not released in solution before reacting with tRNA. The importance of this process can be appreciated by considering that the set of aaRS enzymes, responsible for the association of amino acids with their cognate tRNAs, actually holds the key of the genetic code. The evolutionary path through which adenylates were introduced in the process remains unidentified. In addition of being thermodynamically unfavourable, the spontaneous reaction is indeed very slow in the absence of enzyme 4,5 , so that the emergence of the biochemical amino acid activation pathway remains unexplained before a set of catalysts (very probably ribozymes) could lead to an embryo of the genetic code for prebiotically available amino acids 6 . In spite of this obstacle, the evolution of this pathway from an abiotic process of random peptide formation via the polymerization of a-amino acid mixed anhydrides with phosphate (aa-PMAs) or phosphate esters (aa-PEMAs) and adenylates (aa-AMPs) has prompted much work [7][8][9][10] . However, the abiotic formation of adenylates or their analogues from phosphate anhydrides did not receive any experimental support. As a matter of fact, the claim 11 that ATP is capable of driving the polymerization of aamino acids on clays through aa-AMP intermediates turned out to be non-reproducible 12 . Though the genetic code might have evolved late in the hypothesis of an ''RNA world'' without needing ATP activation as shown by the successful selection of ribozymes capable of aminoacylating RNAs using either amino acid esters 13 or activated RNAs 14 , an early co-evolution involving the chemistries of nucleotides and amino acids is consistent with the comparatively higher abundance of the latter as the products of abiotic processes. Therefore, selecting the co-

evolutionary_importance_of_the_intramolecular_pathways_of_hydrolysis_of_phosphate_ester_mixed_anhydr

<!>Results<!>2<!>Discussion<!>Methods

<p>evolutionary option, the elucidation of the potential evolutionary process through which aa-AMPs could have been introduced requires the identification of simple pathways capable of leading to these intermediates. A likely possibility is the reaction of a-amino acid N-carboxyanhydrides (NCAs) with inorganic phosphate 15 and its esters including adenylates that takes place spontaneously at moderate pH 16,17 (Fig. 2a). This possibility is supported by the role of NCAs deduced from the literature 2 and the disclosure of realistic abiotic pathways for their formation during the last decade 18,19 . Since the activation of the C-terminus in peptides has recently been identified as a plausible prebiotic pathway and involves the formation of 5(4H)-oxazolone intermediates 20 , it is reasonable that similar mixed anhydrides with phosphates involving acylated amino acids (acyl-aa-PEMAs) or peptides (peptidyl-PEMAs) could be formed by reaction of the energy-rich cyclic intermediate (Fig. 2b). The occurrence of abiotic pathways leading to aa-PEMA or peptidyl-PEMA must have preceded their involvement in chemical evolution. However, the low stability of these mixed anhydrides and the availability of highly reactive cyclic intermediates prone to polymerize more easily renders their role in early abiotic processes of peptide formation highly questionable.</p><p>The kinetic stability of aa-AMPs and of other aa-PEMAs has been studied in aqueous solution leading to contradictory results in the literature [21][22][23][24] . Of particular interest with regard to an evolutionary context is the description of a highly efficient CO 2 -catalyzed path of hydrolysis [21][22][23] . No definitive mechanism has been proposed but the intermediacy of NCAs is highly probable 2,25,26 since other activated amino acids (nitrophenyl esters, thioesters) proved to undergo conversion into NCAs in hydrogen carbonate buffers 25 . This analysis casts doubts on the possibility that aa-AMPs constitute efficient monomers for the abiotic formation of peptides in aqueous solutions 2,3,26 since most early Earth aqueous environments are likely to have contained CO 2 or HCO 3 2 . The present investigations were aimed at providing data on the efficiency of the CO 2 -promoted pathway (Fig. 3a) in aqueous solution at neutral pH and in the presence CO 2 concentrations compatible with early Earth environments and at clearly identifying the NCA as an intermediate. They address both the issues of the stability of aa-AMPs and of other aa-PEMAs and that of the path of peptide formation. They demonstrate the prevalence of the CO 2 -promoted pathway in the hydrolysis of adenylates. More importantly, using model amino amide reactants, they additionally demonstrate that peptide bond formation takes place predominantly from the cyclic intermediates rather than directly from the mixed anhydrides ruling out any possibility of considering the latter as direct peptide precursors at early stages of chemical or bio- chemical evolution. Lastly, considering NCAs as likely precursors of aa-AMPs and aa-PEMAs, the hypothesis of an abiotic formation of non-coded peptides through these mixed anhydrides becomes unnecessary. The evolution of translation must then have proceeded through a pathway independent from abiotic polymerization. This work also addresses the more general goal of understanding the stability of phosphate mixed anhydrides of amino acids and peptides in aqueous media at moderate pH. As a matter of fact, though Nacylation is an obvious way to prevent CO 2 participation, another intramolecular path of breakdown through 5(4H)-oxazolones is possible in the case of acyl-aa-PEMAs (Fig. 3b). Therefore, the issues of the importance of the NCA and 5(4H)-oxazolone pathways in the reactions of the corresponding mixed anhydrides (Fig. 3) are raised as well as that of the potential role of these cyclic intermediates as potential prebiotic precursors of these mixed anhydrides (Fig. 2). The consequences of these chemical pathways as factors determining early biological evolution of amino acid activation processes and their constraints on the contemporary biochemistry of adenylates will also be discussed.</p><!><p>Experiments were carried out from model systems derived from Omethylated tyrosine 5 (Fig. 4) likely to be representative of the reactivity of usual amino acid derivatives. The UV-absorption of the tyrosine side chain (l max 5 273 nm) was selected to monitor reactions by HPLC at a reasonably low (0.05-1 mM) concentration range in which activated intermediates have a lifetime sufficient for their behaviour to be determined. Furthermore, phenol methylation was introduced to simplify analyses by avoiding any side-reaction of this group. Reactions were carried out in non-nucleophilic MES or MOPS buffers at pH values of 6.5 or 7.5, respectively, whereas 50 mM phosphate or methyl phosphate buffers were used for studying the transient formation of mixed anhydrides. Analyses were performed to monitor the reaction progress of samples stored in the HPLC systems located in a room maintained at the temperature of 20uC. Fast reactions were monitored by withdrawing 1 mL samples from the reaction medium and the reaction was blocked by addition of a formic acid solution to bring the pH to a value below 4 (Supplementary information).</p><p>NCAs as intermediates of aa-PEMA reactions promoted by CO 2 .</p><p>The hydrolysis of methyl phosphate mixed anhydride 1b was studied in buffered solutions in the presence of varying contents of CO 2 / HCO 3 2 . The reaction rates were observed to strongly depend on the presence of CO 2 as shown by a c.a. 4 fold increase in rate using pH 6.5 MES buffers previously equilibrated with air as compared with a solution flushed with N 2 for 60 min (Fig. 5, panel A). The rates could be reduced by further c.a. 35% by extensive degasification through cycles of freezing at 295uC/gas removal under vacuum/ melting in a closed vessel. Under the conditions of the experiment displayed in the panel A of Fig. 5, the starting material 1b (HPLC retention time, r.t. 4.6 min, method A) disappeared slowly and several species containing the methoxyphenyl moiety (l max 273 nm) were observed, namely the free amino acid 5 (r.t. 8.4 min) representing the main product of hydrolysis but also several peaks corresponding to the dipeptide H-Tyr(Me)-Tyr(Me)-OH (r.t. 22.7 min) and the diketopiperazine cyclo-Tyr(Me)-Tyr(Me) (r.t. 23.6 min), very probably resulting of the cyclization of the mixed anhydride H-Tyr(Me)-Tyr(Me)-OPO 3 Me 2 , which has not been properly identified. The presence of these two products was confirmed by HPLC-MS analysis ([M 1 H] 5 373.2 at r.t. 1.52 min and 355.2 at r.t. 1.88 min, method C). By contrast, the addition of 2 or 10 mM NaHCO 3 to the buffer led to the fast disappearance (#1% after 3 min) of the mixed anhydride 1b as monitored by HPLC analysis (Fig. 5, panel B). An intermediate (r.t. 23.1 min, method A) formed in proportion yields as high as .06 min, respectively, method C). By contrast reduced amounts of diketopiperazine cyclo-Tyr(Me)-Tyr(Me) formed confirming that the starting material lifetime was not sufficient for it to behave as a polymerization initiator leading to a dipeptide mixed anhydride prone to cyclization 27 . Under these conditions involving the presence of HCO 3</p><!><p>, the polymerization into peptides thus proceeds through the NCA rather than directly from the starting material. An NCA intermediate was also observed to form rapidly at pH 7.5 in 100 mM MOPS buffers in the presence of added HCO 3 2 (Supplementary Information, Fig. S1). This behaviour indicates that the formation of long peptides from adenylates reported in the literature 9,10 results probably from the polymerization of NCAs rather than from that of adenylates. The conversion of aminoacyl adenylates into NCA in the presence of CO 2 /HCO 3 2 was investigated starting from the Tyr(Me) derivative 1c (Supplementary Information, Fig. S2). The conversion of 1c into NCA was observed to proceed with rates similar to that observed for mixed anhydride 1b. The release of AMP (r.t. 1.5 min, method A) accompanying the formation of NCA 3 could be detected by HPLC allowing the reaction to be monitored at 50 mM concentrations of reactant 1c (r.t. 6.8 min, method A). The lifetime of the adenylate decreased with increasing concentrations of CO 2 /HCO 3 2 (t 1/2 , 80 min, ,25 min, and ,2 min at pH 6.5 in N 2 -flushed buffer, air equilibrated buffer and in the presence of 500 mM HCO 3 2 , respectively). At pH 7.5 the lifetime of adenylate 1c was reduced to less than 1 min in the presence of 500 mM HCO 3 2 , which means that this mixed anhydride is likely to be converted into NCA within a few seconds at concentrations of CO 2 /HCO 3 2 above 2 mM and at pH value close to neutrality, which are representative of the present day ocean or physiological fluids. It is worth noting that this lifetime is not sufficient for peptides to be significantly formed by a direct reaction with adenylate so that any observation of peptide products under these conditions results for the most part from the intermediacy of NCAs.</p><p>At pH 4, the hydrolysis of mixed anhydride 1b was much slower (t 1/2 5 ca. 550 min) and CO 2 catalysis was not observed (Supplementary Information, Fig. S3). This result is consistent with the results obtained by Kluger from alanyl ethyl phosphate 24 . The protonation of the amino group of 1b increases the electrophilic character of its acyl group and then the rates of nucleophilic attack, but it also prevents any possibility of reaction with CO 2 according the pathway of Fig. 3a. The hydrolysis of the acetylated mixed anhydride 2b was indeed observed to be slower (t 1/2 , 950 min at pH 6.5) and was not affected by addition of 10 mM NaHCO 3 (Fig. 6) in a way consistent with this explanation and with previously reported analyses 22 . However, it is important to emphasize that the CO 2 -catalyzed pathway does not only constitute a process leading to the deactivation and the hydrolysis of mixed anhydrides since peptide formation can be improved significantly by this means. As a matter of fact, with regard to peptide formation, the prevalence of the NCA pathway was demonstrated by studying the model reaction of 1 mM mixed anhydride 1b with 5 mM glycinamide either in a nitrogen-flushed sample or in the presence of 2 mM NaHCO 3 (Fig. 7). Importantly, less than 2 min were sufficient for the starting material to be exhausted in the presence of carbonate, whereas CO 2 removal increased the reaction times to much higher values (t 1/2 , 50 min) and reduced the final yield in dipeptide (Fig. 7). This reaction remained faster than that observed for the acetylated mixed anhyd-ride 2b (t 1/2 , 260 min) unable to undergo the conversion into NCA, but that will be demonstrated below to partly undergo cyclization into 5(4H)-oxazolones. These experiments carried out using glycinamide for mimicking a growing peptide chain show that the polymerization of adenylates and other aa-PEMA is improved in the presence of CO 2 by the occurrence of the NCA pathway owing to both the higher reactivity of the latter intermediate and its ability to suppress diketopiperazine formation.</p><p>The interconversion of 5(4H)-oxazolones and acyl-aa-PEMA and peptidyl-PEMA. The reaction of Ac-Tyr(Me)-OH-derived oxazolone 4 in methyl phosphate-buffered aqueous solution (pH 6.5) at 20uC was monitored by HPLC and compared with the hydrolysis of mixed anhydride 2b in MES buffers (Fig. 6). Comparable rates were observed and the intermediate of the 5(4H)-oxazolone 4 reaction was identified in situ by HPLC-ESI-HRMS (negative mode, calcd for C 13 H 17 NO 7 P 2 , 330.0743; found 330.0747) as the mixed anhydride 2b. A similar behaviour was observed from a reaction of inorganic phosphate (Supplementary Information, Fig. S5). The hydrolysis of mixed anhydride 2b was monitored by HPLC at 20uC in buffered solutions (Fig. 6). The reaction was also carried out in D 2 O to detect any hydrogen/ deuterium exchange resulting from the transient formation of 5(4H)-oxazolone 20,28 and compared to the product of a similar reaction of pure oxazolone 4 (Table 1). The values obtained demonstrate the occurrence of an intramolecular pathway already suspected from the higher rate of conversion of acylated aa-AMPs compared to simple acyl-adenylates 29 . At pH values below 5, the hydrolysis of anhydride 2b (Supplementary Information, Fig. S4) has been observed to become faster in a way similar to the observation made by Lacey's group for Ac-Phe-AMP 22 . The identification of an intramolecular pathway made in the present work strongly suggests that the acid catalysis of acyl-aa-PEMA hydrolysis is the consequence of a facilitated cyclization from a good neutral phosphate leaving group. However, the absence of H/ D exchange from the reaction of neither acyl-aa-PEMA 2b nor 5(4H)-oxazolone 4 at this pH (Table 1) prevented any determination of the actual pathway of hydrolysis of mixed anhydride 2b.</p><p>Similarly, we analyzed the degree of D/H exchange during the reaction of 2b with L-Ala-NH 2 in D 2 O at pH 6.5 (Table 1). The observation of a partial deuteration of the two diastereoisomers of the dipeptide product demonstrates that even when a better nucleophile is present, the a-proton is exchanged to a significant extent before the subsequent reaction of the 5(4H)-oxazolone takes place. The fast reaction of acyl-aa-AMP 29 and other acyl-aa-PEMA results therefore, at least for a noticeable part, from a transient conversion into 5(4H)-oxazolones. Interestingly, the different degrees of deuteration of the two diastereomers indicate that the intramolecular path of Fig. 3b has a higher stereoselectivity as compared to the direct path (the reactants 2b and 4 were prepared under a racemic form 28 ).</p><!><p>As regards aa-PEMA reactions, it is noteworthy that CO 2 catalysis proceeds through a pathway involving induced intramolecularity 30 . This kind of process shares one of the most important components of enzymatic activity, which corresponds to the utilization of binding energy to non-reacting portions of the substrate to bring about catalysis 31 . It was also proposed to constitute the easiest path for enzyme evolution under the name of uniform binding 32 and is moreover necessary for enzymes to exceed a physical limit 33 . Induced intramolecularity has also been used to drive highly stereoselective catalysis in organic synthesis 34,35 . The efficiency of this kind of catalysis relies on the rates of intramolecular reactions 36 . Carbon dioxide present at total concentrations of ca. 30-40 mM in pH 6.5 solutions equilibrated with air (as deduced from the Henry's coefficient of CO 2 37 and the pK a of carbonic acid) brings about a rate increase sufficient to render the catalytic pathway largely predominating, which is remarkable by considering a simple three-atom molecule compared to the efficiency of enzymes 38 . The ease of formation of 5-membered cycles from a-amino acid mixed anhydrides is also demonstrated by the conversion of acyl-aa-PEMA into 5(4H)-oxazolones.</p><p>These experiments demonstrating that the NCA path is prevailing at pH values close to neutrality in solutions equilibrated with air at present atmospheric levels of CO 2 (ca. 0.04%) suggest that the pathway must be overwhelming in natural environments with higher contents. The experiments at 2 mM HCO 3</p><p>2 are representative of present day ocean total concentration of dissolved carbonate 39 showing that the lifetime of aa-PEMA is expressed in tens of seconds in these media at pH 7.5. In biological media, with total carbonate concentrations approaching or exceeding 10 mM, the lifetime of mixed anhydrides would be even shorter. The early atmosphere had a CO 2 content that remains poorly constrained 40 but values similar to the present atmospheric levels 41 , or representing up to hundred times this value 40,42 , are often considered. Under these conditions, aa-PEMAs would be rapidly converted into NCA before any direct conversion into peptides could take place, which discards the earlier proposed contribution of aa-AMPs in the formation of prebiotic peptides [7][8][9][10][11] . Moreover, a less efficient polymerization ability of aa-PEMA and the diketopiperazine side-reaction make them improbable peptide precursors. The possibility that a very low content of CO 2 in the atmosphere could have transiently permitted mixed anhydrides to be stabilized 23 is made unlikely because it would have also required a very efficient removal of the most part of CO 2 in the whole ocean ($2 mM in HCO 3</p><p>2 ). On the contrary, the development of the activation pathway leading to translation must have occurred in an environment in which the role of NCA was unavoidable rather than in a local environment in which the mixed anhydrides were preserved from the presence of CO 2 and HCO 3</p><p>2 by any kind of geochemical processes. NCA can be considered not only as intermediates of the degradation pathway of adenylates but also as precursors of any kind of aa-PEMA mixed anhydrides including adenylates as well as precursors of peptides through a pathway suppressing diketopiperazine side-reaction. From this point of view, the catalysis by carbon dioxide may lead to a fast exchange among different energy-rich species capable of linking activated amino acids to phosphorylating species. This distribution of energy in a reaction network, that may have anticipated the role of ATP as an energy currency, ensured a global far from equilibrium situation that was essential even at early stages of chemical evolution 43 www.nature.com/scientificreports and nucleotide chemistries 44 the CO 2 -catalyzed pathway may then constitute a key-element in the systemic integration of the two subsystems 45 .</p><p>The fast conversion of adenylates, and more generally mixed anhydrides aa-PEMAs, into NCAs at low concentrations of CO 2 in water questions the way through which the biochemical amino acid activation evolved. As a matter of fact, aa-AMPs, possibly produced from ATP through ribozyme activity 46 , would rapidly be converted into NCAs impeding the evolution of translation. Conversely, the catalytic activity of aaRSs might have evolved by acting on the thermodynamically favourable reverse reaction of aa-AMPs (formed spontaneously from NCAs) as a primitive pathway to produce ATP 2,3 . One could argue that the NCA pathway of Fig. 3a is still active in living cells but this speculation is not supported by any experimental data. However, the mechanism of pretransfer editing of misactivated aaRSs (through which adenylates are hydrolyzed) remains uncertain 47 . Any possible release of adenylates from the active site to solution 48 during this step would lead to the formation of the corresponding NCA within seconds. Whatever NCA is actually or not a biochemical metabolite, the present results indicate that living organisms probably had to limit the importance of the release of adenylates into solution after translation evolved since a conversion into NCA would certainly lead to random aminoacylation of pending amino groups likely to be harmful to protein functional integrity. From this point of view, the N-formylation of methionine needed to initiate ribosomal peptide synthesis in bacteria might be considered as a remnant of a period in which NCA could be released in the cytoplasm. Therefore, we conclude that the potential formation of NCAs at least influenced the development of the translation apparatus and that of the aaRS family of enzymes in order to avoid random aminoacylation and that the NCA pathway must be taken into account in evolutionary studies.</p><p>Our analyses confirm the observations made by Lacey that CO 2 is a very efficient catalyst for the conversion of adenylates. However, taking into account the probable role of NCAs and the diversity of processes made available through their intermediacy leads us to the very different conclusion that the process could be favourable to the development and evolution of life rather than solely detrimental to the role of adenylates as intermediates of peptide formation. It is also worth noting that acyl-aa-PEMA that were considered by Lacey as blocked equivalents of aa-AMPs 22,23 does actually not constitute models of the reactivity of their parent compounds since they also undergo a spontaneous cyclization into 5(4H)-oxazolone. The transient formation of 5(4H)-oxazolone intermediates may be responsible for their efficiency in peptide formation 20 . The mixed anhydrides formed from free amino acids as well as peptide segments turn out to constitute unlikely precursors of peptides since their reactions are actually preceded by a very efficient cyclization into uncharged intermediates that thus constitute better electrophilic agents. This observation can be related to the evolutionary advantage of phosphate derivatives 49 that is partly related to their negative charge reducing spontaneous hydrolytic degradation with respect to their enzymepromoted reactions. From this perspective, their involvement required specific and efficient catalysts. However, the fact that NCA and 5(4H)-oxazolone also constitute precursors of mixed anhydrides through spontaneous processes provides a potential path through which these intermediates may have led for example to aminoacyl esters of RNA at predisposed locations 16,23,50 .</p><!><p>Reagents and solvents were purchased from Bachem, Sigma-Aldrich, or Euriso-Top and used without further purification. Starting materials and products samples were prepared according to standard procedures and characterized by 1 H, 13 C and 31 P NMR spectrometry and HRMS (Supplementary Information). NMR analyses were performed on a Bruker Avance 300 apparatus. HPLC analyses were performed on a Waters Alliance 2690 system with a photodiode array detector 996 using a Thermo Scientific BDS Hypersil C18 5 mm 2.1 3 50 mm column; mobile phase: A: H 2 O 1 0.1% TFA, B:CH 3 CN 1 0.1% TFA; flow rate: 0.2 mL/min and two different gradients; method A: 0 min (5% B), to 15 min (15% B), 25 min (60% B) and 26 min (100% B); method B: 0 min (5% B), to 10 min (20% B), 11 min (100% B). HPLC-ESI-MS analyses were carried out on a Waters Synapt G2-S system connected to a Waters Acquity UPLC H-Class apparatus equipped with a Acquity UPLC BEH C18, 1.7 mm 2.1 3 50 mm column; method C: A: H 2 O 1 0.01% formic acid, B: acetonitrile 1 0.01% formic acid; flow rate: 0.5 mL/min; linear gradient 0% to 100% B over 3 min.</p>

Scientific Reports - Nature

Electrochemical Proton Intercalation in Vanadium Pentoxide Thin Films and its Electrochromic Behavior in the near‐IR Region

This work examines the proton intercalation in vanadium pentoxide (V 2 O 5 ) thin films and its optical properties in the near-infrared (near-IR) region. Samples were prepared via direct current magnetron sputter deposition and cyclic voltammetry was used to characterize the insertion and extraction behavior of protons in V 2 O 5 in a trifluoroacetic acid containing electrolyte. With the same setup chronopotentiometry was done to intercalate a well-defined number of protons in the H x V 2 O 5 system in the range of x = 0 and x = 1. These films were characterized with optical reflectometry in the near-IR region (between 700 and 1700 nm wavelength) and the refractive index n and extinction coefficient k were determined using Cauchy's dispersion model. The results show a clear correlation between proton concentration and n and k.

electrochemical_proton_intercalation_in_vanadium_pentoxide_thin_films_and_its_electrochromic_behavio

Introduction<!>Results and Discussion<!>Conclusions

<p>Nowadays there is a high demand for materials with welldefined, tunable optical properties to realize new optical modulation devices, e. g. optoelectronic switches and phase or intensity modulators [1,2] as optical data processing is getting more and more important for communication and information technologies. Silicon photonic integrated circuits (PICs) are getting more and more important in this field in which the near-IR region is of great interest. [3] Recently, self-holding optical actuators for silicon photonic waveguides have been proposed, these activators can maintain the switching state without a constant supply of energy. Different types of materials have been exploited which comprise phase change, [1] insulator-metal phase transition, [4] memristor-like plasmonic structure [5] and electrochromic materials. [6] Electrochromic materials are able to reversibly change their optical properties, i. e. refractive index and absorption coefficient by inserting charge which causes redox reactions in the material. [7] Intercalation is commonly done using lithium cations or protons since excellent reversible coloration of the materials in the visible range can be effected with these ions. Some wellknown electrochromic oxides for the visible range are tungsten trioxide (WO 3 ), [8,9] niobium pentoxide (Nb 2 O 5 ), [10] molybdenum trioxide (MoO 3 ) [11] and vanadium pentoxide (V 2 O 5 ). [12,13,14] V 2 O 5 has been used in a broad field of different applications, for example as catalysts in oxidation reactions, [15] in sensors as gas sensing material, [16] as insertion electrodes for lithium ion batteries [17] and as already mentioned above for smart window applications as electrochromic material. There, the electrochromic effect has only been studied with lithium intercalation and in the visible range of the electromagnetic spectrum. In previous works we examined the electrochromic properties of lithiated V 2 O 5 in the near-infrared (near-IR) region. [18] Nevertheless, there is only few information available regarding insertion of protons. Wruck et al. [14] and Ottaviano et al. [13] describe that the insertion of a proton is possible in V 2 O 5 and that it is similar to Li + -intercalation but they only give details about the lithiation.</p><p>In 1998, a solid-state proton battery was introduced by Pandey et al. [19] where vanadium pentoxide was used as cathode material within a mixture of carbon and lead dioxide. Therefore, it is already known that V 2 O 5 may serve as a reversible proton intercalating material.</p><p>Liu et al. presented insertion of hydrogen in the form of intercalated protons accompanied by excess electrons in the conduction band of vanadium pentoxide by treating it in a hydrogen containing atmosphere. [20] In their work, they investigated the optical properties of V 2 O 5 in the visible range in dependence of the hydrogen content in the gas mixture in a Pd/V 2 O 5 device. Thus, they showed that vanadium pentoxide irreversibly changes in a first formation cycle of insertion and extraction of hydrogen but then remains optically passive for the subsequent cycles. By electrochemical insertion of protons, V 2 O 5 has never been tested before as an optically active and tunable material.</p><p>In 2013, Malini et al. presented electrochromism of thin films of CeVO 4 , a mixed oxide sythesized from cerium dioxide and vanadium pentoxide. [21] They inserted and extracted protons electrochemically via hydrochloric acid containing electrolyte and measured the transmittance in the ultraviolet and visible range in-situ. They showed that there is a decrease of the transmittance with rising H + content, giving a clear hint that V 2 O 5 can act as an electrochromic active material.</p><p>To conclude, previous research has been focused on characterizing the electrochromism of vanadium pentoxide mainly in relation to lithiation, there is still a lack of information about the electrochromic properties of pure vanadium oxide with electrochemical proton intercalation especially in the near-IR region. Accordingly, it is of interest to investigate the electrochromic behavior as a function of concentration of intercalated protons.</p><p>Therefore, we prepared vanadium pentoxide thin films via direct current magnetron sputter deposition and investigated in detail their electrochemical and electrochromic properties in the near-IR region in terms of proton insertion and extraction.</p><!><p>The sputtered and annealed V 2 O 5 films all exhibit an orthorhombic structure well matching literature data. [22] An x-ray diffraction (XRD) spectrum of an as prepared V 2 O 5 film (600 nm thickness) on bare silicon is presented in Figure 1. No representative reflex of vanadium dioxide (VO 2 ) [23] can be found so we conclude that all produced thin films consist of pure crystalline V 2 O 5 .</p><p>In a next step we investigated the electrochemical performance in a proton containing electrolyte. Therefore, we have chosen trifluoroacetic acid in a solution of tetrabutylammonium perchlorate in propylene carbonate for the following reasons: In contrast to many other acid solutions, this composition does not dissolve the V 2 O 5 thin film. Nevertheless, trifluoroacetic acid is a strong acid with a pK a -value of 0.23, [24] its solubility in various organic solvents is excellent and in our case it is serving as proton source. Tetrabutylammonium perchlorate is needed as conducting salt to guarantee good conductivity of our electrolyte system, whereupon especially the cation is too big to be intercalated in the structure of V 2 O 5 . As solvent propylene carbonate was chosen, because it is a well-known solvent for electrolytes in lithium-ion batteries, [25] exhibiting good electrochemical stability.</p><p>According to Ottaviano et al. [13] and Tong et al., [26] in an electrochemical experiment the intercalation and deintercalation mechanism of protons in V 2 O 5 can be described as</p><p>While intercalating a proton, V 5 + is reduced to V 4 + and the proton should coordinate with the oxygen atom to form a hydroxyl species.</p><p>Figure 2 shows a cyclic voltammetry (CV) measurement of a V 2 O 5 thin film (1.2 μm thickness) in the voltage range between 0.1 and 1.3 V versus silver chloride electrode (Ag j AgCl) and a scan rate of 1 mV • s À 1 . A well-defined reduction peak and two oxidation peaks are observable, which indicate that at least the deintercalation of protons is taking place in a two-step mechanism, whereas a second peak in the reduction area could not be observed.</p><p>The overall coulombic efficiency of the system is 89 %, indicating that the intercalation and deintercalation process is not fully reversible. This assumption is supported by the fact that the peak current densities are decreasing with repeated cycling. Regarding the relatively high number of protons For this purpose, a 300 nm thin film of V 2 O 5 was investigated with chronopotentiometry. The film was reversibly loaded with � 0.5 μA to a state corresponding to H 0.05 V 2 O 5 and back to H 0 V 2 O 5 for 40 cycles. After a total of five formation cycles the complete system reacts reversible by constantly ranging between 1.15 V (deintercalated, x = 0) and 0.55 V (intercalated, x = 0.05) vs. Ag j AgCl, as shown in Figure 3. By this means, repeated cycling of vanadium oxide films in proton conducting electrolyte has proven to be fully reversible in a well-defined range of x at least to x = 0.05. Comparable results can also be obtained for higher constant currents up to 50 μA, which enables faster switching between different values of x (in the case of a 300 nm thick 1 × 1 cm film less than one minute), making H x V 2 O 5 a promising system for switches or tuning devices. Graphs of chronopotentiometry measurements done with higher currents than 0.5 μA (5 μA and 50 μA) can be found in the supplementary data.</p><p>After proving the electrochemical functionality of the H x V 2 O 5 system, the optical behavior of the thin films was investigated as a function of the proton content. Therefore, chronopotentiometry was used with a constant current of � 0.5 μA for a well-defined period of time to set an accurate value of x, before and after each experiment the cell voltage was measured until open circuit potential (OCP) was reached (OCP values see Table 1). Between every intercalation and deintercalation the cell was dismounted and the thin film was cleaned with ethanol. The sample was investigated with reflectance spectroscopy before and after the intercalation and additionally after the deintercalation.</p><p>Figure 4 shows two chronopotentiometry graphs, one for the intercalation and one for deintercalation. The belonging reflectance graphs of the same sample with x = 0, x = 0.1 and deintercalated back to x = 0 are also shown in the same figure. It is visible that there is a definite deviation between intercalated and the deintercalated state. Furthermore, a very good reversibility of the intercalation and deintercalation process is evident since the reflectance spectrum after the deintercalation step matches very well to the spectrum measured before intercalation. This also proves the reversibility of the electrochemical process up to x = 0.1.</p><p>Not only the reversibility, but also the stability or the maintaining of the optical properties of the intercalated state is of great importance for use as an optical switch. Therefore, a sample of H 0 V 2 O 5 was intercalated to H 0.1 V 2 O 5 , the sample was cleaned and the reflectance was measured directly afterwards. Then, the sample was allowed to rest in ambient conditions, whereas the reflectance was measured after six, nine and twenty days. In Figure 5 the reflectance curves are shown and it is observable that the reflectance changed a little bit between the freshly intercalated sample and the curve measured after six days. One reason for this could be the non-ideal cell-setup, as it was not possible to contact the whole area of the V 2 O 5 thin film with liquid electrolyte without directly contacting the current collector (see also in Figure 7). It is assumed that shortly after intercalation the layer is not yet in total equilibrium. This is also the reason why we decided not to examine the time response of the thin films with additional chronoamperometric measurements. Nevertheless, the reflectance curves after six, nine and twenty days are in good accordance to each other, no change in the reflectivity can be seen on further storage. Thus, the intercalated films are stable for the whole period of 20 days and are able to maintain their optical properties in the intercalated phase.</p><p>In a next step we investigated if a systematic trend in the change of the optical properties can be seen by varying the concentration of protons. For this reason, the sample was intercalated to a certain amount of x in several steps from x = 0 to x = 1. Between the intercalations the sample was always taken out of the electrochemical cell and the reflectance was measured. We discovered that there is a clear correlation between the number of protons intercalated in the V 2 O 5 thin film and the optical properties which can be seen in Figure 6 (for small proton concentrations, x = 0.02 to x = 0.08 see the supporting information).</p><p>Overall, the reflectance decreases with increasing proton concentration. In addition, the maxima and minima of the reflectance curves are shifted to lower wavelengths on the xaxis indicating a change in the refractive index n value with proton intercalation. The intercalation and deintercalation process is reversible up to a proton concentration of 0.1 < x < 0.2. With higher amounts, the reflectance graph of the proton free state (x = 0) cannot be obtained anymore and is shifted to lower reflectance values (reflectance graphs are shown in the supporting information). Figure 7 gives an impression of the irreversible change of the optical properties in the visible range of the spectrum, showing photographs of a H x V 2 O 5 thin film sample without protons and with a proton concentration of x = 0.2 and x = 0.6. There is a clear color change observable in the whole area where the sample was in direct contact with the electrolyte. It was impossible to reach the initial state again. In the following the focus is concentrated on lower amounts of x, namely in the range between x = 0 and x = 0.2 to stay in the reversible range of intercalation, which is especially interesting for optical devices.</p><p>To determine the refractive index n and the absorption coefficient k of the measured samples, Cauchy's dispersion model was used according to equations (3) and ( 4). Figure 8 shows the Cauchy fit for a reflectance measurement done on a V 2 O 5 thin film with a proton concentration of x = 0.1. As can be seen the fit accuracy is excellent and reaches a goodness of fit value higher than 99 %. The received graphs for the n and k values dependent on the wavelength are presented in the inset. It is observable that the values for the refractive index n are constantly decreasing in a wavelength range between 700 and 1700 nm while the graph of the absorption coefficient k exhibits a maximum at 850 nm. At higher wavelengths, the values are also decreasing. According to this example of fitting all other measured reflectance graphs were evaluated.</p><p>The obtained graphs for n and k are summarized in Figure 9, where it can be seen that there are just results for samples with a number of intercalated protons between x = 0 and x = 0.2. Beyond that no additional values for n and k could be obtained. It is noticeable that the limit of reversibility was determined between 0.1 < × < 0.2 and the reflectance curves can only be fitted up to x = 0.2, whereas the goodness of fit is dramatically decreased for x = 0.2. The shape of the obtained curves of n and k in dependence of the wavelength is comparable for all values of x and a clear trend can be observed. The n curves are decreasing with increasing wavelengths. A maximum for k is observable before the values are also decreasing with increasing wavelength for all obtained data curves of different intercalated amounts of x. In general, the refractive index n is decreasing with higher amounts of x incorporated in the structure of V 2 O 5 and the absorption coefficient k is increased.</p><p>This trend can be seen more clearly by plotting the n and k values against the x values of H x V 2 O 5 for different wavelengths (cf. Figure 10). There, a nearly linear behavior of both n and k is clearly visible. For comparison with literature values of e. g. lithiated V 2 O 5 , it is helpful to determine ~n/ ~x and ~k/ ~x at a distinguished wavelength. According to [18] ~n/ ~x is À 1 and ~k/ ~x is 1.43 for Li x V 2 O 5 at a wavelength of 1550 nm. For our investigated system H x V 2 O 5 ~n/ ~x is round about À 0.93 and ~k/ ~x round about 0.15 at the same wavelength. Comparing both systems, the change in n is a little bit lower for H x V 2 O 5 , but in the same order of magnitude, whereas the change in k is smaller for H x V 2 O 5 .</p><!><p>V 2 O 5 thin films were successfully prepared with dc magnetron sputter deposition and following annealing. It has been proven that V 2 O 5 is an excellent material for electrochemical proton intercalation and that the system H x V 2 O 5 can be used as an electrochromic cathodic material in the near-IR region. The reflectivity and therefore the n and k values of the material can be influenced systematically with proton insertion. The behavior of H x V 2 O 5 is comparable with Li x V 2 O 5 [18] although the absolute change of the optical constants, especially the absorption coefficient k, is smaller with proton intercalation than with lithiation. However, one advantage over Li x V 2 O 5 is that it is easier to handle in non-inert environments, as there is no need for moisture-sensitive materials like metallic lithium. To conclude, H x V 2 O 5 is a material which is excellent for use in future optical devices.</p>

Chemistry Open

Photochemical Tyrosine Oxidation with a Hydrogen-Bonded Proton Acceptor by Bidirectional Proton-Coupled Electron Transfer

Amino acid radical generation and transport are fundamentally important to numerous essential biological processes to which small molecule models lend valuable mechanistic insights. Pyridyl-amino acid-methyl esters are appended to a rhenium(I) tricarbonyl 1,10-phenanthroline core to yield rhenium\xe2\x80\x93amino acid complexes with tyrosine ([Re]\xe2\x80\x93Y\xe2\x80\x93OH) and phenylalanine ([Re]\xe2\x80\x93F). The emission from the [Re] center is more significantly quenched for [Re]\xe2\x80\x93Y\xe2\x80\x93OH upon addition of base. Time-resolved studies establish that excited-state quenching occurs by a combination of static and dynamic mechanisms. The degree of quenching depends on the strength of the base, consistent with a proton-coupled electron transfer (PCET) quenching mechanism. Comparative studies of [Re]\xe2\x80\x93Y\xe2\x80\x93OH and [Re]\xe2\x80\x93F enable a detailed mechanistic analysis of a bidirectional PCET process.

photochemical_tyrosine_oxidation_with_a_hydrogen-bonded_proton_acceptor_by_bidirectional_proton-coup

Introduction<!>Experimental<!>Results and Discussion<!>Conclusions

<p>Tyrosyl radicals are key intermediates in a wide variety of energy conversion processes.1 Tyrosine oxidation has been a subject of significant focus in the literature. Specifically, the proton transfer (PT) component of tyrosine oxidation has been of particular interest both in natural2 and model systems.3,4 Tyrosyl radicals are particularly essential to a biology as diverse as that derived from photosystem II,5,6 cytochrome c oxidase7 and ribonucleotide reductase (RNR).8 We have developed biophysical tools that permit tyrosine radicals (Y•) to be photogenerated in biological systems with emphasis on RNR.9 These photoRNRs have been invaluable for deciphering the PCET mechanism by which radicals are generated and transported in this enzyme.10–13 The value of Y• photogeneration in the development of new biophysical tools for radical enzymology14 provides an imperative for a mechanistic understanding of tyrosine photooxidation.</p><p>Formation of tyrosyl radical from tyrosine requires both deprotonation and one-electron oxidation of the native amino acid. Stepwise mechanisms are thermodynamically demanding and result in high-energy intermediates, implicating a PCET mechanism for this reaction. In natural systems, PCET processes occur with exquisite sensitivity and remarkable selectivity by separating the PT coordinate form the ET coordinate, i.e. by installing a bidirectional PCET pathway. This requirement is due to the relatively large mass of a proton in comparison to an electron. Hence, whereas the electron can tunnel over long distances, the proton cannot;15,16 the control of the PT coordinate over a different length scale to that of the electron is essential for the kinetic feasibility of a PCET reaction and the attendant opportunity to exercise kinetic control over the reaction.</p><p>To this end, well-defined, pre-organized PT pathways are essential for the generation of Y•, and the incorporation of a well-defined proton acceptor is essential for the generation of Y•. A variety of studies have demonstrated the importance of hydrogen bonding on the kinetics of phenol oxidation by PCET.17–22 Herein, we report the use of a bidirectional scaffold for the study of tyrosine oxidation that enables the independent variation of the driving force for both proton transfer (PT) and electron transfer (ET), which are critical determinants of the PCET governing tyrosine photooxidation.23 A pyridyl-amino acid-methyl ester (Py–AA) has been appended to a rhenium(I) tricarbonyl 1,10-phenanthroline core [Re] to yield rhenium–amino acid complexes with tyrosine ([Re]–Y–OH) and phenylalanine ([Re]–F). A PCET network is self assembled by association of [Re]–Y–OH to a base, which is both well-defined and easily varied to permit the PT to be examined in concert with ET to the photoexcited [Re] core. We find that the efficacy of radical generation depends intimately on the strength of the associated base.</p><!><p>The [Re]–AA compounds are synthesized via the route shown in Scheme 1. Syntheses were accomplished by methodologies described in the ESI and products were spectroscopically and analytically characterized in detail to ensure compound identification and purity; these data are also given in the ESI.</p><p>Steady-state spectroscopy was performed using dilute solutions of [Re]–AA (AA = Y or F) complexes. Emission titrations were performed by adding sequential volumes of pyridine (neat) or imidazole (1.0 M in dichloromethane) to a sample of [Re]–Y–OH (50 μM in dichloromethane). Equilibrium constants were determined for the association of [Re]–Y–OH and pyridine or imidazole by monitoring emission quenching as a function of base added. Fitting to determine the equilibrium association constants for [Re]–Y–OH binding to bases was performed according to published methods.24</p><p>Time-resolved absorption and emission experiments were completed by nanosecond flash photolysis, which were performed using a system which was significantly modified from one previously described.25,26 Emission lifetimes were measured for both [Re]–Y–OH and [Re]–F as a function of pyridine and imidazole concentrations. Full experimental procedures, detailed description of experimental apparatus, a complete explanation of data analysis, and a derivation of the rate law for excited-state quenching are provided in ESI. Errors reported for rate constants and equilibrium constants measured are two standard deviations, as calculated from the standard error of the corresponding fit.</p><!><p>The [Re]–AA compounds shown in Scheme 1 were developed as a modular platform where relevant driving forces could be varied for the study of PCET. A similar method of their synthesis has recently been reported.27 The PCET network is self assembled by addition of base (pyridine or imidazole) to a solution of [Re]–Y–OH in dichloromethane. as shown in Fig. 1. This approach for forming the bidirectional PCET network provides a modular and versatile platform for studying tyrosine oxidation. The absence of a hydrogen bond in the [Re]–F system prevents assembly of the PCET network, and hence this system provides a control for kinetic measurements associated with the photogeneration of Y•. Moreover, because F is redox inactive, any disparity in excited-state lifetimes between [Re]–F and [Re]–Y–OH is directly attributable to reactivity at the tyrosine phenol.</p><p>Ground state absorption and steady state emission spectra of [Re]–Y–OH and [Re]–F (Fig. 2) are nearly identical to one another and dominated by the electronic properties of the [Re] centre. Hydrogen-bonded association between the tyrosine phenol proton of [Re]–Y–OH and base is evident from emission spectra. In dichloromethane solution, [Re]–Y–OH is highly emissive from a 3MLCT excited state (3[ReI]*). As shown in Fig. 3, emission from [Re]–Y–OH is quenched significantly upon addition of base (pyridine or imidazole). The equilibrium constants, Kassoc, for association between [Re]–Y–OH and the added base may be measured from the concentration dependence of this quenching. Equilibrium constants were measured for both pyridine (Kassoc = 16 ± 2 M−1) and imidazole (Kassoc = 157 ± 13 M−1) by fitting the concentration dependence of emission intensity as previously reported.24 The stronger binding to imidazole than to pyridine is in accordance with the relative aqueous pKa values. These equilibrium rate constants correspond to reaction free energies of ΔGo = −6.9 kJ mol−1 and ΔGo = −12.5 kJ mol−1.</p><p>Transient absorption spectra indicate that the 3[ReI]* excited state reacts with base by electron transfer. The transient absorption spectra and single-wavelength kinetic data for solutions of [Re]–Y–OH and [Re]–F in dichloromethane are shown in Fig. 4 (top) in the absence of base. Transient spectra exhibit absorption features as expected for compounds of this type.28 Significant growth features are observed at 300 and 450 nm. The transient signals decay monoexponentially; 3[ReI]* excited state in [Re]–Y–OH is slightly shorter than [Re]–F. This is consistent with oxidation of tyrosine by the [Re]–Y–OH. The 3[ReI]* excited state is highly oxidizing and is of sufficient potential to oxidize base (Eo(ReI*/0) = 1.7 V vs. NHE)29. The given value was measured in acetonitrile and provides only an estimate for the present report where experiments are conducted in dichloromethane. Furthermore, previously reported electrochemical experiments show the onset of pyridine and imidazole oxidation at potentials of ~1.6 V30 and ~1.4 V31 vs. NHE, respectively. These values are consistent with oxidation by the 3[ReI]* excited state as well as the relative rates of oxidation for imidazole and pyridine.</p><p>When pyridine or imidazole is added to solutions of [Re]–F and [Re]–Y–OH, the TA signal of the 3[ReI]* excited state is considerably shortened, more so for imidazole than pyridine (Fig. 4, bottom). Concentrations of base were chosen to ensure that at least 95% of [Re]–Y–OH was bound. We note that a transient signal for photo-oxidized tyrosine is not observed, indicating that the intermediate does not accumulate owing to back electron transfer rate that is faster than the forward PCET rate constant for quenching.</p><p>In the absence of direct observation of a transient signal for base owing to fast back reaction, the excited state kinetics were further examined by transient emission spectroscopy. The quenching paths of 3[ReI]* for [Re]–F and [Re]–Y–OH are shown in Figure 5. In this model, we assume that the intrinsic decay processes of and bimolecular processes of [Re]–Y–OH and [Re]–Y–OH---Nbase cannot be distinguished in excited state decay profiles.</p><p>The excited-state reactivity of [Re]–F with base is straightforward; the emission will decay with intrinsic radiative and nonradiative process defined by k0 (= 1/τ0) or react with base by electron transfer, defined by the bimolecular quenching rate constant kq. In a Stern-Volmer process, the overall observed emission rate constant for reaction (= 1/τF) should follow the kinetics,</p><p>As predicted by eq. 1, Stern-Volmer plots are linear with increasing concentration of base. From the natural lifetime of in Fig. 4, bimolecular quenching rate constants kq = 3.2(6) × 105 M−1 s−1 and 3.1(6) × 107 M−1 s−1 for pyridine and imidazole, respectively. These data are summarized in Table 1.</p><p>The excited state decay processes of 3[ReI]* in the [Re]–Y–OH---Nbase assembly are richer. In addition to the natural decay of the 3[ReI*] excited state and its bimolecular quenching by base, two unique unimocular processes arise from electron transfer from Y–OH to 3[ReI]* and PCET. The excited state dynamics of the rhenium center should not be affected by the remote amino acid inasmuch as the it is not conjugated to the ligands of the the rhenium center. Hence k0 and kq, to a first approximation, should be the same for both systems. One possible exception to this approximation may be the difference in reduction potential of the bound base relative to base in solution; however, the large excess of free base is expected to dominate the kinetics of base oxidation by 3[ReI]*. Moreover, lacking the tyrosine phenol, Re–F cannot be oxidized by 3[ReI]*; oxidation of toluene (a suitable approximation to the phenylalanine sidechain) occurs at a potential greater than that of 3[ReI]* (1.8 V32 vs. NHE),. Accordingly, the Re–F center provides a reference for the ET process between the unassociated tyrosine and the excited rhenium center.</p><p>The kET rate constant is given by,</p><p>From the data in Fig. 4 and rate constants summarized in Table 1, a kET = 6.1 × 104 s−1 is determined. Whereas the kinetics associated with ET are straighforward, the PCET process is more complicated. The emission decay dynamics are modulated by the equilibrium between [Re]–Y–OH and Nbase. For this reason, a linear Stern-Volmer relation is not expected. Indeed, the base concentration dependence of the emission lifetime exhibits significant curvature as shown in Figure S1. The rate of excited-state decay for [Re]–Y–OH varies as a function of base concentration according to the following equation,</p><p>Detailed derivations of these rate laws (eqs. 1 and 2) are available in the ESI. The last term of the above equation accounts for the equilibrium between [Re]–Y–OH and base, and the PCET process enabled by that equilibrium. Although variations in the observed rate constants (k0, kET, kq) may occur as a result of hydrogen bonding to the tyrosine phenol, in the context of the final analysis, where the PCET rates exceed kET by a factor of 10 or 100, variation in kET is unlikely to cause significant interference with the calculation of kPCET. The only unkown variable in the above equation is kPCET. Fitting the observed rate constant for excited-state deactivation, kobs (as calculated from the emission lifetime), to the concentration of base furnishes kPCET = 4.1(6) × 105 s−1 and kPCET = 4.8(8) × 106 s−1 and for pyridine and imidazole respectively.</p><p>These PCET rates are comparable to those previously observed in unimolecular systems incorporating hydrogen bonding to phenols within bimolecular17 and unimolecular (tethered)18 frameworks. In these cases, the photoacceptor is ruthenium(II) tris(bipyridine), which is less oxidizing Re(I) polypyridyl excited states. Hydrogen bonding facilitates PCET by substantially decreasing the inner-sphere contribution to reorganization energy from the phenol and by introducing 'promoting' vibrational modes; the thermodynamic strength of the hydrogen bond has been observed to have a lesser impact on PCET rate enhancement.19 This is not the case here. The rateenhancement for phenol oxidation in [Re]–Y–OH---Nbase varies with the strength of the hydrogen bond as has also recently been observed in protein maquettes.33 The correlation may be more apparent for [Re]–Y–OH---Nbase owing to the imilarity of the hydrogen bond type formed by imidazole and pyridine. Previous studies on phenol photooxidations have emphasized hydrogen bonding networks that are formed from carboxylates where differences in the hydrogen-bonded adduct (e.g., six- or seven-membered rings formed in the two cases) likely introduces importatnt vibrational effects and the greater sensitivity to the vibrational modes of the hydrogen bonding network. When this complexity is removed, it appears that the PCET rate for photooxidation follows a more straightforward thermodynamic trend with the strength of the hydrogen bonds within the PCET network.</p><p>With respect to the observed variation of kPCET with respect to the proton acceptor pKa, previous work in a bimolecular system has shown that the as the strength base (proton acceptor) increases, faster rates of PCET are observed for phenol oxidation.34,35 In recent related work, the importance of hydrogen bond distance (rather than strength) on the rate of PCET reactions has been investigated.36,37 Although the present scaffold does not offer a way to directly control the proton transfer distance, the facile variation of hydrogen-bond strength that can be achieved with this scaffold may yield valuable insights of interest to this discussion.</p><!><p>The supramolecular assembly formed between a rhenium–tyrosine complex associated to base estableshes a network for the photogeneration of tyrosyl radical by a PCET mechanism. An equilibrium interaction between the tyrosine phenol proton and bases in solution provides a well-defined proton acceptor, simplifying analysis of PCET kinetics. The observed rates of tyrosine oxidation associated to base are consistent with those reported in the literature for intramolecular model systems. The approach affords for the self-assembly of a modular scaffold for the study of bidirectional PCET by simply varying the nature of the base. To this end, the approach reduces the complexity associated ith the synthesis of tethered networks.</p>

PubMed Author Manuscript

Overexpression of HDAC9 Promotes Invasion and Angiogenesis of Triple Negative Breast Cancer by regulating microRNA-206

Triple negative breast cancer (TNBC) is among the most aggressive breast cancer subtypes with poor prognosis. The purpose of this study is to better understand the molecular basis of TNBC as well as develop new therapeutic strategies. Our results demonstrate that HDAC9 is overexpressed in TNBC compared to non-TNBC cell lines and tissues. Furthermore, we show that HDAC9 overexpression is inversely proportional with miR-206 expression levels. Subsequent HDAC9 siRNA knockdown was then shown to restore miR-206 while also decreasing VEGF and MAPK3 levels. This study highlights HDAC9 as a mediator of invasion and angiogenesis in TNBC cells through VEGF and MAPK3 by modulating miR-206 expression and suggests that selective inhibition of HDAC9 may be an efficient route for TNBC therapy.

overexpression_of_hdac9_promotes_invasion_and_angiogenesis_of_triple_negative_breast_cancer_by_regul

Introduction<!>Breast cancer cell lines and culture<!>Tissue samples and immunohistochemical staining<!>Quantitative real-time RT-PCR<!>Construction of HDAC9 siRNAs and Transfection.<!>Matrigel Invasion Assay<!>Matrigel plug assay and hemoglobin assay<!>Statistical analysis<!>HDAC9 is overexpressed in TNBC and is inversely correlated with miR-206<!>Inhibition of HDAC9 blocks the invasion of TNBC cells in vitro<!>Knockout of HDAC9 inhibits the angiogenesis of TNBC tumors<!>HDAC9 regulates expression of VEGF and MAPK3 through miR-206 miRNA<!>Discussion

<p>Two classes of enzymes impact the acetylation state of histone proteins — histone acetyltransferases (HATs) and histone deacetylases (HDACs) [1, 2]. The HDAC family of enzymes is involved in various biological processes including transcriptional control, growth arrest, and cell death, particularly in tumor development and proliferation [3–7]. A number of HDAC inhibitors have been characterized that inhibit tumor growth in vitro and in vivo at amounts that have little to no toxicity [8–15]. The results from these studies and their proposed mechanisms indicate that HDACs are excellent targets for cancer treatment. However, clinical benefits of selective versus broad HDAC inhibitors are unknown and the appropriateness of inhibitor may depend on tumor HDAC enzyme expression, enzyme selectivity of the inhibitor, and the desired effects. Although there are several published studies [16, 17] and numerous ongoing clinical trials involving HDAC inhibitors [18], very little is known about the roles of individual HDAC enzymes, and a formal assessment of HDAC enzyme expression is not routinely done. Emerging data suggests that the currently used HDAC inhibitors may differ significantly with regard to target selectivity. In addition, the expression of HDAC enzymes may vary considerably between normal and tumor tissues and likely between different phenotypes of tumor tissues. HDAC9 is thought to regulate gene expression through epigenetic modulation of the chromatin structure by catalyzing the deacetylation of histone proteins [19]. HDAC9 is also known to target non-histone proteins, such as forkhead box protein 3, ataxia telangiectasia group D-complementing protein (ATDC), and glioblastoma 1 protein, which are members of pathways implicated in carcinogenesis [20, 21]. More recently, aberrant HDAC9 expression has been observed in several types of cancers, including medulloblastoma [21], acute lymphoblastic leukemia [22], glioblastoma [23], osteosarcoma [24], and breast cancer [25]. HDAC9 has been shown to promote the growth of these tumors.</p><p>Triple negative breast cancer (TNBC) is one of the most clinically aggressive subtypes of breast cancer and is associated with overall poor prognosis [26–28]. TNBC lacks expression of the estrogen receptor (ER), progesterone receptor (PR) or human epidermal growth factor receptor 2 (her2/neu) [29, 30]; as a result, conventional therapies targeting each of these receptors proves to be unsuccessful in TNBC. Therefore, there is a major unmet need to better understand the molecular basis of this type of breast cancer as well as develop new therapeutic strategies. Emerging studies have highlighted microRNAs (miRNAs) as critical mediators of tumorigenesis, as their involvement have been well-established across several types of cancers, including breast, prostate, ovarian, and head and neck cancers [31–34]. Previous work from our group identified miR-206 as a critical tumor suppressor in TNBC and showed that downregulation of miR-206 lead to an upregulation of VEGF, MAPK3, and SOX9, crucial drivers of invasion and angiogenesis [35]. In this study, we demonstrate that overexpression of HDAC9 promotes the invasion and angiogenesis in TNBC by directly modulating miR-206 and provides a selective basis for TNBC treatment.</p><!><p>The human breast cancer cell lines MDA-MB-231, MDA-MB-1739, HCC1395, MDA-MB-361, and SKBR3, were grown in RPMI1640 medium containing 10% FBS, 100 U/ml of penicillin sodium, and 100 µg/ml of streptomycin sulfate at 37 oC in a humidified atmosphere of 5% CO2. MCF-7, an ER-expressing breast cancer cell line, was cultured in DMEM medium containing 10% FBS plus 10 μg/ml of insulin. SKBR3 is a Her2/neu-expressing human breast cancer cell line, and MDA-MB-361 is positive for ER, PR, and Her2/neu. MDA-MB-231, MDA-MB-1739, and HCC1395 are all triple negative cell lines.</p><!><p>A formalin-fixed and paraffin-embedded breast cancer tissue array was obtained from US Biomax (Derwood, Maryland, USA). This is a breast cancer and matched metastatic carcinoma tissue array, including TNM and pathology grade, with ER, PR and Her-2 (neu) IHC results, 50 cases/100 cores. The sources and characteristics of archived breast tumor breast samples are summarized in Table 1. HDAC9 antibody (Abcam, Cat No. ab70954) was applied on slides at 1:500 and incubated for 1 hour at room temperature after deparaffinized, antigen-retrieved (DAKO, Cat No., S1699), and endogenous peroxidase block with 3% Hydrogen Peroxide (DAKO, Cat No., S2003,). Visualization and detection were established using DAKO EnVision+ Dual (mouse and rabbit) Link System-HRP (Cat No., K4061) with an incubation time of 30 minutes. The detailed staining procedure and semi-quantitative method of immunohistochemical staining of these tissue sections for HDAC9 are described in our previous paper [36] (DAKO, Cat No., K4061).</p><!><p>Regular and quantitative RT-PCR were performed following our previous protocol [37]. Primer sequences of miR-206, and U6 snRNA have been described in our previous report [35]. Primer sequences of HDAC9 are as follows: (GeneBank accession number BC152405), 5'- CAGCAACGAAAGACACTCCA-3' and 5'- CAGAGGCAGTTTTTCGAAGG-3'. SYBR Green quantitative PCR reaction was carried out in a 15 μl reaction volume containing 2× PCR Master Mix (Applied Biosystems) per our previous reports [38, 39].</p><!><p>We designed and purchased the small interfering RNA (siRNA) duplexes against HDAC9 (Genbank accession no., BC152405) from ThermoFisher Scientific (Grand Island, NY, USA). The target sequence of HDAC9 siRNA is 5′-UAAAAUCUUCCUGCCCACCdTdT-3′. The nonspecific control siRNA duplexes were purchased from ThermoFisher Scientific with the same GC content as HDAC9 siRNAs. The siRNA or control oligonucleotides were transfected into TNBC MDA-MB-231 cells at a final concentration of 100 nM with Lipofectamine 2000 per the instructions.</p><!><p>The invasion assay was performed by using a Matrigel invasion chamber from Corning Biocoat (Tewksbury, MA) as previously described [40]. 5×104 HDAC9 inhibitor-transfected or control oligonucleotide-transfected TNBC MDA-MB-231 cells were added into the top chambers. The Matrigel invasion chambers were then incubated for 20 hours in a humidified tissue culture incubator. Invading cells at the bottom was determined by counting the H&E-stained cells.</p><!><p>For the in vivo angiogenesis assay (Matrigel plug assay), 2X105 MDA-MB-231 cells were mixed with 0.5 ml of growth factor-reduced Matrigel (Corning, Tewksbury, MA) and implanted subcutaneously into the flanks of nude mice. The following day, six mice in each group were treated with 100 µg/kg HDAC9 siRNAs or control oligonucleotides via daily subcutaneous injections between the two plugs on the back of the mice. For selective HDAC IIa inhibitor, TMP269, mice were administered with TMP269 by subcutaneous injections every other day at 15 mg/kg mice body weight between the two plugs on the back of the mice. The animals were sacrificed and the Matrigel plugs were excised 10 days after Matrigel injection. The excised plugs were homogenized and subjected to measure hemoglobin content with 100 µL of Drabkin's solution (Sigma, St. Louis, MO) following manufactural instruction and our previous description [41].</p><!><p>Real-time RT-PCR reaction was run in triplicate for each sample and repeated at least 2 times, and the data were statistically analyzed with a Student T-test.</p><!><p>Expression levels of HDAC1–11 in three TNBC cell lines, MDA-MB-231, MDA-MB-1739, and HCC70 compared to those in three non-TNBC cell lines, MCF-7, MDA-MB-361, and SKBR3 were profiled with qRT-PCR analysis. Compared to non-TNBC cells, TNBC cells expressed much higher HDAC9 (Fig. 1A). Quantitative RT-PCR results reveal that expression levels of miR-206 are notably lower while HDAC9 levels are inversely higher in TNBC cell lines than those in non-TNBC cell lines (Fig. 1B). Furthermore, we analyzed the expression levels of HDAC9 proteins determined by immunohistochemical staining in breast cancer tissue samples. TNBC tissues express higher levels of HDAC9 compared to non-TNBC tissue samples (Fig. 1C). These results demonstrate that expression levels of HDAC9 are upregulated in TNBC tissues in comparison to non-TNBC tissue samples and are inversely correlated with the levels of miR-206.</p><!><p>To investigate whether selective HDAC9 inhibition blocks the invasion of TNBC cells, TNBC MDA-MB-231 cells were treated with TMP269, a selective class IIa HDAC inhibitor, or HDAC9 siRNA prior to Matrigel invasion assay. The invasive cells from treated groups were determined and compared to their controls by Matrigel invasion assay. Fig 2A shows representatives of invasive cell photographs from individual groups. Matrigel Invasion assay shows that the invasion of TNBC cells treated with selective class IIa HDAC inhibitor TMP269 was only 28% of that of the control (Fig. 2B). Similarly, HDAC9 siRNA treatment significantly blocked TNBC cell invasion. These results suggest that selective HDAC9 inhibition efficiently blocks the invasion of TNBC cells.</p><!><p>To determine the effect of selective HDAC9 inhibition on angiogenesis in vivo, Matrigel plug assay was performed in nude mice. Briefly, a mixture of 2×105 TNBC MDA-MB-231 cells in 0.5 ml of growth factor reduced Matrigel was implanted at two subcutaneous sites. The mice in two groups were treated with 100 µg/kg HDAC9 siRNAs or control oligonucleotides via daily subcutaneous injections. For other two groups, mice received 15 mg/kg TMP269 or vehicle every other day by intraperitoneal injection between the two plugs on the back of the mice. Ten days after the Matrigel implant, the mice were sacrificed. The Matrigel plugs were excised, photographed, and processed to measure hemoglobin content by using Drabkin's solution following manufacturer' instructions. When MDA-MB-231 cells successfully promote neovasculature formation within the Matrigel plug, these neovasculatures allow tumor cells to proliferate much better than those without neovasculatures. Therefore, the control group with better angiogenesis in the Matrigel plug showed more red cells than the treated group (Fig. 3A). Fig. 3B summarizes the quantification of percentage of antiangiogenic efficacy based on hemoglobin content in 12 Matrigel plugs per group. In comparison to their controls, TMP269 treatment or HDAC9 siRNA transfection shows an obvious antiangiogenic effect with 76 % and 72% inhibition of angiogenesis, respectively. These results suggest that selective inhibition of HDAC9 effectively blocks TNBC angiogenesis.</p><!><p>To determine whether HDAC9 regulates miR-206 expression, HDAC9 was silenced with HDAC9 siRNA and then miR-206 expression levels were measured by quantitative RT-PCR analysis. The results show that selective HDAC9 inhibition significantly upregulated miR-206 expression (Fig. 4A). Furthermore, the results of a search for the predicted targets with TargetScan revealed that VEGF and MAPK3 are predicted targets of miR-206 (Fig. 4B). To confirm these results in vitro, TNBC MDA-MB-231 cells were treated with HDAC9 siRNA and then protein levels of VEGF and MAPK3 by Western blot analysis. The results demonstrated that selective inhibition of HDAC9 decreased the expression levels of VEGF and MAPK3 compared to their controls (Fig. 4C). These results demonstrated that selective HDAC9 inhibition increased the expression of VEGF and MAPK3 via upregulating miR-206 expression.</p><!><p>It is now widely accepted that aberrant expression or activity of HDAC enzymes may lead to carcinogenesis and that specific HDAC enzymes are associated with particular malignancies; in turn, the inhibition of HDAC enzymes can result in therapeutic benefits in certain cancer types [42]. However, clinical benefits of selective versus broad HDAC inhibitors are unknown and the appropriateness of inhibitor may depend on tumor HDAC enzyme expression, enzyme selectivity of the inhibitor, and the desired effects. Although there are several published studies [16, 17] and numerous ongoing clinical trials involving HDAC inhibitors [18], very little is known about the roles of individual HDAC enzymes, and a formal assessment of HDAC enzyme expression is not routinely done. Emerging data suggests that the currently used HDAC inhibitors may differ significantly with regard to target selectivity. In addition, the expression of HDAC enzymes may vary considerably between normal and tumor tissues and likely between different phenotypes of tumor tissues. HDAC9 is thought to regulate gene expression through epigenetic modulation of the chromatin structure by catalyzing the deacetylation of histone proteins [19]. More recently, aberrant HDAC9 expression has been observed in several types of cancers, including medulloblastoma [21], acute lymphoblastic leukemia [22], glioblastoma [23], osteosarcoma [24], and breast cancer [25]. HDAC9 has been shown to promote the growth of these tumors. Our results demonstrate that HDAC9 is overexpressed in TNBC compared to non-TNBC cell lines and tissues. Furthermore, our data showed that selective inhibition of HDAC9 repressed the invasion and angiogenesis of TNBC cells. These findings reveal that HDAC9 promotes the invasion and angiogenesis of TNBC cells besides proliferation.</p><p>Several investigations have demonstrated that decreased miR-206 expression is involved in breast cancer proliferation [43, 44]. Our previous studies show that miR-206 is antagonistically involved in TNBC invasion and angiogenesis through modulating VEGF and MAPK3 [35]. Numerous investigations have demonstrated that VEGF actively promotes angiogenesis, metastasis, and chemoresistance [45–47]. MAPK overexpression has been shown to be associated with advanced stages and short survival in patients with some cancers [48, 49]. However, miR-206 has not been characterized as the targets of HDAC9 regulation. Our findings demonstrated that the selective inhibition of HDAC9 in siRNA-transfected TNBC MDA-MB-231 cells not only increased expression of miR-206, but also repressed the expression of VEGF and MAPK proteins and further inhibited TNBC cell invasion and angiogenesis. Here, we report HDAC9 for the first time as a mediator to modulate VEGF-mediated invasion and angiogenesis of TNBC tumor cells via modulating miR-206 expression. The results further confirmed that miR-206 actively regulates the expression of VEGF and MAPK3 in TNBC cells.</p><p>In conclusion, higher expression levels of HDAC9 are inversely correlated with miR-206 expression in TNBC cells. Furthermore, the selective inhibition of HDAC9 not only modulated the expression of miR-206, VEGF and MAPK3, but also particularly inhibited TNBC invasion and angiogenesis. Our findings suggested that HDAC9 overexpression in TNBC cells promotes the invasion and angiogenesis through VEGF and MAPK3 via regulating miR-206. These findings may be beneficial for better understanding TNBC regulation and designing personalized therapies for breast cancer patients.</p>

PubMed Author Manuscript

Quantification of Photocyanine in Human Serum by High-Performance Liquid Chromatography-Tandem Mass Spectrometry and Its Application in a Pharmacokinetic Study

Photocyanine is a novel anticancer drug. Its pharmacokinetic study in cancer patients is therefore very important for choosing doses, and dosing intervals in clinical application. A rapid, selective and sensitive high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the determination of photocyanine in patient serum. Sample preparation involved one-step protein precipitation by adding methanol and N,N-dimethyl formamide to 0.1 mL serum. The detection was performed on a triple quadrupole tandem mass spectrometer operating in multiple reaction-monitoring (MRM) mode. Each sample was chromatographed within 7 min. Linear calibration curves were obtained for photocyanine at a concentration range of 20–2000 ng/mL (r > 0.995), with the lower limit of quantification (LLOQ) being 20 ng/mL. The intrabatch accuracy ranged from 101.98% to 107.54%, and the interbatch accuracy varied from 100.52% to 105.62%. Stability tests showed that photocyanine was stable throughout the analytical procedure. This study is the first to utilize the HPLC-MS/MS method for the pharmacokinetic study of photocyanine in six cancer patients who had received a single dose of photocyanine (0.1 mg/kg) administered intravenously.

quantification_of_photocyanine_in_human_serum_by_high-performance_liquid_chromatography-tandem_mass_

1. Introduction<!>2.1. Experimental Chemicals<!>2.2. Chromatographic Conditions<!>2.3. Mass Spectrometric Conditions<!>2.4. Sample Preparation<!>2.5. Method Validation<!>2.6. Application<!>3.1. HPLC-MS/MS Condition Optimization<!>3.2. Sample Preparation Procedure<!>3.3. Specificity<!>3.4. Linearity and LLOQ<!>3.5. Accuracy and Precision<!>3.6. Extraction Recovery and Matrix Effect<!>3.7. Stability<!>3.8. Analysis of Patient Samples<!>4. Conclusion

<p>Photodynamic therapy (PDT) is a potential model for cancer therapy, which has been used to treat or relieve the symptoms of skin cancer, esophageal cancer, prostate cancer, and non-small cell lung cancer [1–3]. During the PDT procedure, the excited photosensitizer forms highly reactive oxygen species using visible light of an appropriate wavelength, resulting in oxidative damage to cellular membranes and membranous organelles [2, 4–7].</p><p>As one type of the photosensitizer, porphyrins have been approved for PDT in the USA, Europe, Canada, and Japan, but their weak absorption attenuates their optimal application in PDT [8]. Phthalocyanines and their derivatives are also widely used photosensitizers for the PDT of cancer, displaying high absorption of visible light, mainly in the phototherapeutic wavelength window (600–800 nm) [9, 10]. Photosense, Pc4, and CGP55847 are second-generation photosensitizers that have been used in clinical practice [11–14].</p><p>Photocyanine (ZnPcS2P2), a new second-generation PDT drug, was approved for clinical trials as a new medicine in 2008 by the State Food and Drug Administration in China. It is an isomeric mixture of di-(potassium sulfonate)-di-phthalimidomethyl phthalocyanine zinc (Figure 1) [15–17]. The presence of both hydrophobic and hydrophilic groups in photocyanine would improve its tumor selectivity. Clinical trial of a novel synthesized drug requires a reliable method for measuring levels of the drug in biological samples. Because photocyanine is a novel photodynamic drug, few methods have been developed for its quantification. Li et al. [18] also reported an HPLC method to separate the four isomers of a photocyanine mixture from human serum. However, the detection signals from this method display insufficient specificity in the biological samples, because it is difficult to avoid the interference from the matrix or other interferents by ultraviolet detector. Therefore, a method with higher specificity should be established to ensure the validity of the determination.</p><p>No evidence verifies the difference of each isomer of photocyanine in pharmacodynamic studies. Moreover, no standard substances for any of the isomers can be obtained from Fujian Longhua Pharmaceutical Co. Therefore, we developed a new high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method to determine the total concentration of photocyanine in cancer patient serum in a pharmacokinetic study. The method was validated for its specificity, sensitivity, linearity, accuracy, precision, matrix effect, dilution integrity, and stability, and the data established the method as a high-throughput and reliable bioanalytical assay.</p><!><p>Photocyanine (purity > 95%) and the internal standard (IS) mono-β-sulfonated zinc phthalocyanine potassium (purity > 95%) were provided by Fujian Longhua Pharmaceutical Co. (Fujian, China). HPLC-grade N, N-dimethyl formamide (DMF) and methanol were purchased from Tedia Company, Inc., (Fairfield, OH, USA). Aqueous ammonia was obtained from Guangzhou Chemical Reagent Factory (Guangzhou, Guangdong, China). Deionized water was obtained from a Milli-Q analytical deionization system (Millipore, Bedford, MA, USA). Freshly obtained, drug-free human serum was collected from healthy individuals and stored at −80°C before use.</p><!><p>The HPLC system consisted of an LC-20AD solvent delivery system, an SIL-20AC autosampler, a CTO-20AC column oven, and a CBM-20A controller from Shimadzu (Kyoto, Japan). Chromatographic separation of photocyanine and mono-β-sulfonated zinc phthalocyanine potassium was evaluated on an XBridge C18 column (50 mm × 4.6 mm, 5 μm) from Waters (Milford, MA, USA). For method validation and sample analysis, chromatographic separation was conducted by gradient elution using deionized water (adjusted to pH 10.0 with aqueous ammonia) as mobile phase A (MPA) and methanol as mobile phase B (MPB). The HPLC program for gradient elution was as follows: 20% of MPB (0–0.2 min), from 20% to 95% of MPB (0.2–1.3 min), 95% of MPB (1.3–4.0 min), from 95% to 20% of MPB (4.0–4.1 min), and 20% of MPB (4.1–7.0 min). The separation was performed using a flow rate of 0.6 mL/min. The column temperature was maintained at 60°C.</p><!><p>An API 4000 QTRAP system (AB SCIEX, MA, USA) was operated in negative ionization mode with multiple reaction monitoring (MRM) for HPLC-MS/MS analysis. The mass spectrometric parameters were optimized to improve the MRM sensitivity. The instrument parameters for monitoring photocyanine and IS were as follows: vaporizer temperature, 650°C; ion spray voltage, −4,500 V; curtain gas (CUR), nitrogen, 25; nebulizing gas (GS1), 65; heated gas (GS2), 65; declustering potential (DP), photocyanine −140 V, IS −135 V; collision energy (CE), photocyanine −50 eV, IS −64.4 eV; entrance potential (EP), −10 V; collision cell exit potential (CXP), −10 V. The precursor-to-product ion transitions used for the MRM of photocyanine and IS were m/z 526.0 → 146.0 and m/z 655.1 → 591.8, respectively. The mass spectrometer was operated at unit mass resolution for both the first and third quadrupoles.</p><!><p>A 100 μL aliquot of blank human serum, spiked serum, or pharmacokinetic study serum was transferred to a 1.5 mL Eppendorf tube. Then, 200 μL of DMF was added to each tube of serum, and the mixture was vortexed for 1 min. The mixture was then spiked with 300 μL methanol containing 450 ng/mL IS, vortexed, and centrifuged for 10 min at 15,000 rpm at 4°C. The supernatant was collected and filtered. 10 μL of supernatant was injected into the LC-MS/MS system for analysis.</p><!><p>Photocyanine was validated for an HPLC-MS/MS assay. Specificity, the lower limits of quantification (LLOQ), linearity, accuracy, precision, extraction recovery, matrix effect, and stability were evaluated during method validation. The specificity was assessed by testing six independent aliquots of blank serum for exclusion of any endogenous interference at the peak region of photocyanine or IS (Figure 2). LLOQ was defined as the lowest concentration on the standard calibration curve from six different batches, in which both precision and accuracy were ≤20% with a signal-to-noise ratio (S/N) > 10. The linearity of the calibration curve was evaluated over the range of 20 ng/mL and 2000 ng/mL. Calibration curves were constructed via linear least-squares regression analysis by plotting the peak-area ratios (photocyanine/IS) versus the drug concentrations in the serum, and the resulting correlation coefficient (r > 0.99) was considered satisfactory. Precision and accuracy were assessed by the analytes covering the range of the calibration curve, in which the criteria for acceptability are defined as an accuracy ±15% standard deviation (SD) from the nominal values and a precision of ±15% relative standard deviation (RSD). Intrabatch accuracy and precision were evaluated by analyzing the quality control (QC) samples at concentrations of 60, 1000, and 1600 ng/mL with six duplicated levels per concentration on the same day. The interbatch accuracy and precision were assessed over three days. The extraction recovery of photocyanine and IS was determined by calculating the ratio of the peak area of photocyanine and IS spiked in serum before extraction against postextraction spiked photocyanine and IS at the same concentration. The matrix effect was determined by calculating the matrix factor, which was obtained as the ratio of the analyte peak response in the presence of matrix ions to the analyte peak response in the absence of matrix ions by spiking analytes into blank serum extracts and blank water extracts. The stability of photocyanine was compared to the nominal level of photocyanine to determine whether photocyanine was stable in the experiments, including postpreparative stability test, freeze-thaw cycle test, and long-term stability test. If the calculated concentration of photocyanine was less than the nominal concentration by >15%, the analyte was considered to be unstable. Low, medium, and high serum QC samples were determined in six duplicated levels. The stability of the extracts was evaluated by putting them at room temperature for 24 h and then subjecting them to the analytical procedure. Photocyanine maintained at −80°C for 30 days was evaluated by comparing the postfreeze measured concentration with the initial concentration added to the sample. The freeze-thaw stability of the samples was assessed over three freeze-thaw cycles by thawing samples at room temperature, refreezing them for 24 h at −80°C and then analyzing them.</p><!><p>Six patients with cancer were enrolled at the Cancer Center, Sun Yat-sen University. The patients were five males and one female, ranging in age from 37 to 69 years, who had been diagnosed with a primary or metastatic malignancy. All patients provided written informed consent prior to participation. The patients were infused (i.v. administration) with photocyanine (0.1 mg/kg) for 60 min. Blood samples were obtained at 0, 0.5, 1, 2, 4, 6, 8, 12, 24, 72, 120, and 168 h after administration and then placed on ice and kept away from light. The blood samples were centrifuged at 3000 rpm/min for 10 min, and the serum was stored at −80°C until the analysis was conducted. The present study was approved by the Human Subjects Review Committees of the University of Sun Yat-sen University Cancer Center and conducted according to the Declaration of Helsinki.</p><p>Noncompartmental pharmacokinetic parameters were calculated using WinNonlin (Version 5.0, PUMCH Clinical Pharmacology Research Center). The maximum serum concentration (C max⁡) and time to reach it (T max⁡) were determined directly from the data. The terminal-phase rate constant (λ z) was calculated as the negative slope of the log-linear terminal portion of the serum concentration-time curve using linear regression with at least four last concentration-time points. The terminal-phase half-life (t 1/2) was calculated as 0.693/λ z. The area under the curve from time zero to the last observed time (AUC0-t) was calculated by the linear trapezoidal rule for ascending data points. The total area under the curve (AUC0-∞) was calculated as AUC0-t + Ct/λ z, where Ct is the last measurable concentration. The apparent volume of distribution associated with the terminal phase (Vz) was calculated as Vz = CL/λ z, and the apparent total body clearance (CL) was calculated as CL = dose/AUC.</p><!><p>HPLC-MS/MS operation parameters were carefully optimized for the determination of photocyanine. Photocyanine is a typical sulfonate compound, which is usually ionized in negative mode. We tuned the mass spectrometer in both positive and negative ionization modes with ESI for photocyanine and found that both the signal intensity and ratio of signal to noise obtained in negative ionization mode were much greater than those in positive ionization mode, which was consistent with previous study on another sulfonate compound [19]. In the precursor ion full-scan spectra, the most abundant ions were protonated molecules with m/z ratios of 526.0 and 655.1 for photocyanine and IS, respectively. Parameters such as desolvation temperature, ESI source temperature, capillary voltage, and flow rate of desolvation gas and cone gas were all optimized to obtain the highest intensity of protonated analyte molecules. The product ion scan spectra showed high abundance fragment ions at m/z values of 146.0 and 591.8 for photocyanine and IS, respectively. Multiple reaction monitoring (MRM) using the precursor→product ion transition of m/z 526.0→m/z 146.0 and m/z 655.1→m/z 591.8 was used for the quantification of photocyanine and IS, respectively.</p><p>The efficiency of the chromatographic separation of photocyanine and IS was evaluated using tests with different chromatographic columns and mobile phases. Photocyanine is amphoteric and relatively hydrophobic. We found that photocyanine displayed a very strong retention on BDS or XDB C18 columns, and little retention on C8 or SCX columns, which resulted in broad peak or substantial carry over. These columns have been tested without success. It has been shown that good chromatographic profiles of photocyanine and IS are obtained using a Waters XBridge C18 column (50 mm × 4.6 mm, 5 μm), with retention times of 2.33 and 2.59 min, respectively. The total analysis time per sample is 7 min, which is much shorter than that of 45 min in previous study [18]. We also optimized the column temperature by observing the chromatographic peak and resolution and found that XBridge C18 column displayed well column performance at 60°C. Optimization of the mobile phase is important for improving peak shape and detection sensitivity and for shortening the run time. We tested methanol, acetonitrile, and a mixture of the two as the organic modifier of the mobile phase, and we found that the peaks were more symmetric when methanol was used. Moreover, the chromatographic behavior of photocyanine subjected to mobile phases of different pH values was investigated, and we observed that deionized water (adjusted to pH 10.0 with aqueous ammonia) improved the peak shape and significantly increased the signal intensity of the analyte. In order to further optimize the chromatographic condition, the peak symmetry factor of photocyanine was calculated in different percentage of MPB at the elution period 1.3–4.0 min. Symmetry factor of a peak is calculated by dividing W 0.05 by two-fold f, where W 0.05 is the width of the peak at 5% height and f is the distance from the peak maximum to the leading edge of the peak, the distance being measured at a point 5% of the peak height from the baseline. As shown in Table 1, the optimal value was 95%. Finally, the optimized gradient elution with deionized water (adjusted to pH 10.0 with aqueous ammonia) and methanol at a flow rate of 0.6 mL/min was established in this study.</p><!><p>Sample preparation is important for the HPLC-MS/MS assay. Liquid-liquid extraction (LLE) and solid-phase extraction (SPE) are techniques often used in the preparation of biological samples due to their ability to improve assay sensitivity [20, 21]. SPE columns, including Strata, Strata-X, and Strata C18-E from Phenomenex (Torrence, CA, USA), OASIS WAX Cartridge, and Sep-pak C18 from Waters (Milford, MA, USA), were used for sample preparation in this study. However, photocyanine exhibited no elution due to its strong adsorption to the SPE columns. We also carried out LLE with ethyl acetate, n-butyl alcohol, and mixtures of these organic solvents with n-hexane; however, we obtained low recovery and reproducibility using this procedure. Because HPLC-MS/MS quantification is highly specific and sensitive, protein precipitation (PPT) from the sample preparations was tried in the present study. We found that PPT was not only simple and efficient but also applicable to pharmacokinetic studies in which only 100 μL of serum was used to obtain bioanalytical results. In addition, we observed that the linearity of photocyanine concentration in human serum with DMF was significantly improved than that of without DMF, indicating that DMF is conductive to maximize the release of photocyanine in PPT by inhibiting the binding of the drug to serum proteins, but there is no related report so far about the mechanism of DMF in PPT or drug release. Finally, we added 200 μL DMF to the sample, and the effect of DMF here is consistent to that in previous study [18].</p><!><p>Specificity was determined by comparing the chromatograms of six different batches of blank human serum with the corresponding spiked serum. No interference from endogenous substances was observed at the respective retention times of photocyanine and IS (data not shown).</p><!><p>The linear calibration curves were determined from the peak-area ratios (peak-area analyte/peak-area IS) versus concentration in human serum using a weighting factor (1/x 2), varying linearly over the concentration range tested (20–2000 ng/mL). As shown in Figure 3, the typical equation for the calibration curve for photocyanine was y = 0.421x + 0.00439 (r = 0.9965). The slopes of the equations were consistent with the calibration curves prepared on three separate days. The LLOQ in this study was 20 ng/mL for photocyanine, in which the S/N was >10, and the precision of repeat injections was 8.26%. Our method displayed a little higher sensitivity than the previous method, in which the LLOQ was 30 ng/mL [18].</p><!><p>The accuracy and precision of the method were determined by analyzing QC samples at three concentrations in six replicates. The intrabatch accuracy ranged from 101.98% to 107.54% at three concentrations, with precisions between 1.29% and 4.91%. The interbatch accuracy varied from 100.52% to 105.62%, with precisions ranging from 4.72% to 8.53% (Table 2). Thus, the present method has satisfactory accuracy, precision, and reproducibility.</p><!><p>The extraction recoveries from QC samples at low, intermediate, and high concentrations ranged from 31.64% to 43.53% at three tested concentrations. We extracted the analyte from serum using protein precipitation and DMF in the present study, providing a simple and rapid method for the bioanalysis of photocyanine. In terms of matrix effects, the MF ranged from 61.51% to 77.03% at the three concentrations tested (Table 3), indicating that the coeluting substance only slightly influenced the ionization of the analyte.</p><!><p>The results from the stability tests are presented in Table 4, and the data demonstrate a good stability of photocyanine throughout the steps of the determination. The method is therefore applicable to routine analysis.</p><!><p>The validated HPLC-MS/MS method described here was successfully applied to a pharmacokinetic study in 6 cancer patients following i.v. administration of 0.1 mg/kg photocyanine. A mean plasma concentration-time curve of a single dose of photocyanine is shown in Figure 4. This result revealed that our method was sufficiently sensitive to determine the photocyanine concentration in the serum of patients. The parameters of the pharmacokinetic analysis are shown in Table 5. The time of maximum plasma concentration (T max⁡) was 1.83 ± 0.41 h, the maximum plasma concentration (C max⁡) was 2465.3 ± 723.0 ng/mL, the half-life of drug elimination at the terminal phase (t 1/2) was 74.62 ± 13.29 h, the area under the plasma concentration-time curve from 0 h to the time of the last detectable concentration (AUC0-t) was 53137.2 ± 20210.6 ng·mL−1 ·h, the area under the plasma concentration-time curve from 0 h to infinity (AUC0-∞) was 62634.6 ± 25398.6 ng·mL−1 ·h, the volume of distribution (V d) of photocyanine was 11.29 ± 4.12 L, the total clearance (CL) was 0.11 ± 0.04 L/h, and the mean residence time (MRT) was 40.16 ± 4.32 h.</p><!><p>A selective, sensitive, and rapid HPLC-MS/MS method for measuring photocyanine in human serum is described. In comparing this method with the analytical methods reported previously [14, 18], the present method proved superior with respect to higher simplicity of sample preparation, higher selectivity and sensitivity, and shorter chromatographic analysis time. The present description is the first to utilize the HPLC-MS/MS method for the pharmacokinetic study of photocyanine given by injection to cancer patients. 100 μL human serum is sufficient for obtaining results in our pharmacokinetic study, indicating that the present method is applicable to human phase I clinical trials.</p>

PubMed Open Access

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Our previous studies have indicated that de novo ceramide synthesis plays a critical role in ethanol-induced apoptotic neurodegeneration in the 7-day-old mouse brain. Here, we examined whether the formation of sphingosine 1-phosphate (S1P), a ceramide metabolite, is associated with this apoptotic pathway. Analyses of basal levels of S1P-related compounds indicated that S1P, sphingosine, sphingosine kinase 2, and S1P receptor 1 increased significantly during postnatal brain development. In the 7-day-old mouse brain, sphingosine kinase 2 was localized mainly in neurons. Subcellular fractionation studies of the brain homogenates showed that sphingosine kinase 2 was enriched in the plasma membrane and the synaptic membrane/synaptic vesicle fractions, but not in the nuclear and mitochondrial/lysosomal fractions. Ethanol exposure in 7-day-old mice induced sphingosine kinase 2 activation and increased the brain level of S1P transiently 2-4h after exposure, followed by caspase-3 activation that peaked around 8h after exposure. Treatment with dimethylsphingosine, an inhibitor of sphingosine kinases, attenuated the ethanol-induced caspase-3 activation and the subsequent neurodegeneration. These results indicate that ethanol activates sphingosine kinase 2, leading to a transient increase in S1P, which may be involved in neuroapoptotic action of ethanol in the developing brain.

ethanol_triggers_sphingosine_1-phosphate_elevation_along_with_neuroapoptosis_in_the_developing_mouse

Introduction<!>Animals<!>Experimental Procedure<!>Lipid analysis<!>Immunohistochemistry<!>Subcellular fractionation<!>Immunoblotting<!>Sphingosine kinase assay<!>Statistics<!>S1P metabolism in the developing brain<!>Ethanol-induced alterations in the levels of ceramide, sphingosine, and S1P<!>Ethanol transiently increased SphK2 activity<!>Subcellular localization of SphK2<!>The effects of DMS on ethanol-induced caspase-3 activation and neurodegeneration in the P7 mouse brain<!>Discussion

<p>Ethanol triggers apoptotic neurodegeneration in the newborn rodent brain during the period of rapid synaptogenesis that corresponds to the last trimester of pregnancy in humans (Ikonomidou et al., 2000; Olney et al., 2002), and causes long-lasting neuronal loss and impaired neurobehavior as observed in human fetal alcohol spectrum disorders (FASD). This rodent model for FASD has been widely utilized to elucidate mechanisms of ethanol-induced apoptotic neurodegeneration (Carloni et al., 2004; Han et al., 2006; Young et al., 2005). We have previously demonstrated that ethanol-induced neurodegeneration in 7-day-old (postnatal day 7; P7) mice is accompanied by increases in several brain lipids (Saito et al., 2007a). Among them, de novo ceramide synthesis appears to play a vital role in ethanol-induced apoptosis, because ceramide elevation is associated with ethanol-induced caspase-3 activation, and because inhibitors of serine palmitoyltransferase, a rate-limiting enzyme in sphingolipid synthesis, rescue ethanol-induced apoptosis (Saito et al., 2010a). However, we cannot rule out the possibility that ceramide metabolites, specifically sphingosine and sphingosine 1-phosphate (S1P), are also involved in the ethanol-induced apoptotic pathway because these lipids have been implicated as regulators of the cell survival/death pathways.</p><p>It is generally postulated that ceramide and sphingosine induce growth arrest or apoptosis (Ogretmen and Hannun, 2004), while S1P promotes cell proliferation and cell survival (Spiegel and Milstien, 2003), and the balance between these bioactive lipids, termed `sphingolipid rheostat', determines cell fate (Spiegel and Milstien, 2003). This sphingolipid rheostat is mainly regulated by two isoforms of sphingosine kinases, sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2), which phosphorylate sphingosine to form S1P. It has been indicated that SphK1, a cytosolic protein, is translocated to the plasma membrane after activation (Johnson et al., 2002; Pitson et al., 2005) and exerts a pro-survival influence, whereas SphK2, a predominantly nuclear protein, inhibits cell growth and enhances apoptosis (Igarashi et al., 2003). While most of the S1P effects are mediated by the interaction of S1P with five G-protein-coupled cell surface receptors termed S1P receptor 1–5 (Spiegel and Milstien, 2003; Snider et al., 2010), intracellular actions of S1P have also been reported (Olivera and Spiegel, 1993). Induction of apoptosis by overexpressed SphK2 is independent of activation of S1P receptors (Liu et al., 2003), and S1P produced by SphK2 in the nucleus (Igarashi et al., 2003) as well as S1P produced by SphK2 in the endoplasmic reticulum (ER) (Maceyka et al., 2005; Hagen et al., 2009) have been reported to exert apoptotic action.</p><p>In the nervous system, S1P has a critical role in neural development. Dysfunction of SphK1/2 in SphK1/2 double knockout mice leads to embryonic lethality (Mizugishi et al., 2005). S1P plays roles in neurogenesis, neurite formation, and neuroprotection (Shinpo, et al., 1999; Okada et al., 2009; Agudo-Lopez et al., 2010), and may also be involved in astrocyte proliferation (Pebay et al., 2001; Malchinkhuu et al., 2003; Sorensen et al., 2003;Yamagata et al., 2003; Lee et al., 2010) and microglial activation (Nayak et al., 2010). Most of the SphK activity in the brain appears to be due to SphK2, which is localized in neurons, while SphK1 is localized primarily in astrocytes (Blondeau et al., 2007). Whereas activation of the SphK1/S1P axis signaling appears to be related to proliferation of astrocytes (Wu et al., 2008; Lee et al., 2010), protection of oligodendrocyte progenitors from apoptosis (Saini et al., 2005), and microglial activation (Nayak et al., 2010), SphK2 has been implicated to cause apoptosis through intracellular targets in cerebellar granule neurons derived from S1P lyase-deficient mice (Hagen et al., 2009). However, in some animal models of brain ischemia, SphK2 activation is considered neuroprotective (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011; Yung et al., 2012).</p><p>While S1P is thought to play important roles in the developing brain, profiles and functions of the S1P system have not been well studied in the early postnatal brain. Here, we examined S1P metabolism with a particular focus on SphK2 under the basal and ethanol-treated conditions in the P7 mouse brain and evaluated the possibility that S1P is involved in ethanol-induced apoptotic neurodegeneration.</p><!><p>C57BL/6By mice were maintained at the Animal Facility of Nathan S. Kline Institute for Psychiatric Research. All procedures followed guidelines consistent with those developed by the National Institute of Health and the Institutional Animal Care and Use Committee of Nathan S. Kline Institute.</p><!><p>C57BL/6By mice were subcutaneously injected with saline (control) or ethanol at P7 as described previously (Olney et al., 2002; Saito et al., 2007a, b) except that one-time injection with 25 μl/g body weight of ethanol (5.0 g/kg, 20% solution in saline) or saline was performed instead of two-time injections of 2.5 g/kg ethanol with a 2h-interval, because the present study included short-term (less than two hours) treatment conditions. It has been reported that blood ethanol levels obtained by this one-time injection protocol are similar to those of two-time injections (Ieraci and Herrera, 2006). The effects of d-erythro-N,N-dimethylsphingosine (DMS, a SphK inhibitor) on ethanol-induced caspase-3 activation and the subsequent neurodegeneration were examined using both one-time and two-time ethanol injection paradigms. In both cases, DMS (3 μg in 1.5 μl DMSO) was administered 0.5h before the first ethanol injection via intracerebroventricular injection as described (Sadakata et al., 2007). Ethanol-induced caspase-3 activation at 8h after the first ethanol injection was indirectly assessed by measuring increases in cleaved caspase-3 (CC3), and cleaved tau (Ctau) levels by immunoblotting. We have previously shown that Ctau formation is mainly catalyzed by caspase-3 and detected noticeably in degenerating axons/dendrites (Saito et al., 2010b). Ethanol-induced neurodegeneration was assessed by Fluoro-Jade C (Millipore, Billerica, MA, USA) staining using brain sections from mice perfusion-fixed 19h after the first ethanol injection. Except for brief periods of time for injections, mice were kept with dams until sacrificed 1–24h after the saline/ethanol injection. For developmental studies, P1, 4, 7, 10, 13, 16, 19, 25, and 31 naïve mice were used. Three to ten animals were used for each data point.</p><!><p>Ceramide and sphingomyelin were separated from total lipids using high performance thin layer chromatography, and the amounts were measured as described previously (Saito et al., 2010a). Determination of S1P and sphingosine content was performed according to the method of He et al. (2009), modified as described in detail in Appendix S1.</p><!><p>Eight and 19 hours after the ethanol injection, mice were perfusion-fixed with a 4% paraformaldehyde solution, and vibratome sections (50 μm) of the fixed brains were prepared and immunofluorescence-labeled as described previously (Saito et al., 2007b, 2010a) using antibodies against SphK2, NeuN, and Na+,K+-ATPase (see Table S1 for details of the antibodies used). In order to check the specificity of anti-SphK2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the antibody was incubated with the blocking peptide (Santa Cruz) for 1h at room temperature prior to incubation with brain sections. For some experiments, the above SphK2 antibody was further affinity-purified as follows: P2 (mitochondria/synaptosome) fraction of P7 brain homogenates was isolated as described below in the subcellular fractionation section, and was separated on SDS gel electrophoresis and blotted on nitrocellulose membranes. A strip of the membrane containing a band corresponding to SphK2 was blocked with 5% BSA in Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 and incubated with the antibody in the blocking buffer overnight at 4°C. After rinsing, the antibody was eluted with 50 mM glycine/150 mM NaCl (pH 2.4) and neutralized. The antibody thus purified gave a single band when analyzed by immunoblotting. For Fluoro-Jade C staining, brain sections were processed according to the manufacturer's instruction. The extent of neurodegeneration was expressed as the number of Fluoro-Jade C-positive cells per square millimeter in the cingulate and retrosplenial cortices as described previously (Saito et al., 2010a) using four brains per treatment group. Photomicrographs were taken through a 20X and a 100X objective with a Nikon Eclipse TE2000 inverted microscope attached to a digital camera DXM1200F.</p><!><p>Nuclei from the P7 mouse forebrain were isolated according to the method of Block et al. (1992) and Koppler et al. (1993) with modification by Wu et al. (1995). For separation of P1 (crude nucleus), P2 (mitochondria/lysosome/synaptosomal pellet), and P3 (microsomal pellet) fractions, and for further fractionation of P2, the combined method of Rajapakse et al. (2001) and Kiebish et al. (2008) was used. P3 was further fractionated using OptiPrep (60% w/v iodixanol in water, Sigma-Aldrich Chemical Company, St. Louis, MO, USA) density gradient centrifugation according to the methods of Araki et al. (2003), Li and Donowitz (2008), and OptiPrep instruction manual with some minor modifications. Additional detailed subcellular fractionation protocols are available in Appendix S1.</p><!><p>Immunoblotting was performed as described previously (Saito et al., 2010a). Tissue samples or subcellular fractions described above (30 μg of protein) were boiled in SDS-sample buffer, separated on 10% or 15% SDS-PAGE, and blotted onto nitrocellulose membranes. The membranes were then blocked with Odyssey blocking buffer containing 0.1% Tween 20 and probed with various antibodies (see Table S1 for details of the antibodies used). Either mouse monoclonal anti-β-actin antibody, or mouse monoclonal anti-β-tubulin antibody was included as loading control. Antigens were detected by the Odyssey infrared imaging system using secondary antibodies, IR dye 680 conjugated goat anti-rabbit IgG, IR dye 680 conjugated donkey anti-goat IgG, and IR dye 800 conjugated goat anti-mouse IgG, and analyzed by Multi Gauge ver.2.0 (Fujifilm USA Medical Systems). For the quantification analyses, the intensity of each protein band was normalized by the corresponding β-actin intensity. Due to the close molecular weights of S1P1-receptor (S1P1-R; ~44 kD) and β-actin (~43 kD), β-tubulin (~50 kD) was used for S1P1-R quantification. In the developmenal studies (Fig. 1), intensities of bands obtained from samples with the same protein amounts were directly compared among various developmental stages, because amounts of β-actin and β-tubulin changed significantly during development (data not shown). In order to check the specificities of anti-SphK1, anti-SphK2, and anti-S1P1-R antibody, antibodies were pre-incubated with corresponding peptides for 1h at room temperature prior to probing. Blocking peptides used were SphK2-Blocking Peptide (Cat No. SC-22704P, Santa Cruz Biotechnology), unphosphorylated SK1 (Ser-225) Peptide (Cat No. SX1645, ECM-Biosciences), and S1P1-Blocking Peptide (Cat No. 10006616, Cayman Chemicals). The amount of protein was measured by a BCA method (Pierce, Rockford, IL, USA).</p><!><p>SphK1 and SphK2 activities in the P7 forebrain were measured according to the methods of Billich and Ettmayer (2004), Wacker et al. (2009), and Liu et al. (2000) except that NBD [ω-(7-nitro-2-1,3-benoxadiazol-4-yl)-D-erythro]-S1P formed was extracted according to the method of Matyash et al. (2008) using methyl tert-butyl ether. Fluorescence was measured using a fluorometer (BioTek Instrument Inc, Winooski, VE, USA) at excitation wavelength 485 nm and emission wavelength 528 nm. Values were expressed as percent of control.</p><!><p>Values in figures are expressed as mean ± Standard Error of Mean (SEM) obtained from 4–10 samples. Statistical analysis of the data was performed by two-tailed Student's t test, one sample t test, and ANOVA with Bonferroni's post hoc test using the SPSS 11.0 program. A p value of <0.05 was considered significant.</p><!><p>Prior to studies of the effects of ethanol on S1P metabolism in the P7 mouse brain, basal levels of S1P-related lipids and proteins (SphK1, SphK2, and S1P1-R) were examined during the early postnatal brain development. First, we measured changes in brain levels of ceramide, sphingosine and S1P. Results showed that levels of S1P gradually increased between P4 and P31, while sphingosine reached maximum at P19, and ceramide peaked around P13 (Fig. S1). Developmental changes in protein levels of S1P1-R, SphK2, and SphK1 in the forebrain were also examined by immunoblot analyses (Fig. 1). The band in Fig. 1A indicated by an asterisk (*) was considered non-specific because pre-incubation of anti-SphK1 antibody with a blocking peptide solution (as described in Materials and Methods) did not reduce the intensity of this band, while the band below disappeared completely. Bands shown for SphK2 and S1P1-R were considered specific to each antibody, based on experiments using corresponding blocking peptides (data not shown). Fig. 1B shows quantitative results expressed as fold changes compared to P1 values. Levels of SphK2 gradually increased between P1 and P25 in a manner similar to that of S1P (Fig. S1C), while the trace amounts of SphK1 found in the P1 forebrain did not increase during the developmental period. Fig. 1 also shows that S1P1-R, a major S1P receptor isoform in the brain (Brinkmann, 2007), increased during early postnatal days as observed in levels of S1P (Fig. S1C). Thus, the neonatal brain contains significant levels of S1P, sphingosine, SphK2, and S1P1-R, although the levels were lower than those at later developmental stages.</p><p>In Fig. 2, protein levels of SphK2, SphK1, and S1P1-R in the P7 brain were compared with those in other organs by immunoblot analyses. SphK2 protein was high in the forebrain and moderate in the liver, whereas SphK1 protein was very low in the forebrain, moderate in the brainstem, and high in the heart and the liver. S1P1-R protein was high in the brain, especially in the brainstem.</p><p>We also examined the cellular localization of SphK2 in the P7 brain by immunohistochemistry. Brain sections from saline-treated (control) P7 mice were dual immunofluorescence-labeled with anti-SphK2 antibody and anti-NeuN antibody (Fig. 3B). The image shows the cingulate cortex region. In the lower panels, anti-SphK2 antibody was pretreated with blocking peptides. These results indicate that SphK2 is localized primarily in neurons. The affinity-purified anti-SphK2 antibody (prepared as described in Materials and Methods) also gave similar staining in neurons (data not shown). Immunohistochemistry using anti-SphK1 antibody gave faint staining mainly in astrocytes (data not shown). Also, S1P1-R expression was mostly limited to astrocytes (manuscript in preparation) in agreement with previous studies on the human brain (Nishimura et al., 2010).</p><!><p>P7 mice were exposed to ethanol (5 g/kg) once as described in Materials and Methods. This treatment induced caspase-3 activation in the forebrain 8h after ethanol injection (Fig. 4). Caspase-3 activation was assessed by cleaved caspase-3 (CC3) and cleaved tau (Ctau) formation using immunoblotting. Under these ethanol treatment conditions, time course studies on the effects of ethanol on levels of ceramide, sphingosine, and S1P in the brain were performed (Fig. 5). Ethanol exposure in P7 mice significantly increased ceramide levels 8h after ethanol exposure, and the increase was maintained for at least another 16h (Fig. 5A). The result was similar to the effects of two-time ethanol injections (2.5 g/kg each) with a 2 h interval (Saito et al., 2007a). Sphingosine levels, which increased significantly 8h after ethanol injection, were also maintained for another 16h (Fig. 5B). In contrast, S1P increased 2.5 times 4h after ethanol injection and decreased to the basal level within the following 4h (Fig. 5C). The level of sphingomyelin, one of the potential precursors for ceramide/sphingosine/S1P, was not altered by ethanol treatment (data not shown).</p><!><p>Ethanol transiently increased SphK2 enzyme activity (P<0.01 after Bonferroni's correction) at 2h (Fig. 6A). On the other hand, SphK1 enzyme activity, which was roughly one fourth of SphK2 enzyme activity, remained unaffected (data not shown). This transient increase of SphK2 activity after ethanol treatment was not due to an increase in the SphK2 level, because the level measured by immunoblot analyses was unaltered (Fig. 6B, C). The level of S1P1-R was also unchanged after ethanol treatment (Fig. 6B, C). Immunohistochemical analyses indicated that ethanol treatment did not change the cellular distribution of SphK2 (data not shown).</p><!><p>It has been reported that subcellular localization of SphK2 is important for exerting its cellular functions (Igarashi et al., 2003; Hait et al., 2009; Wattenberg, 2010; Strub et al., 2011). P7 mouse brains decapitated 2h after saline/ethanol injection were homogenized and fractionated into P1 (crude nucleus), P2 (mitochondria/lysosome/synaptosome), P3 (microsome), and soluble (cytosol) fractions, and SphK2 levels in these fractions were analyzed by immunoblotting (Fig. 7A). We observed that SphK2 was highly expressed in the P2 and P3 (Micro) fractions while it was not detected in the P1 and very low in the cytosol fraction. Subcellular distribution of SphK2 was not significantly different between saline and ethanol-treated brains. Because the presence of SphK2 in the nucleus has been reported previously (Igarashi et al., 2003; Sankala et al., 2007; Ding et al., 2007; Hait et al., 2009), P1 fraction was further purified as described in Appendix S1 to increase the ratio of SphK2 to other proteins, if SphK2 is enriched in the nucleus. However, SphK2 was not detected in the purified nuclear fraction [Nuc (P)] (Fig. 7B). The purity of the nuclear fraction was confirmed by the abundant presence of acetyl histone and the absence of other subcellular marker proteins, synaptophysin (synaptic vesicle), PSD95 (synaptic membrane), voltage-dependent anion channel (VDAC, mitochondria), Complex IV (COX IV, mitochondria), Na+,K+- ATPase (plasma membrane), and β-glucosidase (lysosome). In order to better understand the subcellular localization of SphK2 found in the P2 fraction, P2 was further fractionated by different density gradient centrifugations, followed by Western blot analyses of each fraction containing 30 μg of protein (Fig. 8A). As predicted from a previous report (Rajapakse et al., 2001), Mito (ns) and Mito (s) fractions were enriched in VDAC, a mitochondrial marker. Also, Mito (ns) contains LAMP1, a lysosomal/late endosomal marker. Synap1 and Synap2 were enriched in PSD95 (a synaptic membrane marker) and synaptophysin (a synaptic vesicle marker), respectively. These synaptosomal fractions also contained Na+,K+-ATPase (a plasma membrane marker) and flotillin-1 (a lipid raft marker). Fig. 8A indicates that SphK2 was absent from mitochondrial/lysosomal fractions and predominantly localized in synaptosomal vesicle- and synaptosomal membrane-enriched fractions. The results shown in Fig. 7 also indicated that SphK2 was present in the P3 (microsomal) fraction. To examine the subcellular localization of SphK2 in this fraction, components of the microsomal pellet were separated by iodixanol density gradient centrifugation and probed with antibodies against different organelle markers. As shown in Fig. 8B and C, SphK2 showed similar distribution to that of flotillin-1, Na+,K+-ATPase, and Rab5 (an early endosomal marker), indicating that SphK2 was enriched in plasma membrane regions. This notion also agrees with the enrichment of SphK2 in synaptic membrane and synaptic vesicle fractions in the P2 pellet (Fig. 8A). Syntaxin6, a Golgi marker, and ERp72, an endoplasmic reticulum (ER) marker, showed different distribution pattern from that of SphK2, although the presence of SphK2 in the ER cannot be excluded because of the small difference in the densities. In Fig. 8D, brain sections from saline-treated (control) P7 mice were dual immunofluorescence-labeled with anti-SphK2 antibody and anti-Na+,K+-ATPase antibody. The image shows the cingulate cortex region. The SphK2 antibody used here was affinity-purified as described in Materials and Methods. The results indicated partial co-localization of SphK2 with Na+,K+-ATPase, which was consistent with the subcellular fractionation results.</p><!><p>In order to evaluate the involvement of S1P in the ethanol-induced apoptotic pathway, DMS, a SphK inhibitor, was administered into P7 mice via intracerebroventricular injection 0.5h before the ethanol injection, and cleaved caspase-3 formation at 8h after ethanol injection was analyzed by Western blotting. Fig. 9 shows the effects of DMS on cleaved caspase-3 (CC3) formation induced by the two-time ethanol injections. The results indicated that DMS alone did not affect caspase-3 activation, but DMS attenuated ethanol-induced caspase-3 activation. Similar effects of DMS were observed using the one-time ethanol injection protocol (data not shown). It has been shown that caspase-3 activation in the P7 mouse brain that peaks around 8h after the ethanol injection leads to robust neurodegeneration detected by silver staining (Olney et al., 2002) and Fluoro-Jade staining (Ieraci and Herrera, 2006; Saito et al., 2010a). In order to assess if DMS attenuates ethanol-induced neurodegeneration, the effects of DMS on Fluoro-Jade C staining were examined in the cingulate and the retrosplenial cortex. Fig. 10A shows representative images of Fluoro-Jade C staining in the cingulate cortex region from control (Ctr), DMS, ethanol (Eth), ethanol+DMS (Eth+DMS) mice, and Fig. 10B shows the quantified results calculated from the images and expressed as Fluoro-Jade C-positive cell number per square millimeter. ANOVA with the Bonferroni's post hoc test showed that the "Eth+DMS" group was significantly different from all other groups, indicating that DMS treatment partially blocked ethanol-induced neurodegeneration assessed by Fluoro-Jade staining.</p><!><p>Our studies showed that ethanol treatment transiently increased SphK2 activity and S1P content in the P7 mouse brain prior to peak caspase-3 activation, and that pretreatment with DMS (an inhibitor of SphK) attenuated the caspase-3 activation and the subsequent neurodegeneration. As far as we know, this is the first report describing the effects of ethanol on the endogenous S1P metabolism in the brain. Under the present conditions, ethanol increased ceramide, sphingosine, and S1P in the P7 mouse brain (Fig. 5) along with inducing robust caspase-3 activation (Fig. 4). While the time course of sphingosine elevation was similar to that of ceramide, the elevation of S1P occurred transiently 4h after ethanol exposure. The similar transient activation of SphK2 by ethanol (Fig. 6A) shortly before the elevation of S1P levels strongly suggests that ethanol-induced SphK2 enzyme activation mediates S1P elevation. Since protein levels of SphK2 remained unaltered (Fig. 6B, C), post-translational modifications, such as phosphorylation described previously (Ding et al., 2007; Hait et al., 2007), may cause ethanol-induced SphK2 activation. The contribution of SphK1 to ethanol-induced elevation of S1P is expected to be minimal, because the SphK1 level was low in the brain (Fig. 1, 2), and the SphK1 enzyme activity, which was one fourth of the SphK2 activity, did not show any change by ethanol treatment. Our observation that SphK2 is the major SphK isoform in the mouse brain (Fig. 1, 2) agrees with previous studies (Blondeau et al., 2007, Pfeilschifter et al., 2011). The immunohistochemical data (Fig. 3) suggest that SphK2 is localized mainly in neurons as indicated in a previous study (Blondeau et al., 2007), while SphK1 seems mainly expressed in astrocytes (data not shown). The localization of SphK1 in astrocytes and microglia has also been reported by others (Lee at al., 2010; Nayak et al., 2010; Fischer et al., 2011). These results indicate that ethanol triggers S1P elevation via activation of SphK2 in neurons in the P7 mouse brain, although we cannot exclude the possibility that S1P is derived from other cell types, such as erythrocytes, microglia, and endothelial cells.</p><p>S1P regulates a wide variety of cellular processes, including growth, survival, differentiation, cytoskeletal rearrangements, angiogenesis, and immunity, and the level of S1P is mainly controlled by the SphK activity (Spiegel and Milstien, 2003). In contrast to the proliferative and anti-apoptotic effects of S1P produced by SphK1, S1P generated by SphK2 has been implicated to cause apoptosis and other cellular functions through intracellular targets in several cultured cells (Igarashi et al., 2003; Maceyka et al. 2005; Okada et al., 2009). Our subcellular fractionation studies showed that SphK2 was localized mainly in the P2 and P3 fractions but not in the nuclear fraction, and the localization was unchanged by ethanol treatment (Fig. 7). Further fractionation of P2 and P3 indicated that SphK2 was enriched in synaptic vesicles (identified by synaptophysin), synaptic membranes (identified by PSD95), and the plasma membrane (identified by Na+,K+-ATPase and flotillin) fractions (Fig. 8), although we cannot rule out the possibility that SphK2 was also localized in endosomes (identified by Rab5), endoplasmic reticulum (identified by ERp72), and other organelles which have similar densities. To the best of our knowledge, this is the first in depth subcellular fractionation study reporting the localization of SphK2 in the brain. It appears that the subcellular localization of SphK2 in the P7 brain is different from that reported mainly in cultured cells, where SphK2 is detected in the nucleus (Igarashi et al., 2003; Sankala et al., 2007; Ding et al., 2007; Hait et al., 2009), ER (Maceyka et al., 2005; Hagen et al., 2009), and mitochondria (Strub et al., 2011). Although the localization of SphK2 in the plasma membrane has been reported in some cell types (Maceyka et al., 2005), apoptotic functions of SphK2 are generally associated with the presence of SphK2 in the nucleus (Igarashi et al., 2003; Okada et al., 2005) and ER (Maceyka et al., 2005; Hagen et al., 2009). Interestingly, our results indicated that DMS, a SphK inhibitor, significantly attenuated ethanol-induced caspase-3 activation (Fig. 9) and neurodegeneration (Fig. 10), although further studies are necessary because DMS is not a specific inhibitor for SphK2 (French et al., 2010). Nonetheless, our results suggest that SphK2 primarily localized in or near the plasma membrane in neurons is activated by ethanol, produces S1P, and induces or enhances apoptotic action of ethanol. Because our previous studies have shown that inhibitors of serine palmitoyl transferase, a rate limiting enzyme for sphingolipid synthesis, attenuate ethanol-induced apoptosis (Saito et al., 2010a), de novo ceramide synthesis may be involved in the S1P formation catalyzed by SphK2. Also, the partial blocking of ethanol-induced neurodegeneration by DMS (Fig. 10) suggests that ceramide/sphingosine may enhance neurodegeneration independent of S1P action. It has been shown that S1P metabolism is affected under pathological and stressful conditions in the nervous system. For example, increases in the expression levels of SphK1 have been shown in kainic acid-treated hippocampal astrocytes (Lee et al., 2010) and lipopolysaccaride-treated cultured microglia (Nayak et al., 2010), whereas increases in SphK2 expression or activity have been reported in cerebral microvessels under hypoxic preconditioning (Wacker et al., 2009), in the ischemic brain (Blondeau et al., 2007), and in the brains of patients with Alzheimer's disease (Takasugi et al., 2011). While S1P elevation produced by SphK2 is considered apoptotic in the cerebellar granule neurons derived from S1P lyase-deficient mice (Hagen et al., 2009) as well as in other non-neural cells (Liu et al., 2003; Okada et al., 2005; Maceyka et al. 2005), SphK2 activation is found neuroprotective in some animal models of brain ischemia (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011; Yung et al., 2012). Whether SphK2 activation leads to neuroprotection or not may depend on the subcellular targets of S1P produced. The efficacy of S1P receptor agonists in neuroprotection in some of these studies (Wacker et al., 2009; Hasegawa et al., 2010; Pfeilschifter et al., 2011) suggests that S1P produced by SphK2 may activate S1P receptors, leading to neuroprotection, or S1P in mitochondria may exert cytoprotection as indicated in a myocardial injury model (Gomez et al., 2011). S1P increased by ethanol treatment in our study may have different targets, inducing or enhancing neuroapoptosis.</p><p>Our observation that S1P1-R, a major S1P receptor in the brain (Brinkmann, 2007), was mainly localized in astrocytes as reported previously (Nishimura et al., 2010) suggests that S1P produced by SphK2 in neurons may exert its function independent of S1P1-R. S1P may interact with other S1P receptor isoforms or may have other targets, such as Na+,K+-ATPase as recently reported (Dakroub and Kreydiyyeh et al., 2012).</p><p>In summary, we demonstrated that ethanol transiently elevated SphK2 activity and S1P levels in the P7 mouse brain, which may be related to ethanol-induced apoptotic neurodegeneration.</p>

PubMed Author Manuscript

Programming hydrogel with classical conditioning algorithm

Living systems are essentially out of equilibrium, where concentration gradients are kinetically controlled by reaction networks that provide spatial recognitions for biological functions. They have inspired life-like systems using supramolecular dynamic materials and systems chemistry. Upon pursuing ever more complex life-inspired systems, mimicking the ability to learn would be of great interest to be implemented in artificial materials. We demonstrate a soft hydrogel model system that is programmed to

programming_hydrogel_with_classical_conditioning_algorithm

<!>Results and discussion<!>Conclusion

<p>algorithmically mimic some of the basic aspects of classical Pavlovian conditioning, the simplest form of learning, driven by the coupling between chemical and physical processes.</p><p>The gel can learn to respond to a new, originally neutral, stimulus upon classical conditioning with an unconditioned stimulus. Further subtle aspects of Pavlovian conditioning, such as forgetting and spontaneous recovery of memory, are also achieved by driving the system outof-equilibrium. The present concept demonstrates a new approach towards dynamic functional materials with "life-like" properties.</p><p>Biological systems have inspired biomimetic materials with fascinating properties, e.g., toughness, structural colors, catalytic activity, and superhydrophobicity 1,2 . In future, materials are foreseen to mimic ever more complex functional and responsive properties of dynamic biological systems. Various model systems for dynamic dissipative out-of-equilibrium self-assemblies have been presented [3][4][5][6] . To allow a major progress towards "life-like" materials, the importance of 2 systems chemistry has been emphasized for the structural and temporal programming of the functionalities by coupled chemical reactions under out-of-equilibrium conditions [7][8][9][10][11] .</p><p>One of the most relevant biological functions deals with the concept of "learning" 12 . In its full biological form, it involves formidable complexity. However, systematic approaches have shed light on its simplest forms, i.e., habituation, sensitization, and classical conditioning 13 .</p><p>Adopting a still more reductionist approach, a generic question could be posed: whether even inanimate materials could be designed to show programmed responses mimicking some elementary aspects of learning, in resemblance to systems based on synaptic electronics or biochemical circuits [14][15][16][17] .</p><p>To address the above question, we explore whether artificial materials could be designed to show responses that algorithmically mimic the classical (Pavlovian) conditioning 18,19 . In Pavlov's seminal experiment, an unconditioned dog salivates (unconditioned response, UR) upon seeing food (unconditioned stimulus, US), while ringing a bell (neutral stimulus, NS) does not lead to salivation. However, upon conditioning by simultaneously ringing the bell and showing the food, a conditioned response (CR) to the neutral signal is associatively learned, after which salivation can be triggered also by the bell. The process involves the association of the two stimuli stored in the memory, so that the original response can be triggered by a new stimulus.</p><p>Hydrogels have been shown to be relevant model systems for out-of-equilibrium and systems chemistry 5,9 . Here we introduce a hydrogel that is programmed to mimic classical conditioning, which allows melting of the gel in response to an originally neutral stimulus (light) upon associating the light with an unconditioned stimulus (heat). Further aspects of the classical conditioning, such as forgetting and spontaneous recovery of memory after extinction, were 3 mimicked by temporally programming the gel response using coupled chemical reactions, which drives the hydrogel out-of-equilibrium.</p><!><p>Programming hydrogel with classical conditioning algorithm. The hydrogel consists of pHsensitive gold nanoparticles (AuNPs) embedded in a soft hydrogel network of agarose, and a merocyanine-based photoacid (Fig. 1a). The photoacid allows reversible photo-switching of the solution pH between 5.4 and 3.8 in the aqueous solution (0.2 mM) upon irradiation in the visible range (Supplementary Fig. 1) 20,21 . The AuNPs are modified by lipoic acid chosen to respond to pH changes caused by the photoacid 22 . The composition of the system, such as the size of AuNPs and the gel concentration, has been optimized for fast response under mild conditions (Supplementary Fig. 2-6). The Pavlovian response of the hydrogel is shown in Fig. 1 b-e. Heating (US) above the melting temperature (Tm ~ 33 °C, Supplementary Fig. 7) induces gel melting as the unconditioned response (Fig. 1b), whereas the AuNPs remain well dispersed as confirmed by TEM imaging and UV-Vis measurement. A clear plasmonic band can be seen at 520 nm before and after heating (Fig. 1b, right panel). This is due to the sufficient electrostatic stabilization of the carboxyl groups at pH above 5, since heating doesn't affect the pH of the gel significantly (Supplementary Fig. 1).</p><p>As the neutral stimulus we used laser irradiation at 635 nm (140 mW cm -2 ), mixed with 455 nm LED light (25 mW cm -2 ). The photoacid absorbs strongly in the range between 380 nm and 460 nm, so that efficient photoacid activation can be achieved by the LED light. Besides, the intensity of the LED is chosen to be just sufficient for the activation of photoacid, in order to avoid contributing significantly to the photothermal heating inside the gel. Under irradiation, the original unconditioned gel does not melt due to the low absorption at 635 nm and thus insufficient heating (Fig. 1c, Supplementary Fig. 8). Note that in this case the interparticle electrostatic repulsion is in 4 fact also reduced upon the pH decrease, but the plasmonic band remains at 520 nm, suggesting dispersed particles due to the stabilizing gel matrix. The presence of the gel network hinders the diffusion of the AuNPs, and thus no significant self-assembly into chains takes place even when the photoacid is activated.</p><p>The crucial step to achieve conditioning is the self-assembly of the AuNPs by simultaneous exposure to light and heat (Fig. 1d). Once the gel melts upon heating, the AuNPs regain mobility and self-assemble into linear aggregates triggered by the irradiation-induced pH change (Supplementary Fig. 9, 10). The formation of linear assemblies of AuNPs is dependent on different parameters such as ligand composition, pH, solvent, or salt 23 . In our system, the use of lipoid acid is important to achieve the linear self-assembly. It has been proposed that anisotropic electrostatic repulsion of AuNP dimers formed in the initial aggregation stage accounts for the rather linear configuration of the aggregates 24 . In the gel, the linear self-assemblies of AuNPs remain stable even after the pH recovery (light off) and re-gelation of the agarose (Fig. 1d), which we attribute mainly to interparticle hydrogen bonding and/or van der Waals attraction 25 . The self-assembled AuNPs can be only separated by increasing the pH of the solution to above 8, as we show in the later sections. The resulting spectral change of self-assembly is the appearance of a new plasmonic band around 635 nm due to the coupling of the AuNPs in the linear self-assemblies 26 . Note that non-specific aggregation would not result in a defined new band at longer wavelengths. This spectral change serves as the memory, which enables significantly enhanced photothermal heating at 635 nm due to the thermoplasmonic properties of AuNPs 27,28 , and the gel thus melts upon irradiation (Fig. 1d,e). Hence the system has evolved to a new state, where upon conditioning the AuNPs are self-assembled from individual particles into chains, and the gel melting occurs upon the newly learned stimulus, i.e. light irradiation (CS). For the generalization of the Pavlovian concept, Fig. 2 suggests the underlying logic circuit for the Pavlovian gel. In order to "learn", the material must possess a memory module that can be triggered by external stimuli, and a read-out mechanism that modifies the behavior upon switching of the memory 16 . In the gel, the memory is the spectral change due to the linear self-assembly of AuNPs that can be switched on by conditioning ("learning" AND gate). This AND gate is achieved by the incorporation of the AuNPs/photoacid pair into the gel network, so that the self-assembly of the AuNPs is only possible when light and heat are both present. This ensures that learning is exclusively based on the association of two stimuli. The OR gate is necessary to sustain the memory once it has been switched on, which is accomplished by the stable self-assembly of AuNPs in the gel. On the other hand, the "recalling" AND gate is achieved by the photothermal effect due to the coupled surface plasmon resonance of the AuNPs, through which the material is able to respond to irradiation. Importantly, the Pavlovian gel can be distinguished from the conventional shape memory materials, where the memory is the equilibrium permanent shape that can be recovered from a temporary shape in the kinetically trapped non-equilibrium state. Shape memory materials do not really "learn" to respond to a new stimulus. Timing dependence of the conditioning process. In biological systems undergoing Pavlovian conditioning, the efficiency of learning is highly dependent on the timing between applied stimuli. US and NS may take place simultaneously, the NS may precede the US, or the US may precede the NS, denoted as simultaneous, forward, or backward conditioning, respectively. Forward and simultaneous conditioning are the fastest, while backward conditioning is less effective or even inhibitory 29 . This is presumably because that the NS no longer predicts the appearance of US in the case of backward conditioning, where the NS is applied after the US 29 , so that such an association will not be beneficial to the organism. The association process in the Pavlovian gel shown in Fig. 3a-c is in line with such observations. When the irradiation (NS) precedes or coincides with the onset of heat (US), the "learning efficiency", as manifested by the increase in absorbance at 635 nm of the gel, is comparable in forward and simultaneous conditioning (Fig. 3a,b). In contrast, backward conditioning is less effective, and the increase of absorbance is roughly 70% of that resulting from the simultaneous conditioning process (Fig. 3c). This can be attributed to the viscosity increase of the solution upon removal of the heat 30 , which slows down the diffusion and thus the self-assembly of the AuNPs. Consequently, after backward conditioning the gel does not melt upon irradiation, since the temperature stays below the Tm of the gel, though the photothermal effect is stronger compared to the unconditioned gel (Supplementary Fig. 11).</p><p>The ability to differentiate the temporal relationship between the unconditioned and neutral stimuli is intriguing, reminiscent to the timing dependence of classical conditioning 29 . Forgetting and spontaneous recovery of the memory using out-of-equilibrium processes. The above results allow to mimic some aspects of the classical conditioning in equilibrium state, where the memory stays unchanged after conditioning. In a step further, we expect that programming the time domain of the response and potentially driving the system out-of-equilibrium are needed to mimic more subtle aspects. The classical conditioning may involve several stages, such as acquisition, extinction, and spontaneous recovery 18,31 . In Fig. 4, we show the possibility of temporally programming the memory of the Pavlovian gel to achieve "forgetting" and "spontaneous recovery" using coupled chemical reactions. In Fig. 4a-c, the gel contains additional 20 mM of urea and 5 µg mL -1 of urease as an internal "clock" to trigger the forgetting process. The urease catalyzes the hydrolysis of urea, resulting in the production of 2 eq. of ammonia and 1 eq. of carbon dioxide. The temporal profile of the solution pH can thus be programmed depending on the ratio and concentration of the two components 32 . In the gel, the pH remains almost unaffected by the presence of urea/urease on the time scale of conditioning (~ 10 min), which thus enables acquisition of the memory. Yet the conditioned gel is not in the equilibrium state. The forgetting process takes place as the result of urea hydrolysis, which slowly increases the pH to around 8.8 in 12 hours. The gel was left in the liquid state to facilitate the disassembly of the AuNPs triggered by the pH change, resulting in the drop of absorbance (memory) as shown in Fig. 4b,c. The high pH required for the disassembly could be the result of interparticle van-der-Waals attraction / hydrogen bonding that has to be counter-balanced by strong electrostatic repulsion from the deprotonation of the carboxyl groups on the AuNP surfaces. The recovery of the absorbance at 635 nm is significant, indicating that the AuNPs are well protected by the ligands during the selfassembly process. In addition, the absorbance around 450 nm dropped as a result of the deprotonation of photoacid at high pH. The forgetting profile is reminiscent of the Ebbinghaus' forgetting curve: the memory decreases with time, yet a small portion of it is retained over an extended period 33 . As a result, the gel no longer melts upon irradiation (Supplementary Fig. 11), and can be considered as having forgotten the conditioning. The conditioned memory can also be extinguished by an external stimulus followed by a spontaneous recovery (Fig. 4d-f). The extinction is induced by a chemical cue containing a potassium phosphate buffer (K3PO4) and methyl formate, offering a new possibility to control the memory inspired by the extinction process in biological systems, which is triggered by repeated exposure to the NS without US. After addition of the chemical cue to the conditioned gel, the pH of the melted gel increases instantaneously to 11.8 due to the buffer (20 mM), which leads to disassembly of the AuNPs and thus fast extinction of the memory. Subsequently, the spontaneous hydrolysis of the methyl formate (240 mM) results in the formation of formic acid and thus a controlled decrease of the pH to below 5.0 in 20 hours 34 . As the pH decreases, self-assembly of the AuNPs again takes place spontaneously, where the gel was kept in liquid state to allow diffusion of the AuNPs. The absorbance at 635 nm therefore gradually recovers after the extinction (Fig. 4e). The decrease of the absorbance around 530 nm is due to the protonation of the photoacid during the recovery. This kinetically controlled pH change using phosphate buffer and methyl formate thus enables the extinction of the memory and the following spontaneous recovery. After extinction, the gel does not respond to irradiation, but subsequently regains the ability to melt upon irradiation once recovered (Supplementary Fig. 11).</p><p>The memory processes shown in Fig. 4 are essentially out-of-equilibrium, in contrast to the equilibrium "learning" demonstrated in Figs. 1-3, where the memory remains unchanged after conditioning. The acquired memory through conditioning is only in a temporally stable state in Fig. 4a-c, which gradually decays with time due to the kinetically controlled chemical reaction.</p><p>The system reaches equilibrium only after the memory is mostly lost (disassembly of the AuNPs).</p><p>The same applies to the process in Fig. 4d-f, where the memory is temporarily extinguished and then recovers spontaneously. These results demonstrate the possibility to further manipulate the (primitive) memory of the gel system, inspired by real cognitive processes that operates under outof-equilibrium conditions.</p><!><p>Summarizing, we have shown that an inanimate soft hydrogel can be designed to exhibit responses resembling classical conditioning, which has been considered as one of the elementary forms of learning. Therein, the material learns to respond to an initially neutral stimulus (light) through an associative conditioning process, during which the material is exposed to both neutral (light) and unconditioned (heat) stimuli. Forgetting and recovery of memory can be achieved by introducing kinetically controlled coupled chemical reactions to the system, which can be discussed in the context of systems chemistry and out-of-equilibrium processes. The Pavloviantype sol-gel transition in hydrogels may find applications, e.g., in intelligent drug delivery or cell culture, and the concept may be extended to other material systems beyond gels following the demonstrated logic flow diagram, based on other functional groups and fields, e.g., using magnetic fields. Admittedly, the content of learning in the demonstrated Pavlovian hydrogel is prescribed to preselected stimuli when compared to the complex adaptive behavior in biological systems capable of responding to a wide variety of stimuli 35 . Yet our systems offer selectivity towards stimuli, and allow considerable flexibility for new properties and functions to be engineered (e.g., forgetting/recovery). The possibility to program Pavlovian conditioning in a hydrogel, even including forward and backward conditioning, as well as forgetting/recovery under out-ofequilibrium conditions, is conceptually intriguing. We envision that designing complex conditioning behaviors coupled with engineered physical properties of materials may provide</p>

ChemRxiv

Green tea polyphenol epigallocatechin-3-gallate suppresses melanoma growth by inhibiting inflammasome and IL-1\xce\xb2 secretion

Epigallocatechin-3-gallate (EGCG), the major polyphenolic component of green tea, has been demonstrated to possess anti-inflammatory, antioxidant, anti-mutagenic and anti-carcinogenic properties. The anti-melanoma effect of EGCG has been previously suggested, but no clear mechanism of action has been established. In this study, we demonstrated that EGCG inhibits melanoma cell growth at physiological doses (0.1\xe2\x80\x931 \xce\xbcM). In the search for mechanisms of EGCG-mediated melanoma cell suppression, we found that NF-\xce\xbaB was inhibited, and that reduced NF-\xce\xbaB activity was associated with decreased IL-1\xce\xb2 secretion from melanoma cells. Since inflammasomes are involved in IL-1\xce\xb2 secretion, we investigated whether IL-1\xce\xb2 suppression was mediated by inflammasomes, and found that EGCG treatment led to downregulation of the inflammasome component, NLRP1, and reduced caspase-1 activation. Furthermore, silencing the expression of NLRP1 abolished EGCG-induced inhibition of tumor cell proliferation both in vitro and in vivo, suggesting a key role of inflammasomes in EGCG efficacy. This paper provides a novel mechanism for EGCG-induced melanoma inhibition: inflammasome downregulation \xe2\x86\x92 decreased IL-1\xce\xb2 secretion \xe2\x86\x92 decreased NF-\xce\xbaB activities \xe2\x86\x92 decreased cell growth. In addition, it suggests inflammasomes and IL-1\xce\xb2 could be potential targets for future melanoma therapeutics.

green_tea_polyphenol_epigallocatechin-3-gallate_suppresses_melanoma_growth_by_inhibiting_inflammasom

1. Introduction<!>2.1. Cell culture and EGCG treatment<!>2.2. Cell viability assay<!>2.3. Lactate dehydrogenase (LDH) cytotoxicity assay<!>2.4. NF-\xce\xbaB activity determination<!>2.4.1. IL-1\xce\xb2 and IL-18 production and secretion determinations<!>2.4.2. Western blotting analysis<!>2.4.3. Short hairpin RNA (shRNA) transfection<!>2.4.4. In vivo study<!>2.4.5. Statistics<!>3.1. EGCG inhibits proliferation of human metastatic melanoma cells in vitro<!>3.2. NF-\xce\xbaB activity is suppressed by EGCG in human metastatic melanoma cells<!>3.3. EGCG reduces IL-1\xce\xb2 secretion but not production, and downregulates NLRP1 inflammasome in human metastatic melanoma cells<!>3.3.1. Growth inhibition by EGCG is abolished after NLPR1 knockdown<!>4. Discussion

<p>Melanoma is the most deadly type of skin cancer and its prevalence continues to rise [1], making it crucial to search for effective therapeutics. It is increasingly recognized that cancer, including melanoma, represents an inflammatory condition [2]. Human melanoma cells secrete many inflammatory cytokines and chemokines that are associated with cancer invasiveness and aggressiveness [3]. Therefore, targeting inflammation may provide us with a new line of therapeutics for melanoma. Among the many inflammatory mediators involved in melanoma development and progression, IL-1β has been shown to be one of the critical cytokines mediating tumor growth, progression, immunosuppression and chemoresistance [4,5]. In metastatic melanoma cells, IL-1β is constitutively secreted and activated, mediating macrophage chemotaxis, angiogenesis, and sustained melanoma growth [6]. IL-1β is synthesized as an inactive precursor (pro-IL-1β) which is cleaved to the biologically active and secreted form by caspase-1 [7]. Caspase-1, in turn, is regulated by a multi-protein complex termed the inflammasome. The inflammasomes are responsible for the recruitment and activation of caspase-1 [8]. Each inflammasome complex consists of a nucleotide oligomerization domain-like receptor (NLR) component, an ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)) component, and caspase-1. Assembly of the inflammasomes is triggered by pathogen or danger-associated molecular patterns, which cause oligomerization of NLRs. Oligomerized NLRs interact with ASC, which in turn cleaves pro-caspase-1 into active caspase-1 [9]. NLRP (NACHT-, LRR- and pyrin-domain-containing protein) 1 and NLRP3 inflammasomes are two of the best characterized human inflammasomes. NLRP1 contains a CARD domain that can directly interact with inflammatory caspases-1, and thus ASC may not be required for NLRP1-mediated caspase-1 activation [10]. In melanoma cells, NLRP3 inflammasome is constitutively assembled and activated, suggesting its importance in tumorigenesis [6].</p><p>(−)-Epigallocatechin-3-gallate (EGCG) is the major polyphenol component of green tea responsible for its biological effects. It has been shown to inhibit various inflammatory enzymes and cytokines, including iNOS, COX2, MMPs, IL-6, IL-8, IL-12 and TNFα, all of which are induced by secreted active IL-1β [11-13]. It has also been suggested that EGCG has an anti-tumor effect in melanoma cells, but previous reports have utilized doses of EGCG (20–100 μM) much higher than the physiological concentrations[14,15]. Consuming 5–6 cups of green tea a day leads to a serum concentration of EGCG of only about 1 μM [16], and drinking up to 8–16 cups of green tea a day is known to be safe [17].</p><p>In this study we investigated whether physiological concentrations of EGCG (0.1–1 μM) can inhibit melanoma cells. We present evidence that physiological doses of EGCG have an inhibitory effect on the proliferation of human metastatic melanoma cell lines. Furthermore, we demonstrated that EGCG suppresses NF-κB activity and reduces IL-1β secretion. The decreased IL-1β is associated with downregulation of NLRP1, a component of the inflammasomes, and reduced caspase-1 activation. The inhibitory effect of EGCG on tumor proliferation was abolished by silencing NLRP1, suggesting a key role of inflammasomes in the tumor-inhibitory effect of EGCG in human melanoma cells.</p><!><p>Human metastatic melanoma cell lines 1205Lu and HS294T were obtained from the American Type Culture Collection. 1205Lu cells were maintained in RPMI 1640 medium (Mediatech, Inc.), and HS294T cells in Dulbecco's modified Eagle's medium (Mediatech, Inc.). Both media were supplemented with 10% fetal bovine serum (FBS, Mediatech, Inc.), 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM l-glutamine. Cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.</p><p>EGCG (≥95% purity, Sigma–Aldrich) was dissolved in dimethyl sulfoxide (DMSO) to a stock concentration of 100 mM (45.837 mg/ml). EGCG was then further diluted in phosphate-buffered saline (PBS) to varying concentrations (0.1, 1, or 10 μM, which are 0.046, 0.46 and 4.6 lg/ml, respectively) prior to use in cell culture. Due to the short half-life of EGCG in vitro, EGCG was added every 8–10 h. DMSO diluted in PBS (0.01% DMSO, corresponding to the amount contained in 10 μM EGCG) was used for vehicle control.</p><!><p>About 4 × 103−5 × 103 cells/well were plated in flat-bottom 96-well plates and treatment with EGCG (0.1, 1, or 10 μM) was started 4 h after plating. Cells were incubated for up to 96 h. Cell viability was determined by the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega).</p><!><p>2.5 × 104 Cells/well were plated in 24-well plates and treatment with EGCG (0.1, 1, or 10 μM) was started 4 h after plating. Cells were incubated for up to 96 h. Supernatants and lysates were then transferred to 96-well plates to complete the assay. Cytotoxicity was determined using the Cytotox96® Non-Radioactive Cytotoxicity Assay (Promega). Cytotoxicity was calculated as follows: %cytotoxicity = 100% × (experimental LDH − spontaneous LDH)/(maximum LDH − spontaneous LDH).</p><!><p>2.5 × 104 Cells/well were plated in 24-well plates. After an overnight incubation, cells were transfected with a control vector, pMetLuc2-Reporter (Clontech) or an NF-κB vector, pNFκB-Met-Luc2 Reporter (Clontech) in Opti-MEM® (Invitrogen). After 4 h, medium was changed to supplemented RPMI 1640, and cells were treated with EGCG (0.1, 1, or 10 μM). EGCG was added every 8 h. After 24 h, supernatants were collected and luciferase activity was measured using the Ready-to-Glow™ Secreted Luciferase Reporter Assay (Clontech).</p><!><p>1.2 × 104 Cells/well were plated in 48-well plates. After an overnight incubation, medium was changed to Opti-MEM® (Invitrogen), and EGCG (0.1, 1, or 10 μM) was added every 8 h. After 24 h, supernatants were collected to assess secreted IL-1β. To assess synthesized IL-1β, cells were lysed with 0.5% Triton X-100 in PBS and subjected to a freeze-thaw cycle.</p><p>Supernatants and cell lysates were analyzed using the Human IL-1β/IL-1F2 DuoSet ELISA Development System (R&D Systems) and Human IL-18 ELISA Kit (MBL International Corporation).</p><!><p>Cells were incubated with EGCG (1 μM) for 24 h. EGCG was added every 8 h. Supernatants were collected for analysis of secreted protein. Cells were lysed on ice in 1X SDS sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromophenol blue) supplemented with protease inhibitor mixture (Roche Applied Science). Lysates were heated to 95 °C for 5 min. About 20 μg lysates were then loaded into SDS–PAGE gel (Invitrogen), and proteins were separated by electrophoresis. Proteins were transferred onto polyvinylidene difluoride membranes (0.4 μm) in 25 mM Tris, 192 mM glycine, and 20% methanol at 60 V for 1.5 h. Blots were incubated with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies. Blots were then developed with horseradish peroxidase substrate (West Femto Solution from Thermal Fisher Scientific, Inc.) and analyzed using Gel-Doc 200 (Bio-Rad).</p><!><p>1205Lu cells were transfected with shRNA Lentiviral Particles against control and NLRP1 (Santa Cruz Biotechnology, Inc.) in supplemented RPMI 1640 containing 5 μg/ml Polybrene (Santa Cruz Biotechnology, Inc.). Following overnight transduction, cells were incubated in supplemented RPMI 1640 with 1 lg/ml puromycin to select for stable clones expressing transduced shRNA. Transfected 1205Lu cells were maintained in supplemented RPMI 1640 with 1 μg/ml puromycin at 37 °C.</p><!><p>Six-week-old female athymic nu/nu mice were obtained from the National Cancer Institute. Animals were kept under specific pathogen-free conditions, according to National Institutes of Health Animal Care Guidelines. Experimental protocols were approved by the Institutional Animal Care and Use Committee of University of Colorado Denver. 1205Lu cells transfected with control shRNA or NLRP1 shRNA were re-suspended in Matrigel (BD Biosciences) 1:1 diluted with PBS. Mice were injected subcutaneously with the melanoma cells, with two implantations (1 × 106 cells/implantation) in each mouse. Tumors were allowed to grow to a size of 100 mm3, at which time mice were randomized into two groups and treated with vehicle (10% DMSO in saline) or EGCG (20 mg/kg dissolved in 10% DMSO in saline) intraperitoneally daily for 17 days. Tumor size was evaluated three times a week by caliper measurements using the following formula: tumor volume = (longest diameter) × (shortest diameter)2/2. Relative tumor growth was calculated by tumor volume of treated mice divided by tumor volume at the initiation of therapy.</p><!><p>Statistically significant differences were determined using Student's unpaired t-test. Differences were considered statistically significant if p < 0.05.</p><!><p>Two different human metastatic melanoma cell lines, 1205Lu and HS294T cells, were treated with varying concentrations of EGCG (0.1, 1, or 10 μM) for 24, 48, 72, and 96 h, followed by cell viability/proliferation analysis. In both 1205Lu (Fig. 1A) and HS294T (Fig. 1B) cells, cell proliferation was significantly inhibited by increasing concentrations of EGCG in a dose-dependent manner (*p < 0.05 relative to control, * denotes every value below it is significantly different from control). In addition, LDH levels were not significantly different from control following treatment with different concentrations of EGCG (data not shown), suggesting that EGCG suppresses melanoma cell growth in vitro via a mechanism other than cell death.</p><!><p>Since NF-κB plays a major role in melanoma cell proliferation [18], the activity of NF-κB was assessed following EGCG treatment. Both melanoma cell lines, 1205Lu cells and HS294T cells, after being transfected with pNFκB-MetLuc2 Reporter Vector, were treated with varying concentrations of EGCG (0.1, 1, or 10 μM) followed by NF-κB activity determination. In both transfected 1205Lu (Fig. 2A) and HS294T (Fig. 2B) cells, treatment with EGCG led to dose-dependent inhibition of NF-κB activity (*p < 0.05).</p><!><p>IL-1β is constitutively secreted by metastatic melanoma cells and plays a significant role in tumor development and progression [6]. Because IL-1β signaling leads to activation of NF-κB, we determined whether EGCG can decrease the activity of IL-1β. As indicated earlier, secreted IL-1β is the active form. 1205Lu cells and HS294T cells were treated with different concentrations of EGCG (0.1, 1, 2 or 10 μM) for 24 h, and IL-1β levels were determined in both lysates and supernatants. Whereas EGCG treatment did not change the synthesis of IL-1β, it significantly decreased the secretion of IL-1β in melanoma cells (1205Lu cells represented in Fig. 3A and B, and HS294T cells in Fig. 3C and D, *p < 0.05), suggesting that the secretion mechanism was altered by EGCG treatment.</p><p>As IL-1β secretion is tightly controlled by inflammasomes, we investigated inflammasome components (NLRP1, NLRP3, ASC and caspase-1) following EGCG treatment. 1205Lu cells were treated with 1 μM EGCG for 24 h, and lysates and supernatants were subjected to western blot analysis. EGCG treatment led to decreased levels of NLRP1 in both lysates and supernatants (Fig. 3E). Furthermore, this downregulation of NLRP1 was shown to be specific, as NLRP3 and ASC levels were not changed. To determine whether the decreased NLRP1 was associated with functional impairment of inflammasome, the levels of its downstream molecule, caspase-1, were determined. Full-length caspase-1 (p45) is cleaved to p10 to be active. As shown in Fig. 3E, EGCG reduced active p10 in the cell lysates and supernatants, suggesting that EGCG-mediated NLRP1 reduction was accompanied by decreased inflammasome function. Because active caspase-1 processes pro-IL-1β and pro-IL-18 to the bioactive forms, we investigated levels of synthesized and secreted IL-18 in melanoma cells in response to EGCG. Intracellular levels of IL-18 were found to be ≥100 times less than those of IL-1β in melanoma cells (1205Lu and HS294T cells), and EGCG did not change the intracellular IL-18 levels. In addition, secreted IL-18 was undetectable in these cells (data not shown), suggesting a role of inflammasome-mediated IL-1β but not IL-18 in the tumor-inhibitory effect of EGCG in human melanoma cells.</p><!><p>To determine whether EGCG-induced inhibition of proliferation is due to inflammasome downregulation, 1205Lu cells were transfected with NLRP1 shRNA to silence NLRP1 (Fig. 4A). The transduced 1205Lu-NLRP1-shRNA cells no longer showed growth inhibition by EGCG, suggesting that the inhibitory effect of EGCG was inflammasome-dependent (Fig. 4B and C, *p < 0.05 relative to control, * denotes every value below it is significantly different from control).</p><p>We then determined whether the same effect can be observed in vivo. Nude mice were injected with 1205Lu-control-shRNA cells or 1205Lu-NLRP1-shRNA cells and treated with EGCG at 20 mg/kg intraperitoneally (i.p.) daily. This dose is physiologic as plasma EGCG concentration measured an hour after i.p. injection of 10 mg/kg EGCG was 138 ± 44 ng/ml (0.3 ± 0.1 μM) [19]. EGCG treatment significantly suppressed tumor growth in mice injected with 1205Lu-control-shRNA cells, but not in mice injected with 1205Lu-NLRP1-shRNA cells, confirming that the inhibition of tumor growth by EGCG requires inflammasomes (Fig. 4D, *p < 0.05 relative to vehicle-shControl).</p><!><p>Our recent work indicates that melanoma, especially metastatic melanoma, represents an autoinflammatory state characterized by constitutive activation of inflammasomes and IL-1R signaling [6]. Given the strong link between inflammation and melanoma, and the role of IL-1β in tumor growth, strategies that can inhibit IL-1β secretion would be logical candidates for melanoma therapeutics. In this study, we demonstrated that EGCG, a potent anti-inflammatory chemical, may be one of these candidate molecules. Using doses that are practical, in the 0.1–1 μM range, we found that EGCG decreases metastatic melanoma cell growth, both in vitro and in vivo. In addition, IL-1β secretion is decreased, likely due to specific downregulation of inflammasome molecule, NLRP1, and subsequent inhibition of caspase-1 activity. Furthermore, NLRP1 downregulation was demonstrated to play an important role in EGCG-induced growth inhibition since silencing NLRP1 abolished the EGCG effects on growth. The reduced secretion of IL-1β likely contributes to inactivation of NF-κB, which then leads to decreased cell proliferation. To our knowledge, this is the first publication demonstrating the effect of EGCG on inflammasomes in cancer research. The only other report which examined the impact of EGCG on inflammasomes was a study which demonstrated preventive effects of EGCG in lupus nephritis mice via NLRP3 inhibition [20]. Our study did not demonstrate NLRP3 inhibition; this discrepancy could result from different cell types (renal cortex cells vs. melanoma cells) and different doses of EGCG (120 mg/kg vs. 20 mg/kg in our study).</p><p>The link between inflammasomes and melanoma is only recently beginning to be appreciated. In addition to above-mentioned constitutive expression of inflammasomes in melanoma, it was reported that inflammasomes in the melanoma tumor micro-environment diminishes the endogenous antitumor immune response and facilitates tumor growth [21]. These data further support inflammasome activity as a putative target for melanoma treatment.</p><p>A few other studies have also investigated the effect of EGCG in melanoma cells. Nihal and colleagues demonstrated induction of apoptosis by EGCG [22], which is in discordance with our data which showed that reduced cell viability is due to decreased cell growth but not cell death. This discrepancy could be explained by the higher concentrations of EGCG used in their study (2.2–22 μM). In addition, even though both studies used the HS294T melanoma cells, characteristics of cells may differ depending on the number of passages. Indeed, Ravindranath and colleagues recently demonstrated differential effects of green tea catechins on different melanoma cell lines [14]. To make matters more complex, EGCG has been shown to block apoptosis in other melanoma cell lines [23].</p><p>The mechanisms by which EGCG inhibits inflammasomes are unclear. Previous studies have identified a 67-kDa laminin receptor as a cell surface receptor for EGCG [24], which could play a role in the pathway leading to inflammasome downregulation. Since EGCG is a potent antioxidant [25], modulation of the redox balance in melanoma tumor cells could also potentially lead to changes in inflammasomes.</p><p>Developing EGCG into a practical therapeutic agent may require an interdisciplinary approach in order to modify the structure of EGCG and improve its potency and pharmacokinetic properties. For example, one of the methylation products, 7-OMe EGCG, was shown to have equal efficacy but increased bioavailability when compared to EGCG [26]. Given the aggressive nature of melanomas, effective therapies may most likely involve a combinational approach. In fact, EGCG has been shown to have a stronger anti-melanoma effect when used in conjunction with vitamin A [27], vorinostat [28], interferon [29], DNA vaccination [30], dacarbazine [31], and even red light [32]. This combinational treatment will likely decrease the doses required for either medication, and consequently decrease the adverse effects associated with these therapies.</p><p>In conclusion, this research not only proposes a novel treatment for melanoma, but also provides mechanistic data that can be applied to various other inflammatory and malignant disorders in which IL-1β and inflammasomes play a major role.</p>

PubMed Author Manuscript

Heterogeneous 1H and 13C Parahydrogen-Induced Polarization of Acetate and Pyruvate Esters

Magnetic resonance imaging of [1-13C]hyperpolarized carboxylates (most notably, [1-13C]pyruvate) allows one to visualize abnormal metabolism in tumors and other pathologies. Here we investigate the efficiency of 1H and 13C hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties using heterogeneous hydrogenation of corresponding vinyl, allyl and propargyl precursors in isotopically unlabeled and 1-13C-enriched forms with parahydrogen over Rh/TiO2 catalysts in methanol-d4 and in D2O. The maximum obtained 1H polarization was 0.6 \xc2\xb1 0.2% (for propyl acetate in CD3OD), while the highest 13C polarization was 0.10 \xc2\xb1 0.03% (for ethyl acetate in CD3OD). Hyperpolarization of acetate esters surpassed that of pyruvates, while esters with a triple carbon-carbon bond in unsaturated alcoholic moiety were less efficient as parahydrogen-induced polarization precursors than esters with a double bond. Among the compounds studied, the maximum 1H and 13C NMR signal intensities were observed for propyl acetate. Ethyl acetate yielded slightly less intense NMR signals which were dramatically greater than those of other esters under study.

heterogeneous_1h_and_13c_parahydrogen-induced_polarization_of_acetate_and_pyruvate_esters

Introduction<!>General remarks.<!>Hyperpolarization of ethyl acetate by HET-PHIP-SAH.<!>Hyperpolarization of propyl acetate by HET-PHIP-SAH.<!>Hyperpolarization of allyl acetate by HET-PHIP-SAH.<!>Hyperpolarization of ethyl pyruvate by HET-PHIP-SAH.<!>Hyperpolarization of propyl pyruvate by HET-PHIP-SAH.<!>Hyperpolarization of allyl pyruvate by HET-PHIP-SAH.<!>Efficiency of HET-PHIP-SAH of acetates and pyruvates.<!>Conclusion<!>Materials.<!>PHIP experiments.

<p>Hyperpolarization techniques enable dramatic increase in sensitivity of NMR spectroscopy and magnetic resonance imaging (MRI) techniques through the increase of nuclear spin polarization (P) by several orders of magnitude.[1–6] For example, dissolution Dynamic Nuclear Polarization (d-DNP) technique[7–10] provides nuclear spin polarization of tens % corresponding to NMR signal enhancement by 4–5 orders of magnitude at magnetic fields of several tesla. Compounds hyperpolarized by d-DNP have been successfully employed as molecular contrast agents for metabolic studies in vivo.[11–13] For example, hyperpolarized (HP) [1-13C]pyruvate has demonstrated its utility as a molecular contrast agent for tumor diagnosis[14,15] and monitoring of response to tumor treatment[16,17] due to enhanced pyruvate metabolism in tumor tissues compared to normal tissues (Warburg effect[18]). Moreover, HP [1-13C]pyruvate has been successfully employed in clinical trial MRI of prostate cancer.[19,20] Other molecular contrast agents hyperpolarized by d-DNP are being considered, including carboxylates such as [1-13C]acetate[21,22] and [1,4-13C2]fumarate.[23] [1-13C]acetate has attracted research interest as a potential contrast agent for metabolic investigations in brain,[24,25] kidney,[21,22] liver[26] and muscle.[27] HP [1,4-13C2]fumarate showed the prospects for its application as a noninvasive marker of cellular necrosis in heart disease.[23]</p><p>Despite the success of d-DNP, this technique has a significant drawback of highly expensive and complex hyperpolarization equipment, making its widespread use in the clinical setting challenging. Moreover, d-DNP technique requires relatively long 13C hyperpolarization times of ~1 h. The more affordable alternative is Parahydrogen-Induced Polarization (PHIP) technique which employs nuclear spin order of parahydrogen (p-H2) molecule as a source of hyperpolarization.[1,28,29] In PHIP, p-H2 is added to an asymmetric unsaturated substrate in a pairwise manner, which means that two atoms from the same p-H2 molecule end up in the same reaction product molecule. 1H hyperpolarization in solution usually relaxes with spin-lattice relaxation time T1 of several seconds. To prolong the hyperpolarization lifetime, polarization can be transferred from 1H to heteronuclei such as 13C or 15N with T1 values ranging from tens of seconds to tens of minutes.[30–34] Transfer of hyperpolarization to heteronuclei can be carried out at a high magnetic field of NMR spectrometer using dedicated radiofrequency (RF) pulse sequences,[35–40] or by magnetic field cycling (MFC) of the HP sample through microtesla magnetic fields where protons and heteronuclei of the hyperpolarized molecule come into level anti-crossing (LAC) regime.[41–44]</p><p>A number of biologically relevant compounds have been directly hyperpolarized by PHIP technique so far, including succinate,[45–48] phospholactate,[48–51] and fumarate.[52–54] Moreover, PHIP by means of side arm hydrogenation (PHIP-SAH), the approach developed by Reineri and co-workers,[31] enables 13C polarization of carboxylates which cannot be hyperpolarized by PHIP directly due to the lack of corresponding unsaturated precursors. In PHIP-SAH the starting material is a 13C-labeled ester containing unsaturated alcoholic moiety which is hydrogenated with p-H2. Next, polarization is transferred from 1H to 13C nuclei of the reaction product either using RF pulse sequences[40,55] or MFC.[31,56,57] Subsequent cleavage of the formed ester via alkaline hydrolysis provides 13C HP carboxylate, e.g., [1-13C]pyruvate or [1-13C]acetate.</p><p>Most PHIP studies employ transition metal complexes as homogeneous hydrogenation catalysts to implement pairwise p-H2 addition.[58,59] These catalysts yield P of up to ~60% for the resultant HP reaction product.[55] However, metal-based catalysts are toxic and thus should be separated from the HP compound before its administration to a subject. Reineri et al. demonstrated that the use of an immiscible with water organic solvent (e.g., chloroform) in PHIP-SAH experiments enables separation of a 13C HP carboxylate (which resides in aqueous phase after ester hydrolysis) and catalyst.[31] An alternative approach is the use of heterogeneous (HET) hydrogenation catalysts which can be easily filtered out of the solution[60,61] or can be used in a continuous vapor phase hydrogenation of an unsaturated ester with subsequent hydrolysis downstream.[62] Moreover, HET-PHIP catalysts can be potentially recycled.[63] Heterogeneous catalysts provide somewhat lower polarization levels compared to homogeneous catalysts due to lower contribution of pairwise hydrogen addition route to the overall hydrogenation mechanism.[64–66] Nevertheless, the HET-PHIP catalyst development remains a hot area of research with the key goals to expand the scope of amenable substrates[67–69] and improve polarization yields.[66,70,71] In PHIP-SAH studies, heterogeneous Rh/TiO2 catalyst allowed obtaining 13C polarization (P13C) of 0.035% for ethyl [1-13C]acetate in organic solvents[72] and P13C = 0.011% in aqueous media.[61] Ligand-capped Pd and Rh nanoparticles provided higher 13C polarization levels of up to 1.3% for ethyl [1-13C]acetate, but at the expense of lower chemical conversion of the reactant vinyl [1-13C]acetate to reaction product.[33,73] Also heterogeneous PHIP-SAH approach was utilized to produce 13C HP amino acids with P13C = 0.29% after side arm cleavage.[74]</p><p>Recently, we presented an efficient synthetic approach for the preparation of 1-13C-labeled acetate and pyruvate esters containing vinyl, allyl and propargyl alcoholic moieties (Scheme 1),[75] and investigated their use as unsaturated precursors for homogeneous PHIP-SAH studies.[76] Here, we employed these compounds for systematic HET-PHIP-SAH investigations using Rh/TiO2 catalysts for pairwise p-H2 addition in methanol and in water aiming to compare the efficiency of HET-PHIP hyperpolarization of acetate and pyruvate esters with different alcoholic moieties.</p><!><p>HET-PHIP-SAH experiments were performed using Rh/TiO2 catalysts (0.97 wt.% or 20 wt.% of Rh metal) in liquid phase with methanol-d4 or D2O as solvents. Rh/TiO2 catalysts were chosen because they previously demonstrated the highest polarization levels among various monometallic supported metal catalysts.[65,77,78] The following PHIP experiments were carried out: PASADENA[29] (hydrogenation with p-H2 at high magnetic field), ALTADENA[79] (hydrogenation with p-H2 at Earth's magnetic field with subsequent transfer of the sample to the high field), and MFC[41] (hydrogenation with p-H2 at Earth's magnetic field with subsequent transfer of the sample to magnetic shield and then to the high field) for polarization transfer to 13C nuclei. Since PHIP experiments were performed with a broadly varied p-H2 fraction (from 58 to 89%), the observed polarizations were recalculated to the highest utilized p-H2 fraction of 89% (see SI). Unless otherwise specified, all reported polarization values correspond to 89% p-H2 fraction.</p><!><p>Heterogeneous liquid-phase hydrogenation of vinyl [1-13C]acetate with p-H2 over Rh/TiO2 catalysts to form HP ethyl [1-13C]acetate was previously investigated by some of us.[61,72] Here we revisited this reaction to compare the obtained polarization levels of ethyl [1-13C]acetate with those of other esters under study at the same experimental conditions. To this end, 0.97 wt.% Rh/TiO2 catalyst was tested in ALTADENA polarization of ethyl [1-13C]acetate in methanol-d4 (Figure 1b,c) and in D2O (Figure 1d,e), yielding 1H polarizations (P1H) of 0.5 ± 0.2% and 0.12 ± 0.04%, respectively. The obtained P1H in methanol-d4 was greater than that in the previous study by a factor of 17.[72] The 2.5-fold increase of polarization levels should be expected based on the higher p-H2 fraction employed here (89% vs. 50%). The additional ~7-fold increase is probably due to the fact that in this study we employed 0.97 wt.% Rh/TiO2 catalyst instead of 23.2 wt.% Rh/TiO2. Catalysts with higher metal loading provide greater chemical conversion of the reactant; however, the resultant polarization levels tend to be lower.[72,80] Here, we obtained ~40% total conversion of the reactant, although ~16–30% of the reacted vinyl acetate formed ethylene, ethane and acetic acid due to C–O bond hydrogenolysis process (Table S1). Next, MFC experiments were performed for the transfer of polarization to 13C nuclei of ethyl [1-13C]acetate. The obtained P13C were 0.10 ± 0.03% in methanol-d4 (Figure 1g,h) and 0.07 ± 0.02% in D2O (Figure 1i,j)—also several times greater than in the previous studies.[61,72]</p><!><p>Next, allyl acetate was employed as a PHIP precursor to HP propyl acetate. In PASADENA experiments with 0.97 wt.% Rh/TiO2 catalyst in methanol-d4 P1H = 0.3 ± 0.1% was obtained (Figure S1b,c), while ALTADENA experiments under the same conditions yielded P1H = 0.6 ± 0.2% (Figure 2b,c). In D2O, 0.97 wt.% Rh/TiO2 catalyst provided lower 1H polarization levels of 0.25 ± 0.08% and 0.28 ± 0.08% in PASADENA and ALTADENA experiments, respectively (Figure S1d,e and Figure 2d,e). 9.79 wt.% Rh/TiO2 catalyst in D2O provided PASADENA P1H = 0.09% at slightly lower conversion level than in the case of 0.97 wt.% catalyst (Table S2 and Figure S2b,c). Next, we tested 20 wt.% Rh/TiO2 catalyst for allyl acetate hydrogenation with p-H2 in D2O which allowed to increase conversion to 65%, although at the expense of significantly diminished polarization levels (P1H = 0.02 ± 0.01% in PASADENA experiment, Figure S2d,e). Therefore, the use of the catalyst with higher Rh loading for hyperpolarization of propyl acetate seems impractical for future applications of HP compound. Hydrogenation of allyl acetate with p-H2 over 0.97 wt.% Rh/TiO2 catalyst in methanol-d4 with subsequent MFC allowed detecting 13C HP propyl acetate even at natural abundance of 13C nuclei (Figure 2g). Corresponding P13C was estimated as 0.09 ± 0.03% using 0.74 M solution of vinyl [1-13C]acetate in methanol-d4 as an external reference. Hydrogenation of allyl [1-13C]acetate over 0.97 wt.% Rh/TiO2 in D2O with MFC yielded P13C = 0.03 ± 0.01% (Figure 2i,j). It should be noted that only ~60% of the reacted allyl acetate formed propyl acetate upon hydrogenation in both solvents, while the rest ~40% formed propylene, propane and CH3COOH as a result of C–O bond hydrogenolysis (Table S2).</p><!><p>When propargyl acetate was hydrogenated with p-H2 over 0.97 wt.% Rh/TiO2 catalyst in methanol-d4, formation of both allyl and propyl acetates was observed (Table S3). Also, C–O bond hydrogenolysis took place leading to formation of propylene, propane and acetic acid. As a result of lower conversion of the reactant (~17%) and selectivity of its hydrogenation to allyl acetate (~40%), only ~7% of the propargyl acetate was converted to allyl acetate after 10–20 s of H2 bubbling through the solution (Table 1). PASADENA and ALTADENA P1H were 0.16 ± 0.05% and 0.22 ± 0.07%, respectively (Figure S3b,c and Figure S4b,c). When propargyl [1-13C]acetate was hydrogenated with p-H2 over 0.97 wt.% Rh/TiO2 in methanol-d4 with subsequent magnetic field cycling, P13C of ca. 0.01% was observed for resulting allyl [1-13C]acetate (Figure S5). Taken together, HET-PHIP-SAH of allyl acetate in methanol-d4 was significantly less efficient than that of ethyl and propyl acetates in terms of both polarization and conversion levels. In aqueous phase hydrogenation of propargyl acetate with p-H2 over 0.97 wt.% Rh/TiO2, only ~1% conversion of the reactant was obtained (Table S3). Nevertheless, it was possible to detect 1H PASADENA and ALTADENA polarization of allyl acetate with P1H of ca. 0.6% and ca. 0.3%, respectively (Figure S3d,e and Figure S4d,e). Because the corresponding thermal 1H NMR signals were almost at the noise level, these estimates are not particularly accurate. While 9.79 and 20 wt.% Rh/TiO2 catalysts provided a higher conversion of ~5% in D2O, the resultant 1H PASADENA signals were lower in intensity due to lower P1H = 0.14 ± 0.05% (Figure S6). All attempts to observe 13C polarization of allyl acetate in D2O using 0.97 wt.% Rh/TiO2 and MFC were unsuccessful despite the use of the 13C-enriched precursor.</p><!><p>Next, heterogeneous PHIP-SAH of ethyl pyruvate was investigated. Hydrogenation of vinyl pyruvate with p-H2 over 0.97 wt.% Rh/TiO2 catalyst in methanol-d4 provided HP ethyl pyruvate with P1H = 0.11 ± 0.03% in both PASADENA and ALTADENA experiments (Figure S7 and Figure S8). Note that in methanol the pyruvate esters are present in two forms (ketone and hemiketal) with the prevalence of the latter. 1H chemical shifts of alcoholic moieties of vinyl and ethyl pyruvates are similar for both forms. Therefore, the estimated P1H corresponds to the sum of both ketone and hemiketal forms. Conversion of vinyl pyruvate to ethyl pyruvate was ~15% (Table S4). Because of the lower conversion and polarization levels for ethyl pyruvate compared to those for allyl acetate, it was not possible to detect 13C hyperpolarization of ethyl pyruvate at natural abundance of 13C nuclei in MFC experiments (13C-enriched precursor was not available to us due to inefficient vinyl pyruvate synthesis[75]). Aqueous phase hydrogenation of vinyl pyruvate was also unsuccessful—no reaction product was detected when either 0.97 or 20 wt.% Rh/TiO2 was utilized.</p><!><p>Hydrogenation of allyl pyruvate with p-H2 over 0.97 wt.% Rh/TiO2 catalyst in methanol-d4 under PASADENA conditions yielded HP propyl pyruvate with P1H = 0.25 ± 0.08% (Figure S9). In ALTADENA experiment in methanol-d4, P1H = 0.3 ± 0.1% was observed (Figure 3b,c). However, the selectivity of allyl pyruvate hydrogenation to propyl pyruvate was only ~22%—the rest of reacted precursor molecules converted to propylene, propanol, propane and pyruvic acid as a result of C–O bond hydrogenolysis (Table S5). Hydrogenation of allyl pyruvate over 0.97 wt.% Rh/TiO2 catalyst in D2O gave similar 1H polarization levels of 0.21 ± 0.07% and 0.3 ± 0.1% in PASADENA and ALTADENA experiments, respectively (Figure S10 and Figure 3e,f). However, total conversion of the reactant was only 8–10%—taking into account the similarly low selectivity to formation of propyl pyruvate, conversion of allyl pyruvate to the hydrogenated ester was only ~2%. In spite of a low amount of propyl pyruvate formed upon hydrogenation and modest P1H, it was possible to detect 13C HP propyl pyruvate in MFC experiments in methanol-d4 with P13C = 0.01% (Figure S11). Note that propyl and allyl pyruvates were present in two forms in both methanol-d4 and D2O solvents, similar to ethyl and vinyl pyruvates. P1H values were estimated for combination of both forms of propyl pyruvate which had similar chemical shifts, while P13C was calculated using signal of hemiketal form only because it was not possible to detect thermal 13C NMR signal of ketone form of propyl pyruvate.</p><!><p>Hydrogenation of propargyl pyruvate with p-H2 over 0.97 wt.% Rh/TiO2 catalyst in methanol-d4 produced HP allyl pyruvate with PASADENA P1H = 0.06 ± 0.02% (Figure S12) and ALTADENA P1H = 0.11 ± 0.03% (Figure S13). These polarization values were estimated for combination of both ketone and hemiketal forms of allyl pyruvate present in methanolic solution. Similar to the case of allyl and vinyl pyruvates hydrogenation, significant amount of the reacted precursor produced propylene, propane and pyruvic acid due to C–O bond hydrogenolysis (Table S6). The total conversion of propargyl pyruvate was lower than in the case of allyl pyruvate hydrogenation (~15%). Considering low activity of the Rh/TiO2 catalyst in production of allyl pyruvate from propargyl pyruvate and low 1H polarization, it is not unexpected that 13C NMR signals of HP allyl pyruvate were not detected in MFC experiments even when 13C-enriched precursor was utilized. The 0.97 wt.% Rh/TiO2 catalyst showed no activity in aqueous phase hydrogenation of propargyl pyruvate—even the NMR signal enhancement provided by the use of p-H2 did not allow to detect allyl pyruvate. However, when 20 wt.% Rh/TiO2 was employed, PASADENA and ALTADENA signals of HP allyl pyruvate were detected with P1H of ca. 0.1% and ca. 0.08%, respectively (Figure S14 and Figure S15). Nevertheless, conversion of propargyl pyruvate was only ~1% (Table S6).</p><!><p>The obtained 1H and 13C polarization levels and NMR signal intensities of HP acetate and pyruvate esters are summarized in Table 1, along with conversions of the unsaturated precursors to the desired esters. The difference in PASADENA and ALTADENA polarizations is not unexpected due to different protocols of corresponding experiments. While in ALTADENA experiments significant part of polarization is lost because of relaxation during transfer of the sample from the Earth's magnetic field to the NMR spectrometer, in PASADENA experiments the observed NMR signal is diminished due to anti-phase multiplet structure of PASADENA line.[81] Three important trends can be deduced from the analysis of the data presented in Table 1. First of all, the efficiency of HET-PHIP-SAH approach for hyperpolarization of acetates significantly surpassed that of pyruvates as a result of the fact that in the former cases the polarization levels, conversions and selectivities to hydrogenated ester tend to be greater than those in the latter cases. The reasons behind these observations are not clear. One can speculate that the carbonyl group in pyruvate moiety may coordinate to the metal surface, thus facilitating hydrogenolysis of C–O bond which leads to lower selectivity towards hydrogenated esters. The bulkiness of pyruvate moiety may be a reason of lower catalytic activity in case of pyruvates. The ability of pyruvates to form hemiacetals and heminal diols can also have an important effect on the catalysts' performance in hydrogenation of these substrates with p-H2. Nevertheless, to the best of our knowledge this is the first demonstration of HET-PHIP hyperpolarization of pyruvate esters, and further catalyst optimization can make this approach more efficient. Second, the performance of Rh/TiO2 catalysts in hydrogenation of propargyl esters with p-H2 was inferior to that in case of hydrogenation of esters with a C=C bond. This is in agreement with results of gas-phase hydrogenation of propyne and propylene with p-H2 over Rh/TiO2 catalyst.[82] In that study the intensities of 1H NMR signals of HP propane were higher than those of HP propylene under otherwise similar reaction conditions. Therefore, Rh/TiO2 catalyst is apparently more efficient in hydrogenation of double bonds with p-H2 than in hydrogenated of triple bonds. Third, HET-PHIP-SAH in methanol-d4 was significantly more efficient than in D2O. This is not unexpected because hydrogen solubility in methanol is greater than in water, leading to higher conversion of the reactant. As a result of the aforementioned trends, the most intense 1H and 13C NMR signals among HP esters under study were obtained for propyl acetate and ethyl acetate. This observation is in contrast with results of PHIP-SAH of the same six esters using cationic complex [Rh(NBD)(dppb)]BF4 (NBD = norbornadiene, dppb = 1,4-bis(diphenylphosphino)butane) as homogeneous hydrogenation catalyst, where the highest hyperpolarization efficiency was demonstrated for allyl pyruvate produced by pairwise p-H2 addition to propargyl pyruvate.[76] In HET-PHIP with Rh/TiO2 catalysts this ester yielded the least intense NMR signals; however, it is possible that other heterogeneous hydrogenation catalysts can be more efficient for hyperpolarization of this compound.</p><!><p>To conclude, we systematically investigated hyperpolarization of acetate and pyruvate esters with ethyl, propyl and allyl alcoholic moieties produced by heterogeneous hydrogenation of the corresponding unsaturated vinyl, allyl and propargyl precursors with p-H2 over Rh/TiO2 catalysts in methanol-d4 and D2O solvents. 1H polarization levels of up to 0.6 ± 0.2% were demonstrated in ALTADENA experiments, while magnetic field cycling allowed to hyperpolarize 13C nuclei with polarization of up to 0.10 ± 0.03%. The hydrogenation of corresponding unsaturated precursors was accompanied by hydrogenolysis of the C–O bond leading to formation of alkene and alkane hydrocarbons from side arm alcoholic moiety and carboxylic acids from carboxylic moiety. As a result, the efficiency of conversion of unsaturated precursors to the hydrogenated esters was diminished. The most intense 1H and 13C NMR signals in both methanol-d4 and D2O solvents were obtained for HP propyl acetate. Hyperpolarization of acetate esters was superior to that of pyruvates, while hydrogenation of the C=C bonds in vinyl and allyl moieties with p-H2 was more efficient than hydrogenation of the C≡C bond in propargyl moiety. Further optimization of reaction conditions and catalyst nature may increase the efficiency of heterogeneous PHIP-SAH approach for the preparation of HP biocompatible compounds for possible MRI applications.</p><!><p>Commercially available methanol-d4, D2O (Sigma-Aldrich-Isotec) and ultra-high pure (UHP) hydrogen (>99.999%) were used as received. Unsaturated precursors (vinyl acetate, allyl acetate, propargyl acetate, vinyl pyruvate, allyl pyruvate and propargyl pyruvate, see Scheme 1) were synthesized according to previously reported procedures.[75] These compounds were employed in both unlabeled and 1-13C-labeled (~98% 13C enrichment in the carboxyl group) forms, with the exception of vinyl esters (13C-enriched vinyl acetate and unlabeled vinyl pyruvate were used). The catalysts preparation procedure is described in the Supporting Information (SI).</p><!><p>p-H2 enrichment was achieved using custom-built ParaSun p-H2 generator based on cryocooler module (SunPower, P/N 100490, CryoTel GT; a detailed description of the generator was published elsewhere[75,83]). The generator was operated at 40–68 K, resulting in ca. 89–58% p-H2 enrichment. For sample preparation, 20 mg of Rh/TiO2 catalyst was placed at the bottom of a medium-wall 5 mm NMR tube (Wilmad glass P/N 503-PS-9) tightly connected with ¼ in. outer diameter PTFE tube. Next, 0.5 mL of 80 mM substrate solution in deuterated solvent was added.</p><p>The scheme of experimental setup is presented in Figure S16. The samples were pressurized up to 70 psig and preheated up to 55 °C (for CD3OD samples) or to 65 °C (for D2O samples) using NMR spectrometer temperature control unit. Hydrogen gas flow rate (140 standard cubic centimeters per minute, sccm) was regulated with a mass flow controller (SmartTrak 50, Sierra Instruments, Monterey, CA); duration of p-H2 bubbling can be found in SI, Tables S1–S6. In PASADENA[29] experiments the samples were located inside the probe of the NMR spectrometer. In ALTADENA[79] experiments the samples were located at the Earth's magnetic field during p-H2 bubbling (in some experiments they were additionally heated to 80–85 °C in a beaker with hot water). After termination of p-H2 flow, the samples were transferred to the NMR spectrometer for detection. MFC procedure was similar to ALTADENA but in this case the samples were placed inside the MuMETAL magnetic shield after cessation of p-H2 flow, and then slowly (~1 s) pulled out of the shield and quickly placed inside the probe of the NMR spectrometer. The total sample transfer time in this case was ~8 s after the termination of H2 gas bubbling. The magnetic field inside the shield was adjusted using additional solenoid placed inside the previously degaussed three-layered MuMETAL shield (Magnetic Shield Corp., Bensenville, IL, P/N ZG-206). This previously calibrated solenoid was powered by a direct current (DC) power supply (GW-Instek, GPR-30600), and the DC current was attenuated by a resistor bank (Global Specialties, RDB-10) to achieve the desired magnetic field inside the MuMETAL shield. The MuMETAL shield provides an isolation of approximately 1200 according to the manufacturer's specification; therefore, the use of the shield in the Earth's magnetic field results in the maximum residual magnetic field of ca. 40 nT. The magnetic fields employed for MFC are presented in Table S7.</p><p>NMR spectra were acquired on a 9.4 T Bruker NMR spectrometer using π/4 RF pulse for PASADENA experiments and π/2 RF pulse for ALTADENA and MFC experiments. The 1H ALTADENA and 13C PHIP spectra were recorded as pseudo-two-dimensional (2D) sets consisting of 64 1D NMR spectra (acquisition time 1 s) to avoid delays between placing the sample into the probe and starting the acquisition. The acquisition of these pseudo 2D data sets was always initiated before the sample was placed inside the NMR probe.</p>

PubMed Author Manuscript

SP174 ANTIBODY LACKS SPECIFICITY FOR NRAS Q61R AND CROSS-REACTS WITH\nHRAS- AND KRAS-Q61R MUTANT PROTEINS IN MALIGNANT MELANOMA

HRAS, KRAS and NRAS, highly homologous proteins, are often mutationally activated in cancer. Usually, mutations cluster in codons 12, 13 and 61 and are detected by molecular genetic testing of tumor DNA. Recently, immunohistochemistry with SP174 antibody has been introduced to detect NRAS Q61R mutant protein. Studies on malignant melanomas showed that such an approach could be a viable alternative to molecular genetic testing. This investigation was undertaken to evaluate the value of SP174 immunohistochemistry for detection of NRAS Q61R mutant isoform. Two hundred ninety-two malignant melanomas were evaluated using Leica Bond-Max automated immunostainer. Twenty-nine tumors (10%) showed positive immunoreactivity. NRAS codon 61 was PCR amplified and sequenced in 24 positive and 92 negative cases using Sanger sequencing, qPCR and next generation sequencing approaches. A c.182A>G substitution leading to NRAS Q61R mutation was identified in 22 tumors. Two NRAS-wild type tumors revealed c.182A>G substitutions in H- and K-RAS codon 61, respectively. Both mutations were detected by next-generation sequencing and independently confirmed by Sanger sequencing. None of 85 NRAS codon 61-wild type tumors and 7 NRAS mutants other than Q61R showed immunoreactivity with SP174 antibody. Thus, SP174 antibody was 100% sensitive in detecting NRAS Q61R mutant isoform in malignant melanoma, but not fully specific as it cross-reacted with HRAS and KRAS Q61R mutant proteins. Therefore, molecular testing is needed to determine which RAS gene is mutated. The rarity of H-and K-RAS Q61R mutants in malignant melanoma let previous investigations erroneously conclude that SP174 is specific for NRAS Q61R mutant protein.

sp174_antibody_lacks_specificity_for_nras_q61r_and_cross-reacts_with\nhras-_and_kras-q61r_mutant_pro

INTRODUCTION<!>Study material and design<!>Immunohistochemistry<!>Molecular studies<!>RESULTS<!>DISCUSSION<!>

<p>HRAS, KRAS and NRAS, highly homologous proteins, are located on the inner surface of the cell membrane. These enzymes represent GDP/GTP-regulated switches that transfer extracellular signals and play a fundamental role in signal transduction regulating cell proliferation, and apoptotic cell death.1</p><p>In cancer, RAS proteins are often pathologically activated. Typically, gain-of-function RAS mutations cluster in codons 12, 13 and 61. In Caucasian population approximately 20% of malignant melanomas carry NRAS mutations with codon 61 being the most commonly involved "hot spot". Specifically, the c.182A>G substitution which leads to Q61R mutation accounts for about 40% of all NRAS mutants.2</p><p>For the last several decades, molecular genetic assays such as the melting curve analysis, Sanger sequencing, pyrosequencing and qPCR have been commonly used to detect RAS mutations. Lately, immunohistochemistry with SP174 antibody was introduced to pinpoint NRAS Q61R mutant protein, an equivalent of NRAS c.182A>G mutation. Recent studies on malignant melanomas showed that such immunohistochemical approach could be a valuable alternative to molecular genetic testing.3–9 This investigation was undertaken to verify value of SP174 immunohistochemistry in search for NRAS Q61R mutant protein in malignant melanomas.</p><!><p>Two hundred ninety-two anonymized, well characterized, malignant melanomas from Europe and the United States were analyzed. Malignant melanoma diagnosis was based on clinicopathologic data, histology and S100, HMB45 and Melan A immunohistochemistry. Clinicopathologic characteristic of analyzed cases is summarized in Table 1. The cohort included 96 primary tumors, 187 metastatic lesions and 9 cases in which primary tumor versus metastatic status was unclear. SP174 immunohistochemistry was performed in the Laboratory of Pathology on multitissue blocks build as previously reported.10 DNA was extracted from formalin-fixed paraffin-embedded tumor tissues following published procedure.11 Screening for RAS mutations was completed independently and blindly without knowledge of the results from immunohistochemical studies in three different institutions. Sanger sequencing in the Laboratory of Pathology, qPCR in the Department of Biology and Genetics, University of Gdansk, Gdansk, Poland and Ion Torrent™ next-generation sequencing in the Department of Molecular Diagnostics, Holycross Cancer Center, Kielce, Poland.</p><!><p>A rabbit monoclonal antibody, clone SP174 (Spring™ Bioscience, Pleasanton, CA) and Leica Bond-Max automatic immunostainer (Leica, Bannockburn, IL) with Leica Refine detection kit were used in this study. The primary antibody was incubated for 30 min, followed epitope retrieval using Leica H2 buffer (25 min). The 1:100 dilution of primary antibody was selected as the lowest dilution yielding a strong signal in NRAS Q61R mutant while giving no staining in NRAS wild type tumor. Either Diaminobenzidine or Fast Red were used for visualization following protocols provided by Leica. The immunostainings were scored arbitrarily by three pathologists (S.I., J.L., M.M.) as negative (no staining), weakly positive and strongly positive.</p><!><p>HRAS, KRAS and NRAS codon 61 sequences were PCR amplified and the amplification products were evaluated by Sanger sequencing as previously reported.11 Primer sequences and PCR conditions for each reaction are listed in Table S1 in supplementary data. NRAS and KRAS qPCR assays were performed using RAS Mutation Analysis Kits (NRAS- and KRAS-RT50) and Rotor-Gene Q Software version 1.7 following the manufacturer's instructions (EntroGen, Inc., Woodland Hills, CA). These tests detect spectrum of NRAS and KRAS substitutions leading to NRAS Q61H, -K, -L, and -R, and KRAS Q61H-L, and R mutations at the protein level. Next-generation sequencing was completed using the Ion Torrent™ next-generation sequencing platform and Ion AmpliSeq™ Cancer Hotspot Panel v2 Kit following the manufacturer's instructions (Life Technologies/Thermo Fisher Scientific, Waltham, MA) and previously published procedure.12</p><!><p>Twenty-nine (10%) of 292 analyzed malignant melanomas revealed positive immunostaining with SP174 antibody. In all cases, delicate granular perimembranous and cytoplasmic staining was seen. Example of SP174 immunostaing is shown in Figure 1.</p><p>Twenty-four SP174-positive cases with tissue available for DNA extraction were evaluated by Sanger sequencing. A c.182A>G substitution leading to NRAS Q61R mutation at the protein level, was identified in 11 of 24 analyzed cases. In the remaining SP174 positive cases: 1) PCR amplification was unsuccessful (n=4), 2) Sanger sequencing chromatograms showed a small mutant peak and the possibility of c.182A>G substitution could not be ruled out (n=7), 3) only wild type NRAS codon 61 sequences were detected (n=2). Subsequently, these cases (n=13) were blindly evaluated using qPCR and Ion Torrent™ next-generation sequencing. NRAS codon 61 mutants were identified in all but 2 tumors. The latter revealed c.182A>G substitution in H-and K-RAS codon 61 (Q61R mutation), respectively. The next-generation sequencing results were later confirmed by PCR amplification of HRAS and KRAS codon 61and Sanger sequencing.</p><p>In 3 cases, Ion Torrent™ next-generation sequencing detected c.182A>G NRAS substitutions with moderate to low (35%, 27% and 6%) frequency. In these cases, the material submitted for Sanger sequencing was re-examined and either inadequate sampling, dominance of normal stromal cells and lymphoid elements, or low tumor cell content was identified to explain negative results in mutation analysis. Representative examples of histology, immunohistochemistry and next-generation sequencing are shown in Figure 2. Representative examples of histology, immunohistochemistry and molecular genetic testing are shown in Figure 3.</p><p>None of 85 NRAS codon 61-wild type tumors and 7 NRAS mutants other than Q61R showed immunoreactivity with SP174 antibody. However, weak SP174 immunostaining was noticed in the normal sebaceous glands in a few cases. Representative images are shown in Figure S1 in supplementary data.</p><!><p>HRAS, KRAS and NRAS proto-oncogenes are often mutated in cancer including malignant melanoma. The frequency of the mutant isoforms defined by distinctive point mutations in RAS codons 12, 13 and 61 varies significantly between different types of tumors.2 In malignant melanoma, NRAS Q61R is the most common RAS mutation while KRAS Q61R is extremely rare as reported by the catalog of somatic mutations in cancer (cancer.sanger.ac.uk/cosmic). However, HRAS Q61-mutants are distinctive feature of Spitz tumors with benign or unknown malignant potential.13</p><p>In vitro studies based on colorectal cancer cell lines showed that oncogenic potential and sensitivity to inhibitor treatment can depend on type of RAS mutant isoform expression. Thus, in the future, RAS mutation status might be a key element necessary for pinpointing right therapeutic strategy in malignant melanoma.14</p><p>Recently published investigation showed that immunohistochemistry with SP174, a rabbit monoclonal antibody, detects NRAS Q61R mutant protein with high sensitivity and specificity, and because of that it may have clinical utility and substitute molecular genetic testing.3–9 Although this study confirmed high (100%) sensitivity of SP174 immunohistochemistry in detection of NRAS Q61R mutant isoform, specificity of this antibody has to be questioned because of cross-reactivity with HRAS- and KRAS-Q61R mutant proteins. This cross-reaction can be attributed to the high sequence homology of the amino-terminal catalytic domain (amino acid 1–165) of RAS proteins.15 Previously emphasized 100% specificity of this antibody was based on the presumption rather than experimental evidence because no malignant melanoma HRAS or KRAS Q61R mutants were evaluated.5 During the preparation of this manuscript cross-reactivity of SP174 with H-, and K-RAS Q61R mutants has been reported in colorectal and thyroid cancer.16,17</p><p>Weak non-specific staining of normal cells including adipocytes, bronchial epithelial cells, endothelial cells macrophages and plasma cells with SP174 antibody was reported in previously published studies.4,6,8 In this study, only normal sebaceous glands showed weak SP174 positivity in a few analyzed cases. Such cross-reactivity may be attributed to the technical factors including platform used for the immunohistochemistry. A cross-reactivity between mutant-specific antibody and normal cell epitopes was previously reported for VE1, BRAF V600E mutant-specific antibody.11,18</p><p>For the last several decades Sanger sequencing has been considered the gold standard for DNA mutation detection. However, sensitivity of this methodology doesn't allow discovery of the mutations in the tissue samples with low tumor cell content. Although laser-based micro dissection could be employed to enriched tumor cell population, the procedure is time consuming and can't be easily used in clinical setting. Next-generation sequencing used in this study is a powerful tool allowing detection of the low-copy mutant alleles in partially degraded DNA from formalin-fixed-paraffin-embedded tissue.19</p><p>This study showed that prescreening with SP174 immunohistochemistry followed by next-generation sequencing offers effective and precise detection of different RAS codon 61 mutants. Significant cost reduction could be an additional advantage of such strategy. Although availability of next-generation sequencing technology is still restricted to research/academic centers, recently developed smaller, less expensive instruments with smaller targeted mutation panels may be more applicable for clinical testing.20</p><p>This study showed that immunohistochemistry with SP174 antibody allowed identification of three different H-, K- and N-RAS Q61R mutant proteins. Although this antibody is 100% sensitive for RAS Q61R, molecular genetic testing is necessary to determine which RAS gene is mutated.</p><!><p>Example of positive immunoreactivity to SP174, NRAS Q61R mutant-specific antibody in malignant melanoma.</p><p>Two malignant melanomas, one with low tumor cell content, second with prominent stromal cells and lymphoid elements (LCA staining). Next generation sequencing revealing NRAS c.182A>G substitution predicted to cause NRAS Q61R mutation.</p><p>Two malignant melanomas with positive immunoreactivity to SP174 (wild type for NRAS codon 61 by Sanger sequencing) carry HRAS or KRAS c.182A>G substitutions documented by next generation sequencing and sanger sequencing, respectively.</p><p>Summary of clinicopathologic data.</p><p>Age based on 228 cases;</p><p>Sex o based on 236 cases</p>

PubMed Author Manuscript

Catalytic and Inhibitor Binding Properties of Zebrafish Monoamine Oxidase (zMAO): Comparisons with human MAO A and MAO B

A comparative investigation of substrate specificity and inhibitor binding properties of recombinant zebrafish (Danio rerio) monoamine oxidase (zMAO) with those of recombinant human monoamine oxidases A and B (hMAO A and hMAO B) is presented. zMAO oxidizes the neurotransmitter amines (serotonin, dopamine and tyramine) with kcat values that exceed those of hMAO A or of hMAO B. The enzyme is competitively inhibited by hMAO A selective reversible inhibitors with the exception of d-amphetamine where uncompetitive inhibition is exhibited. The enzyme is unreactive with most MAO B-specific reversible inhibitors with the exception of chlorostyrylcaffeine. zMAO catalyzes the oxidation of para-substituted benzylamine analogues exhibiting Dkcat and D(kcat/Km) values ranging from 2\xe2\x80\x938. Structure-activity correlations show a dependence of log kcat with the electronic factor \xcf\x83p with a \xcf\x81 value of +1.55 \xc2\xb1 0.34; a value close to that for hMAO A but not with MAO B. zMAO differs from hMAO A or hMAO B in benzylamine analogue binding correlations where an electronic effect (\xcf\x81 = +1.29 \xc2\xb1 0.31) is observed. These data demonstrate zMAO exhibits functional properties similar to hMAO A as well as exhibits its own unique behavior. These results should be useful for studies of MAO function in zebrafish models of human disease states.

catalytic_and_inhibitor_binding_properties_of_zebrafish_monoamine_oxidase_(zmao):_comparisons_with_h

1 Introduction<!>2.1 Materials<!>2.2 Determination of steady state kinetic parameters<!>2.3 Data analysis<!>3.1 Steady State Kinetic Properties of zMAO<!>3.2 Binding of reversible MAO A and MAO B inhibitors to zMAO<!>3.3 Quantitative structure-activity relationships for zMAO oxidation of para-substituted benzylamine analogues<!>4.1 Functional properties of zMAO<!>4.2 Mechanistic and structural interpretation of the QSAR data<!>5 Conclusions

<p>The elucidation of the separate gene sequences for human monoamine oxidases (EC 1.4.3.4) A and B (MAO A and MAO B) (Bach, et al. 1988) has provided unequivocal proof for the existence of two isoforms of these membrane-bound flavoenzymes that catalyze the oxidation of amine neurotransmitters. Gene sequences for both forms are available for a number of mammals that are commonly used as animal models for MAO inhibitor development studies (Edmondson, et al. 2009). In contrast, available gene sequence data on teleosts show the existence of only a single gene encoding MAO. The initial sequence was published for trout by Shih's group (Chen, et al. 1994). Recently, the gene sequence of zebrafish (Danio rerio) has been elucidated (Setini, et al. 2005; Anichtchik, et al. 2006). This organism exhibits a number of characteristics to make it a good animal model system for drug development studies (Kokel et al.,2010; Rihel, et al. 2010) and initial studies have shown MAO to be important in serotonergic biological processes (Sallinen, et al. 2009a; Sallinen, et al. 2009b). A comparative study to determine the functional properties of zMAO is reported here to facilitate further work and to provide a basis for comparison to human MAO's. Previous work from this laboratory has shown the successful expression, purification, and partial characterization of recombinant zMAO (Arslan and Edmondson, 2009). This laboratory has also expressed, purified, and characterized human and rat MAO A and MAO B (Newton-Vinson, et al. 2000; Li, et al. 2002; Upadhyay and Edmondson 2008; Wang and Edmondson, 2009). Therefore, the tools are in place for a detailed comparative functional study of zMAO with those of human MAO A and MAO B.</p><p>Previous studies have demonstrated that zMAO exhibits inhibitor binding properties that overlap those of human MAO A and of MAO B (Setini, et al. 2005; Anichtchik, et al. 2006; Sallinen, et al. 2009). The results presented in this manuscript provide a more in-depth approach to verify this suggestion. Investigations of the structure and function of hMAO A and of hMAO B demonstrate the two enzymes differ in active site structures (Binda, et al. 2002; Son, et al. 2008), in inhibitor binding (Youdim,et al. 2006), substrate specificities (Edmondson, et al. 2007), and in analysis of the influence of para-substituents of benzylamine analogues on catalysis (Walker and Edmondson 1994; Miller and Edmondson 1999). Crystallographic studies show hMAO B contains a bipartite active site with an entrance cavity (290 Å3) and a substrate cavity (~400 Å3) separated by an Ile199 gate residue (Hubálek, et al. 2005) MAO A contains a monopartite single cavity of ~550 Å3 (DeColibus, et al. 2005) These differences in cavity structures account for specificity of MAO B binding of reversible inhibitors such as 8-(3-chlorostyryl)-caffeine, trans-trans-farnesol, diphenyl-2-butene and safinamide (Hubálek, et al. 2005; Binda, et al. 2007) . The rates of oxidation of para-substituted benzylamine analogues by MAO A exhibit a strong dependence on the electron withdrawing capacity of the para-substituent with a Hammett plot exhibiting a ρ value of + 1.89 (Miller and Edmondson 1999) while the rate of MAO B-catalyzed oxidation of this class of substrate analogues exhibits no detectable electronic dependence (Walker and Edmondson 1994, M.Li, PhD Disseration, Emory University). For a detailed discussion of the published differences between the human enzymes, the reader is referred to a recent review article (Edmondson, et al. 2009).</p><p>With these demonstrated differences between hMAO A and hMAO B functional behaviors, we report here a comparative investigation of the substrate and inhibitor binding properties of zMAO. The results confirm and extend previous suggestions in the literature that this teleost MAO exhibits functional properties that are more similar to those of MAO A rather than those of MAO B.</p><!><p>Zebrafish (Danio rerio) monoamine oxidase was expressed in Pichia pastoris and purified as previously described (Arslan and Edmondson, 2009). Reduced Triton X-100, glycerol, HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, isatin, benzylamine, kynuramine, serotonin, 1,4-diphenyl-1,3-butadiene, methylene blue, and 1,4-diphenyl-2-butene were purchased from Sigma-Aldrich (St. Louis, MO, USA). Safinamide was a gift from Newron Pharma (Milan, Italy). 8-(3-Chlorostyryl)-caffeine and trans-trans-farnesol were gifts from Dr. N. Castagnoli, Department of Chemistry, Virginia Tech. University. Harmane, pirlindole mesylate and tetrindole mesylate were purchased from TOCRIS Bioscience (Ellisville, MO, USA). All other commercially available reagents were used without further purification. The structures of the MAO A and MAO B specific inhibitors used in the study are shown in Figure 1. All benzylamine analogues used in this study were synthesized in this laboratory as previously described (Walker and Edmondson, 1994; Miller and Edmondson 1999).</p><!><p>Spectrophotometric enzyme assays were determined using either a Perkin-Elmer Lambda 2 UV-Vis or a Varian Cary 50 UV-Vis spectrophotometer. Assays were conducted at 25 °C in 50 mM potassium phosphate, pH=7.4 buffer containing 0.5% (w/v) reduced Triton X-100 unless otherwise stated. All buffers were equilibrated for 30 min at 25 °C before use. Assays using benzylamine and their α,α-2[H] analogues used the Amplex-red peroxidase coupled assay (Invitrogen) for increased detection sensitivity (Δεm560 = 54,000 M−1cm−1). MAO substrates containing phenolic oxygens such as serotonin, dopamine and tyramine were assayed polarographically by measuring the rate of O2 uptake since they are also substrates for the horseradish peroxidase used in the coupled Amplex Red assay leading to erroneous kinetic results. The rate of oxygen consumption was determined using a Model 782 Oxygen Meter interfaced to a PC (Strathkelvin Instruments Ltd. North Lanarkshire, Scotland). One unit of enzyme activity is defined as the amount of enzyme required to oxidize 1 μmol of substrate in one minute. Inhibition assays for zMAO were performed with kynuramine as substrate. Kinetic measurements at saturated O2 concentrations required sparging the solutions with O2. Oxygen concentrations in the reaction buffers were determined polarographically. Enzyme functionality was determined from spectral measurements of the level of enzyme-bound flavin reduction on the anerobic addition of substrate compared to the level of reduction observed on the addition of excess sodium dithionite.</p><!><p>Steady state kinetic data (kcat and Km values) were determined from fits to the Michaelis-Menten equation using GraphPad Prism 5.0. Inhibition constants (Ki) were determined from analysis of steady state kinetic values measured with at least 4 inhibitor concentrations using GraphPad Prism 5.0 software. Inhibition data with Methylene Blue were analyzed using methodology for tight binding inhibitors described by Morrison (1968). Values of substituent parameters (σ, the Hammett electronic parameter; and π, the hydrophobicity parameter) were obtained from Hansch (Hansch, et al. 1995) and VW values (the substituent van der Waals volumes) from Bondi (1964). The values for substrate binding affinities to zMAO (Kd) were calculated from steady state deuterium kinetic isotope effect data as described (Klinman and Matthews, 1985). Apparent Kd values were corrected to reflect the concentration of deprotonated amine in solution at the pH value of the assay since previous studies on mammalian MAO have shown the deprotonated form of the amine substrate is specifically bound (McEwen, et al. 1969; McEwen, et al. 1968). Multi-component statistical analyses of correlations tested were performed using StatView software to determine contributions of various para-substituent parameters with kinetic and binding constants. The F value is a statistical term relating the residuals of each point to the fitted line to the residuals of each point to the mean value. F is weighted for the number of variables in the correlation and the number of data points. The higher the F value, the better the correlation. The significance is calculated from the F value and represents the fractional chance that the correlation is meaningless.</p><!><p>To compare the substrate specificities and kinetic properties of zMAO with those of human MAO A and B, a number of commonly used substrates were tested. Since the available kinetic data on the human enzymes have been collected at 25 °C, the same temperature was used in this study for comparative purposes. As shown in Table 1, zMAO oxidizes serotonin (a MAO A-specific substrate) with a kcat value of 187 min−1, which is equivalent to the value exhibited by human MAO A and ~6 times higher than the kcat value for hMAO B; zMAO oxidizes kynuramine and dopamine with kcat values of 75 min−1 and 242 min−1, respectively. Thus, zMAO is a more effective catalyst for dopamine oxidation than either hMAO A or hMAO B. The kcat value for zMAO catalyzed oxidation of tyramine (467 min−1) is greater than the values determined for hMAO A (182 min−1) or for hMAO B (343 min−1).</p><p>Among the substrates tested, 4-phenylbutylamine and 3-aminomethylpyridine provide interesting distinctions between MAO A and MAO B. 4-Phenylbutylamine functions as a competitive inhibitor for hMAO A (Nandigama and Edmondson 2000) but is a substrate for hMAO B (kcat/Km = 5.8 × 106 M−1min−1) and is also found to be a poorer substrate for zMAO (kcat/Km= 6.7 × 105 M−1min−1) (Table 1). 3-Aminomethylpyridine is a substrate for hMAO B but not for hMAO A (Li, M, 2006) nor for zMAO. p-Carboxybenzylamine is a substrate for zMAO and for hMAO A, but is neither a substrate nor a competitive inhibitor with hMAO B (Table 1). These observations suggest a functional diversity of zMAO in MAO A or MAO B substrate specificities with properties closer to those of hMAOA.</p><!><p>Inhibition studies further support a "MAO A-like" behavior of zMAO. zMAO catalytic activity is competitively inhibited by a number of MAO A inhibitors (Tables 2 and 3). Of the reversible inhibitors tested, methylene blue is exceptional in that it binds to zMAO with a 4nM Ki value which is 6-fold tighter than observed with hMAO A and ~103 tighter than measured with hMAO B. Tetrindole mesylate and pirlindole mesylate are tetracyclic anti-depressants that selectively target and reversibly inhibit the catalytic activity of MAO A (Andreeva, et al. 1992). These indole analogues inhibit zMAO with 6–7 fold weaker affinities than observed with hMAO A under identical conditions. The tricyclic hMAO A inhibitor, harmane, exhibits a similar Ki value with zMAO as it does with hMAO A.</p><p>A variety of MAO B-specific inhibitors were tested with zMAO (Table 3). No observable inhibition is observed with a number of MAO B-specific inhibitors that have been shown to occupy both the substrate and entrance cavities of the human enzyme (Binda et al. 2003). 8-(3-Chlorostyryl)-caffeine inhibition of zMAO is the only exception and is the only "dual cavity spanning" MAO B reversible inhibitor observed to bind. The reversible MAO inhibitor d-amphetamine (Green and El Hait 1980; Sowa, et al. 2004) functions as an uncompetitive inhibitor of zMAO catalyzed kynuramine oxidation although it competitively inhibits human MAO A (Vintém et al., 2005) and can function either as a competitive or as a mixed inhibitor of human MAO B depending on the substrate (Pearce and Roth, 1985). The sensitivity of zMAO to amphetamine inhibition shows it differs from hMAO A in that inhibition occurs by binding to either E or ES (or EP) forms of zMAO rather than solely to the free enzyme. This behavior then differs with those exhibited by hMAO A and is closer to those exhibited by hMAO B.</p><!><p>A major difference in the catalytic properties of human MAO A and MAO B is the influence of para-substituents on the rates of oxidation of benzylamine analogues. hMAO A demonstrates a strong electronic contribution (ρ = + 1.89) (Miller and Edmondson, 1999) from para-substitution in this class of substrate analogues but no electronic effect is observed with bovine MAO B (Walker and Edmondson 1994) or with human MAO B (Li,2006) . Given the similarities of zMAO to MAO A, the influence of para-substitution on the steady state rates of benzylamine analogue oxidation were determined and the kinetic data are shown in Table 4.</p><p>In agreement with published data on hMAO A (Miller and Edmondson, 1999), on bovine MAO B (Walker and Edmondson, 1994), and on human and rat MAO B (Newton-Vinson, et al.,2000;Li, 2006; Upadyhay and Edmondson, 2008), zMAO exhibits deuterium kinetic isotope effects on benzylamine analogue oxidation with Dkcat and D(kcat/Km) values ranging from 2 to 8 (Table 4). These data demonstrate the α-C-H bond cleavage step contributes to the rate limitation in catalysis as is found with MAO A and B from various mammalian sources (Edmondson, 2009). Therefore, substituent effects on rate reflect an influence on the rate of the hydrogen-transfer step in catalysis.</p><p>Correlations between kcat and electronic, steric and hydrophobicity parameters of the para-substituents were estimated with the electronic parameter (σ) exhibiting the major contribution. Figure 2a shows that zMAO exhibits a linear correlation of log kcat with σ, a property also seen with hMAO A (Miller and Edmondson,1999).</p><p>This linear relation is best described by the equation: logkcat=1.55(±0.34)s+1.1(±0.09)(F1,7=20;p=0.006)</p><p>Analysis including additional substituent parameters such as van der Waals volume (VW) or hydrophobicity (π) did not result in any statistical improvement in the correlation. This equation shows that the rate of turnover increases with increasing electron withdrawing power of the para-substituent as shown previously with human MAO A (Miller and Edmondson, 1999) . The calculated ρ value for zMAO (+ 1.55) is close to that (+ 1.89) observed for hMAO A (Miller and Edmondson,1999). These results indicate that the bound benzylamine substrate has a conformation in the substrate binding site that allows transmission of the para-substituent electronic effect to the benzyl carbon as found for hMAO A but not in MAO B. These steady state kinetic measurements were performed at air saturation (240 μM O2) which is approximately twice the KmO2 for zMAO (Arslan and Edmondson, 2009). The same correlations are obtained when the kcat values were determined under conditions where [O2] ~1 mM (not shown); providing additional support that a-C-H bond cleavage is rate-limiting in catalysis.</p><p>The corrected binding affinities of deprotonated para-substituted benzylamine analogues to the active site of zMAO were analyzed to determine whether any correlations with steric substituent parameters could be observed as found with hMAO A (Miller and Edmondson, 1999). No correlations are observed with either steric or hydrophobicity substituent parameters and binding affinity; however, a reasonable correlation is observed for binding affinity (log Kd) and the substituent electronic parameter (σ) (Figure 2b). This observation differs from the correlations reported for hMAO A and hMAO B where steric parameters dominate. The observed ρ value (+1.3) shows that electron withdrawing groups on the benzylamine substrate analogue increase the binding affinity to zMAO according to the following relationship: logKd(corr)=1.29(±0.31)σ+5.86(±0.08)(F1,7=17.1;p=0.009)</p><p>Correlation analysis with two substituent parameters do not improve the statistics although more than seven different examples are required for a more rigorous 2-component analysis. This correlation shows that electron withdrawing groups facilitate benzylamine binding to the active site of zMAO. Since the binding data are already corrected for any substituent effects on benzylamine analogue pKa values, this correlation indicates that electron withdrawal from the benzenoid ring of the substrate facilitates binding to the active site of zMAO. The structural basis for this effect on binding is currently unknown but suggests the presence of a positively charged residue in the active site of zMAO which is positioned to interact with the aromatic ring of the benzylamine substrate (possibly a π-cation interaction). Despite the high sequence similarity with MAO A in the substrate-binding region, zMAO does exhibit unique properties in its substrate binding site.</p><!><p>The amino acid sequence of zMAO is ~70% identical with either hMAO A or hMAO B. The sequence of zMAO in the substrate binding domain is identical with hMAO A and ~70% identical with hMAO B. The C-terminal domain of zMAO associated with the membrane-binding transmembrane helix of hMAO A and hMAO B exhibits the lowest identity (30% with hMAO A and 20% with hMAO B). A residue that has been shown to function as a gate separating the bipartite cavity of hMAO B (Ile199) and involved in inhibitor binding specificity (Hubálek et al. 2005) is a Phe in zMAO as it is in hMAO A.</p><p>The limited number of publications investigating the functional behavior of zMAO suggests the enzyme exhibits properties of both human MAO A and MAO B, with more similarities to hMAO A (Setini, et al. 2005, Sallinen, et al. 2009a). The finding that deprenyl (an irreversible MAO B inhibitor and clorgyline (an irreversible MAO A inhibitor) inhibit zMAO with similar IC50 values (Setini, et al. 2005, Arslon and Edmondson, 2010) documents this unique behavior of zMAO. Deprenyl treatment in vivo results in a decrease in serotonin levels indicating zMAO inhibition (Sallinen, et al. 2009) as expected since this acetylenic inhibitor functions irreversibly. The studies reported here for the recombinant form of zMAO provide further validation for the dual functional specificity of zMAO. Since teleosts contain only a single form of MAO, it is reasonable that this MAO would be able to function in both capacities of the mammalian counterparts.</p><p>The inhibition profile of zMAO is also further defined in this study. Previous results on hMAO B show the Ile199 gate residue between the entrance and substrate cavities to play an important role in the binding of 8-(3-chlorostyryl)-caffeine, 1,4-diphenyl-2-butene, farnesol, and 1,4- diphenyl-1,3-butadiene (Hubálek, et al. 2005) since none of these inhibitors bind to or inhibit hMAO A. The absence of binding to zMAO by the majority of these MAO B specific inhibitors suggests an active site structure of zMAO closer to that of hMAO A. A detailed understanding of these differences must await the determination of the zMAO structure by x-ray crystallography.</p><!><p>Previous studies of human MAO A and MAO B substrate binding properties using para-substituted benzylamine analogues showed that the two isoforms are influenced differently by the para-substitution (Walker and Edmondson 1994; Miller and Edmondson 1999). In the case of catalytic turnover, hMAO A prefers analogues with strong para electron-withdrawing groups while hMAO B catalysis is unaffected by this parameter. Substrate binding to hMAO A is favored by larger para-substituents while hMAO B prefers smaller substituents. These properties were originally interpreted to assign a larger binding site to hMAO A and a smaller more hydrophobic binding pocket to hMAO B. The published crystal structures confirm this interpretation (Edmondson, et al. 2007; Son 2008). These correlations also suggest that the conformation of the bound benzylamine substrate determines whether an electronic effect is observed as contributing to enhancement of catalysis in either human isoform [9]. The orientation of the aromatic ring can influence the alignment of the α-C-H with the π-orbitals of the aromatic ring, which allows substituent electronic effects to be transmitted in hMAO A but not in hMAO B where the more restrictive binding site is suggested to preclude a coplanar orientation (Miller and Edmondson, 1999). The steady-state analyses of zMAO with para-substituted benzylamines suggest that zMAO catalytic turnover with these substrate analogues is similar to that observed in hMAO A (Figure 2). The a-C-H bond cleavage step appears also to be a main contributor to the rate-determining step in zMAO catalysis. An electronic effect on binding affinity of para-substituted benzylamine analogues to zMAO (Figure 2) is unique to zMAO as neither hMAO A nor bovine or hMAO B exhibit such contributions. One interpretation for this behavior in zMAO is the presence of a positively charged residue in a close proximity to the aromatic ring of the substrate where it could stabilize binding via a p-cation interaction (Gallivan and Dougherty, 1999). Confirmation of these suggestions requires further structural investigations of zMAO.</p><!><p>The studies presented in this paper demonstrate that purified recombinant zMAO exhibits functional properties that are more close to those of hMAO A than to hMAO B. Structure-activity studies suggest a large binding pocket containing a positively charged amino acid residue. Considering that zebrafish contains only one form of MAO, the dual selectivity property of this enzyme for substrate analogues appears reasonable for the organism to degrade biogenic amines. Additional structural and mechanistic work on zMAO should provide new insights into the molecular basis for the differing functional properties of MAO A and of MAO B.</p>

PubMed Author Manuscript

Semantic science and its communication - a personal view

The articles in this special issue represent the culmination of about 15 years working with the potential of the web to support chemical and related subjects. The selection of papers arises from a symposium held in January 2011 ('Visions of a Semantic Molecular Future') which gave me an opportunity to invite many people who shared the same vision. I have asked them to contribute their papers and most have been able to do so. They cover a wide range of content, approaches and styles and apart from the selection of the speakers (and hence the authors) I have not exercised any control over the content.

semantic_science_and_its_communication_-_a_personal_view

Overview<!>Openness and the choice of BMC as publisher<!>Open Data<!>The semantic vision<!>Semantic reality<!>Chemistry as a community<!>The value of informatics<!>Publishing<!>The need to change publication processes<!>The content of the issue<!>The future

<p>The articles have a common theme of representing information in a semantic manner - i.e. being largely "understandable" by machine. This theme is common across science and many of the articles can and should be read by people outside the chemical sciences, including information scientists, librarians, etc. An emergent phenomenon of the last two decades is that information systems can grow without top-down directions. This is disruptive in that it empowers anyone with energy and web-skills, and is most powerful when exercised in communities of people with similar or complementary skills.</p><p>It is often possible to move very quickly, and in our hackfests (one was prepended to the symposium) we have shown that it is possible to prototype within a day or two. This creates a new generation of scientist-hackers (I use "hacker" as "A person who enjoys exploring the details of programmable systems and stretching their capabilities" [1]). Several of the authors in this issue would regard themselves as "hackers" and enjoy communicating through software and systems rather than written English. This stretches the boundaries of the possible but also creates tension where the mainstream world cannot react on a hacker timescale and with hacker ethics.</p><p>More generally many scientists and information professionals are increasingly frustrated with the conventional means of disseminating science. Most conventional publishers regard scientific articles as "their content" and a very recent article (2011-06-20) from the STM publishers [2] indicates that the publishers believe they have the right to determine how content is, or more often is not, used. As an example most forbid by default indexing, textmining, repurposing, even of factual data to which the scientist has a legitimate subscription. This has an entirely negative effect on information-driven science, preventing even the development of the technology.</p><p>Generally, therefore, there is a culture of bottom-up change ("web democracy") which looks to the modern web and examples of empowerment. (There are also examples of disempowerment such as attacks on Net-neutrality, walled gardens, information monopolies, vendor lock-in, etc. and this contrast activates many in the modern informatics world). There are several articles, therefore, whose main theme is the access to Open information.</p><!><p>I have been critical of many publishers for their stance on closed information, and resolved that the issue reporting the symposium had to be completely Open. This is difficult in chemistry where there are almost no "Open Access" journals (those where by default all articles are Open ("Gold")). The "Green" approach, where articles may be posted free-as-in-beer but not free-as-in-speech (e.g. CC-BY), is useless in science as it is impossible to discover and harvest green articles. Hybrid journals (where articles may be made Open by publication charges) are also of little value as the rights to the contents are usually poorly labelled and a machine cannot discover all "Open chemistry articles".</p><p>While writing this overview and several articles I have become even more convinced that the only way of creating full semantic science is to publish Openly (CC-BY) and to publish completely (i.e. all experimental information (CC0/PDDL)). I believe that most funders now recognise this and are pushing, as hard as they can, to create fully Openly published science. I think this has to come, the question is how long it takes and in what form.</p><p>I now believe that in many cases it is unethical to restrict access to publicly funded science. Lessig, in his CERN talk ("Scientific Knowledge Should Not Be Reserved For Academic Elite" [3]), showed that it would cost 500 USD for him to read the top 10 papers relating to his child's condition. These papers are effectively only available to academics in rich universities. A colleague recently told me he had spent a month researching the literature of his child's condition (to critically effective purpose) and we agreed he could only do this because he was a professor at a University. That is one reason I support the Open Knowledge Foundation and its projects to define and obtain Open information (of which Open Bibliography [4] in this issue is typical).</p><p>As part of this effort four of us (including authors in this issue) developed the Panton Principles for Open Scientific Data. These principles are simple and, we hope, self-evidently worth pursuing and would lead to a greatly increased substrate for the Scientific Semantic Web. We were therefore delighted when BioMed Central not only enthusiastically adopted the idea but took positive steps to implement this as part of their publication process, for example by labelling data items with the OKF's "OPEN DATA" logo. This is valuable not only in making the data repurposable, but also by promoting the concept - many readers will now be familiar with the logo. BMC have also encouraged authors (and editors) to highlight outstanding examples of data publication (and done me the honour of asking me to present their awards).</p><p>It is therefore a real pleasure to work with a publisher who understands my, and my co-authors', intentions and is prepared to work to make them happen. The article explores many new types of publications and BMC have undertaken, as far as technically possible, to implement them as examples of a new generation of publication technologies. I and others have been critical of PDF as a publication format - it destroys semantics and innovation, but we must "eat our own dogfood" [5] and this is shown by several articles. Henry Rzepa creates all his molecules as semantic objects, while in Open Bibliography [4] we use our newly developed BibJSON and ScholarlyHTML to create and publish the article.</p><p>I am confident that because of the Openness, the readership of these articles will be much larger than if they were published in a closed access manner, however apparent the prestige of the closed access publisher. It is easy for a mature scientist, such as myself, to publish in an Open Access journal as it is unlikely to affect my career. I'd like to pay credit to all young people who have decided to publish in OA journals despite the possible current (irrational) view that this is detrimental to how they are regarded. I believe that their faith will be justified and that in a very short time the work published here will have higher visibility, and possibly regard, than if it had been published in an apparently more prestigious, closed access journal.</p><!><p>Five years ago the term "Open Data" was unknown (I started a Wikipedia page [6] to collect instances of usage). Now it is ubiquitous. Most of the public funders (Research Councils UK, Wellcome Trust, NIH, NSF and other national bodies) are now requiring that researchers make their data Openly available.</p><p>The first challenge is cultural; researchers have to be persuaded that Open Data is not only inevitable but also beneficial to their activities. Even when an author is convinced of the value of publishing Open Data, it is usually not trivial to do so. Unlike a manuscript where a static, human-readable, webpage can be posted and served for all time, data are frequently much more complex. They may be very large (petabytes), complex in both semantics and organisation, and even distributed over several sites. In bioscience, it is becoming commoner to see data published as Excel and other spreadsheets but in chemistry (apart from crystallography) the tradition is still to publish supplemental data as PDF, which destroys much of its semantics. One simple and achievable goal of these publications is to convince chemists that publishing in semantic form is "almost" no effort, compared to the effort of producing the data in the first place. If we were able to persuade researchers in computational chemistry simply to deposit their logfiles (usually less than 5 MB), or the Word documents for their syntheses, machines would be able to revolutionise the practice and understanding of computational and experimental chemistry. Open Access (CC-BY) implies (but may not explicitly state) that articles can be repurposed by machine extraction of data items, e.g. by OSCAR.</p><p>We have also addressed the question of what is Open Data and how do we identify it, both to humans and to machines. For many chemists, this may be the first time that they have had to consider this problem, but it is becoming increasingly required in many fields and for that reason, we have in several papers, discussed the question of licenses and contracts.</p><!><p>I was excited and entranced by chemical informatics in the mid-70s as a result of some of the ground-breaking work done between chemists and computer scientists. The visions of LHASA, CONGEN, DENDRAL and others opened up the prospect of a chemical world where machines were seen as valuable allies of humans. This vision was also held in the world of chess, and indeed many chemical informatics processes are similar to the operations required in 'artificial intelligence'. Chess has succeeded. Machines can now beat any human on the planet. For whatever reasons, chemistry turned its back on AI and there have been few developments in the last three decades. A necessary condition is the Open availability of semantic data, and if this comes about then there will be a major discontinuity in the way we practice chemistry.</p><p>In 1994, Henry Rzepa and I attended the first WWW conference in CERN. It was a remarkable occasion where a number of very early adopters showed what was possible with web technology and gave a vision of how this would change the way that science was not only reported but also done. There was a feeling that we were entering a new frontier where anything was possible and where new rules would evolve to fit the vision of cyberspace. The final session, where Tim Berners-Lee showed how semantic operations altered the real world was one of the seminal events of my last 20 years.</p><!><p>Not surprisingly, semantic progress has turned out very differently from our original visions. We have stuck to our view that science must adopt semantic technologies including both the formal description of objects and the links between them. Chemistry has been very slow to adopt this, but other subjects have been much more adventurous and in bio- and geo-sciences it is routine to create objects which are derived from, and linked to, other objects.</p><p>Many of the problems are cultural and for that reason several of the papers in this issue address the need to change attitudes as much as the technical requirements for the electronic infrastructure. I believe that it is impossible to do modern science unless the key information is completely Open. This applies, for example, to identifier systems, bibliographic data and much factual data. Chemistry, unfortunately in my opinion, has a strong ingrained culture of possession and sale/licensing of data. For this reason, it is often behind other subjects and, in the recent SOAP report [7] chemistry was highlighted as several years behind bioscience in its approach to Openness.</p><p>For that reason, some of the things we report are prototypes rather than completely established semantic resources. The biosciences have convinced funders that it is valuable to have completely Open access to sequences, structures, ontologies, etc. In chemistry, most of the freely accessible material has been produced by enthusiasts rather than large funded organisations. Indeed, it is the availability of bioscience resources such as ChEBI which to some extent drive the adoption of Open chemical semantics.</p><p>It is also an opportunity for our group to summarise formally several of the projects that they have been working on for several years. It is a feature of information projects that there is often no clear point at which a formal publication is immediately relevant and indeed this highlights the disconnect between publishing necessary information and publishing to acquire a community seal of approval ('a publication').</p><!><p>Many disciplines have a close sense of community (I highlight crystallography which has a real sense of communal practice and goals). Many of the ideas in these articles have been inspired by crystallographic practice, its outstanding scientists, and its International Union - probably the leader in driving semantic approaches.</p><p>Scientific communities are now common on the web (and even have commercial value) and several of the articles emphasise the role of ad hoc and other communities. The web has the great advantage that anyone can, relatively easily, find those people and organizations who share values and goals, amplifying minority or early-adopter initiatives. Their dynamics are unpredictable and most die, but enough survive to provide world-changing mechanisms.</p><p>There is no clear community focus for chemistry overall (though sub sections - such as WATOC (World Association of Theoretical Organic Chemists) may provide one). The main drivers (funding, advancement, commerce) have always been present but the modern era has amplified and often dehumanised them. With growing emphasis on publication to generate the income of learned societies there is a decreasing sense that they act as nuclei for community to grow communal goods.</p><p>Because of this, chemistry has almost no public ontologies, and we have a vicious circle. Without ontologies, authors cannot reasonably be expected to create semantic information, and without a clear need for semantic information, the community will not take on the considerable load of creating ontologies. Several of the articles argue that the creation of lightweight dictionaries and other semantic metadata is affordable by the community and I believe that if the communal will is present, then it would be possible through bodies such as IUPAC and others, to create a full semantic infrastructure for much of the current published chemistry.</p><p>The current legal and contractual restrictions on re-using chemical data are seriously holding chemistry behind other subjects. These articles in this issue are not the place for polemics but we hope that traditional creators of information resources in chemistry will now think carefully about the value of making their data fully Openly available. This will be a considerable act of faith, because it will need a change in business model. Some of those providers have been traditionally held in high esteem by the community and if they use that esteem they have the opportunity to change the practice of chemical informatics.</p><!><p>A major feature underlying all of the papers is to give an insight into the process of creating an information ecology. Some of them represent scientific discoveries (e.g. Rzepa) but most are concerned with building a coherent infrastructure usable by the community. It may be useful to liken this infrastructure to the development of instrumentation in many branches of science. Science depended on the microscope, the telescope, the spectrograph, the Geiger counter and many other types of instrumentation. There is sometimes a modern tendency to discount instrumentation and infrastructure as not being 'proper science'. We hope that this issue will redress that balance.</p><p>As an analogy, Mendeleev required access to other scientists' work to produce his classification, as did Pauling, Woodward and Hoffmann. I believe that the current chemical and related literature contains considerable amounts of undiscovered science, and that with 'information telescopes' we can start to discover this.</p><p>The development of infrastructure is a lengthy process. The web has, perhaps, given us an optimistic idea of the speed at which new ways of working can be implemented. We are still often governed by Planck's observation ("Science progresses one funeral at a time") and this is equally true for some areas of informatics. Several of the articles reflect the difficulty of catalysing change in what is essentially a mature and therefore conservative discipline.</p><p>Henry Rzepa and I were active contributors to the development of XML by running the XML-DEV mailing list (1997). This was a highly successful Open example of true collaboration and for me it culminated in the development of the SAX protocol late that year. XML had been seen as a primarily document- plus typesetting-oriented discipline, but some of us realised its potential for data modelling and transfer, and therefore the need for APIs in XML tools. I nagged continually at the community, and, as a result, Tim Bray, David Megginson and others helped us to develop the SAX protocol, now implemented in every computer on the planet. This protocol was developed in a calendar month and has stood the test of time exceedingly well.</p><p>This, perhaps, gave Henry, myself and other early adopters a false vision of how rapidly we would be able to take these new ideas to chemistry. Over the decade 2000-2010, we have developed and published specifications and software which we believe represent a formal but implementable infrastructure for chemical informatics. The uptake of these has been slow, but unlike some new technologies has not gone through the hype and depression syndrome (Gartner curve). In fact, this timescale is not so unusual. HTML itself has been through nearly 20 years of deployment and only now, with HTML5, does it appear that the community is starting to work together rather than fracturing for organisational and personal advantage. Similarly, semantic MathML is taking many years to become established. It is not that these systems, including CML, have been supplanted by 'better' ways of doing things, but more that the community as a whole is yet to be enlightened about the value of semantics.</p><!><p>Scientific publishing should be a key part of the semantic revolution, but it has so far completely failed to address the vision. This is ironic in that HTML, which catalysed the web, was developed as a way for scientists at CERN to share information, but we have currently regressed to a completely non-semantic (PDF) manner of communication. This has replicated the traditional paper format so well that the only discernable value is to transfer the printing bill from the publishers to the readers. Not only has this held back our imagination, but has actually moulded the new, and I think somewhat unfortunate, values in the publication process. In many cases, authors now publish primarily to attain numerical estimates of worth above communication, validating experiments and other fundamental aspects of the process.</p><p>The web can, and, we hope, will, change this. Where you publish should not matter so long as the material is discoverable and the process of reviewing is understood. I believe that the papers in this issue will be read well beyond the cheminformatics community, because their value will be discerned and communicated by methods supplementary to the formal publishing process.</p><p>A major challenge in this issue is that the timescales for many of the projects is complex. In many lab experiments (such as chemical synthesis or chemical crystallography) the process is clearly bounded. "make this compound", "check success through crystal structure analysis". Each (normally) has a clear endpoint and can be published as a static document.</p><p>In contrast how should we publish software? We use public repositories and these contain a complete record and the current semantic object. If we wish to tell the world about a development we put it on the mailing list. There is no need for a formal publication for those aspects. The motivation is therefore primarily to establish our reputation and there is no simple way to decide when this should be done. JUMBO has had six revisions - should this result in six papers or one or none? (Actually the only JUMBO paper is in 1997 [8]). Six papers would confuse - but after 14 active years it's time for another, I think, which explains the design process. OSCAR3 has its citable publication - a few years back - and we feel it's useful to publish our current ideas, which have more to do with software engineering than new chemical entity recognition.</p><p>Or data? Crystaleye was a spinoff from Nick Day's thesis - it wasn't planned as a separate project - but simply a knowledgebase to use for his calculations. It does not have a formal publication other an archive of a presentation [9]. The system has been running 5 years without serious mishap but the lack of a formal publication makes it difficult to write papers which refer to it. So we shall do this - after the fact. But if we had a semantic publication process it would be "published" by now.</p><!><p>Historically the scientific community has required the following from the publication process:</p><p>• Establishment or priority and authorship</p><p>• Exposure and preservation of the scientific record</p><p>• Communicating the science to one's peers and the wider world</p><p>• Allowing the science to be moderated by peers and others ("reviewing").</p><p>There is perhaps an additional axis in today's bibliometric-obsessed world: allowing the work to receive an official assessment of merit.</p><p>However the publication process is out of sync with the modern web-based world ("Web 2.0") which allows the publication process to encourage and support:</p><p>• Collaborative working (as seen in many projects such as Wikipedia, Open StreetMap, and in science, Galaxy Zoo). Here each contribution is often an atom in a much larger cloud and the publication process is continuous rather than discrete. Wikipedia articles are "never finished" though there are some efforts to provide frozen versions. This is a strong theme of this "issue".</p><p>• Independence of the source of publication. Given the ability of search engines, and the social networks, to discover anything of value it matters less where something is published. Other than the choice of reviewers the primary issues is whether a piece of information is accessible or limited. History has shown that high quality scholarship on the web will usually surface regardless of where it is published.</p><p>• Creation of continuous semantic objects. By recording everything we do, annotating it, and revising it, we can maintain a current semantic publication object at all times, including a revisitable history. This should be the object of scientific publication, not the current PDF.</p><p>• The paper (semantic object) as a driver of research. The idea of writing a paper before the research is carried out is valuable and not novel (e.g. George M. Whitesides [10,11]). Here, however, we extend the paper to semantic objects (programs, spreadsheets, molecules, bibliography, etc.).</p><p>Several of the papers in the article have adopted these later ideas. This has been most obvious in Open Bibliography [4] where effectively the whole concept and technology has been driven during the 6 weeks of "writing the paper". We started with a blank page and four people (William Waites, Mark MacGillivray, Ben O'Steen, Peter Murray-Rust) and during the writing process brought in new authors (Jim Pitman, Peter Sefton, Richard Jones) and communally created the design, technology and "paper". The introduction of Scholarly HTML made this paper self-referential. The Quixote paper [12] has also dramatically driven the design of Quixote, particularly the social aspects.</p><!><p>Several of the articles (CML [13], OSCAR [14], OPSIN, dictionaries [15], WWMM [16]) in this issue cover a decade of work. We hope this will be useful to scientists and scholars who wish to implement new ideas and to give them some idea of what works, and what, more commonly, does not work. Sometimes only the passage of time and persistence achieves some level of success. Again, the short-termism of many infrastructural projects militates against developing a good platform for the future.</p><p>The long timescales highlight the difficulty of conventional publication. The world knows of these projects through blogs, online resources, user communities and so on, and a conventional learned paper has little value in communicating or preserving. Its prime merit is to achieve a traditional numeric merit for the work, often delayed by several years through the citation mechanism. I believe that it is important to change the values that we use in our assessment of on-going scientific endeavours, and avoid ritual publication.</p><p>Some of the articles (Wilbanks [17], Neylon [18]) discuss the philosophy and practice of new models of scientific endeavour and communications. Some of the articles have a retrospective look (CML [13], Zaharevitz [19]) but the fundamental principles are still as important today as when the work was started. A number represent growing points whose development is highly unpredictable. These include the WWMM [16], where the vision of a distributed peer-to-peer knowledge resource has had to wait a decade until it could be implemented. The Quixote project is only months old but takes this vision and has already built an impressive prototype, which I expect to set the model for computationally-based knowledge repositories. These projects rely heavily on community, and this is most clearly shown in the Blue Obelisk movement [20] which aims to, and has largely succeeded in, creating an Open infrastructure for cheminformatics. A major motivation for this has been not just that software and data should be universally available but also that this is the only manner in which science can be reputably validated both by humans and machines. An example of the need for such validation is shown in Henry Rzepa's article [21].</p><p>The OpenBibliography project represents a socio-political imperative whose time has come, and for which the technology is appropriate. A year ago the JISC-funded OpenBibliography project could not point to a significant amount of open resources, but in the last year we have helped to catalyse the release of both library data (BL, CUL and several others), and also of scientific bibliography. It is impossible to find Open resources for scientific bibliography but we believe that in a year's time, readers can look back and see this as a key starting point. It is worth noting that the very process of writing this article has generated a great deal of new formalism and tools in Open bibliography, and effectively given major impetus to the BibJSON approach.</p><p>Other articles (OSCAR, Open patents [22], dictionaries, CML and CMLLite [23]) describe the design and implementation of information systems. In general, there is little funding for developing scientific software, though we have been fortunate to receive some from eScience and from JISC. We have taken this responsibility very seriously and our group has installed many of the cutting-edge ideas and tools for building high-quality systems. Members of the group collaborate and use common servers for their work (as far as possible on Open sites). Software libraries are used and re-used between group members, and we have developed a culture of communal ownership and responsibility. By using the continuous integration system (Jenkins), a failure in one library can immediately be highlighted and corrected before it impacts on other projects. Where funding is available, and where the culture allows it, we would very strongly recommend these practices in other groups. Again, many of these systems have taken over a decade to evolve from initial concepts to mature libraries, but we believe that almost all the systems reported in this article have been heavily re-factored and, within the academic environment, represent an attainable level of quality.</p><!><p>Several articles are growing points, perhaps none more than AMI [24] where we explore the human-cyber interface in a laboratory, a "memex" which may ultimately replace some (but hopefully not all) of the role of the chemistry laboratory. In the same way Quixote represents a memex for computational chemistry. There is no clear pathway for AMI (and I predict that this will be largely influenced by what happens in the domestic arena).</p><p>The relative stagnation of chemical informatics suggests that change is unlikely to happen from within chemistry. As progress occurs in other areas (retail, bioscience etc.) chemistry may be dragged into the semantic world regardless. If chemists wish to retain control over their own systems they will be wise to start investing in Open semantic environments, because otherwise the rest of the world will do it for them.</p><p>How can chemical informatics survive and prosper? I think the most likely model will be Open publishing, not just of texts but data and other resources, mandated and paid for by funders. Those publishers which are able to adopt an Open model rather than continuing to maintain their own walled gardens, will ultimately triumph, and probably more rapidly than we expect.</p>

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