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Facile Solid-Phase Synthesis and Assessment of Nucleoside Analogs as Inhibitors of Bacterial UDP-Sugar Processing Enzymes

The privileged uptake of nucleosides into cells has generated interest in the development of nucleoside-analog libraries for mining new inhibitors. Of particular interest are applications in the discovery of substrate mimetic inhibitors for the growing number of identified glycan-processing enzymes in bacterial pathogens. However, the high polarity and the need for appropriate protecting group strategies for nucleosides challenges the development of synthetic approaches. Here we report an accessible, user-friendly synthesis that branches from a common solid phase-immobilized uridinyl-amine intermediate, which can be used as a starting point for diversity-oriented synthesis. We demonstrate the generation of five series of uridinyl nucleoside analogs for investigating inhibitor structure-activity relationships. This library was screened for inhibition of representative enzymes from three functional families including a phosphoglycosyl transferase, a UDP-amino sugar acetyl-transferase, and a glycosyltransferase. These candidates were taken from the Gram-negative bacteria Campylobacter concisus and Campylobacter jejuni, and the Gram-positive bacterium Clostridium difficile, respectively. Inhibition studies show that specific compound series preferentially inhibit selected enzymes, with IC 50 values ranging from 35 ± 7 μM to 174 ± 21 μM. Insights from the screen provide a strong foundation for further structural elaboration, to improve potency, which will be enabled by the same synthetic strategy. The solid-phase strategy was also used to synthesize pseudouridine analogues of lead compounds. Finally, Abstract Nucleoside analogs, including both complex natural products and simpler synthetic derivatives, represent an important class of compounds with significant potential as selective tools for biological research and as therapeutic agents. Synthetic nucleosides, such as those that cause premature termination of DNA and RNA polymerases, represent a mainstay of current antiviral and antitumor therapy regimes. 1-3 Nucleoside natural products show equally fascinating biological activities, exhibiting, for example, antibacterial activity by inhibition of peptidoglycan and O-antigen biosynthesis. 4 A significant impediment however, to exploiting these useful bioactivities is the complexity of the natural product structures, which makes chemical manipulation to reduce toxicity and improve selectivity and pharmacokinetic properties, a major challenge. Nevertheless, nucleosides show advantageous properties for intracellular delivery. 5 For example, it is known that nucleoside analogs used in the clinic are absorbed across the intestinal epithelium by nucleoside transporters, 6 and many uridine derivatives, such as tunicamycin and mureidomycin, are known to be taken up into bacterial and mammalian cells. 7, 8 Additionally, nucleoside derivatives often show advantageous binding of the constituent nucleobases to target proteins. 9 As a result, nucleosides represent important targets, and their synthetic analogues are recurrent in the structures of antiviral, antibiotic, and antifungal agents based on competition with the binding of uridine-based substrates. 10, glycosylation, and involves transfer which is a luciferin-based assay that reports on the number of viable cells based on quantification of ATP, thus indicating the number of metabolically active cells. For the assay a 96-well plate with 20,000 cells per well was incubated overnight to allow adherence, followed by incubation with 50 μM inhibitor in complete media for 24 or 48 hours. Control wells contained either media, untreated cells, cells treated with 0.5% DMSO (inhibitor vehicle), or cells treated with 1 μg mL −1 doxorubicin (a known DNA-damage agent). The Cell Titer Glo reagent was added to induce lysis, and ATP measured by luminescence. Data was plotted as percentage of cell death compared to cells treated with vehicle (Figure S6). No significant cell death was observed after 48 h with the nucleoside analog library, indicating a lack of cytotoxicity in this cell model. In contrast, 50% of cells treated with doxorubicin were dead at 24 h, with near complete cell death at 48 h. Cells treated with 10% DMSO showed complete rounding and detachment from wells after 24 h. Cells treated with 0.5% DMSO were basically unaffected and used to normalize the data from cells treated with nucleoside analogs. Different sets of plates were used at the 24 h and 48 h time points to maintain sterility, with minimal plate variation observed in reference to controls. Inhibitors a 2 mM stock in DMSO, at a final concentration of 100 μM. TcdB-GTD was pre-incubated in the reaction mixture lacking UDP-Glc for 10 minutes at RT. The reaction rate was pre-determined to be linear over 15 minutes at the given concentrations. Upon addition of UDP-Glc, the reaction was allowed to proceed for 5 min before addition of quenching solution. The reaction mixture was transferred to a 96-well plate (white, nonbinding surface, Corning). The plate was shaken at low speed for 30 s, and incubated for 1 h at RT, and luminescence was

limited. For example, the potent bacterial GT inhibitor moenomycin shows very slow development of antibiotic resistance in in vitro experiments, but its pharmacokinetic properties limit development, and it has been primarily used in animal feed stocks. 15 Many bacterial GTs, phosphoglycosyl transferases (PGTs), and sugar-modifying enzymes utilize uridine diphosphate (UDP)-activated sugar substrates, therefore there is considerable interest in UDP-sugar analogs as substrate mimetics. 16 A review by Gloster and Vocadlo examined classes of carbohydrate-like and medicinal chemistry-like inhibitors for glycosyl hydrolases and transferases, delineating the advantages and disadvantages of various approaches. 17 Polar molecules tend to have reduced nonspecific protein binding, and the carbohydrate-like inhibitors can display excellent potency against glycan-processing enzymes, albeit often accompanied by class promiscuity with off-target effects that can be challenging to overcome. Additionally, high aqueous solubility may limit permeability through cellular membranes. Alternatively, inhibitors derived from library-screening approaches do not depend on the same protein interactions exploited by native substrates and are less likely to display class promiscuity. However, compounds from such screens are often highly lipophilic and poorly soluble, which limits use in cells and in vivo and furthermore, show a higher likelihood of general target promiscuity. 18 As an alternative approach, inhibitor discovery efforts have focused on analogs of donor or acceptor substrates of these enzymes, wherein the negatively-charged phosphate functionality is replaced with an isostere. Additional review articles have compared substrate-like and nonsubstrate-like inhibitors in the context of GTs. 19,20 The screening of modified uridine-based compounds in a variety of organisms and pathways has been the subject of several reports. Approaches developed through solution-phase [21][22][23][24][25][26][27] and solid-phase [28][29][30] chemistry, have resulted in multiple lead compounds. In particular, solid-phase approaches have ranged from small, focused libraries for specific enzymes 29 to large libraries prepared using microscale approaches that were not subject to inhibition analysis. 28,30 Here we present development of a versatile solid-phase synthesis (SPS) platform that can be customized to target nucleoside analog inhibitors of GTs, PGTs and other sugar-modifying enzymes, which builds on advantageous aspects of previous approaches. The SPS methodology accommodates phosphate mimetics, is free from multiple purification steps and protecting group manipulations and is readily adapted to provide compounds for analysis with diverse enzymes that act on UDP-sugars. The SPS approach proceeds through three to five reactions from a common building block. Following assembly, compounds are cleaved from the solid support and purified by reverse phase HPLC (RP-HPLC), yielding desired products in good overall yields (4-25%). Compounds are then assayed in the context of selected glycan-processing enzymes for inhibitory activity, yielding multiple potential leads.

Description of enzyme targets and SPS strategy design.
We present the SPS of five series of uridine analogs and examine activity with three types of bacterial glycan biosynthesis enzymes of interest to the community. GTs such as TcdA and TcdB from C. difficile play crucial roles in toxin release. 31 PGTs such as PglC from C. jejuni are essential for bacterial N-linked protein glycosylation, 32 which is important for adhesion to and invasion into human intestinal epithelial cells. 33 Finally, UDP-aminosugar acetyltransferases such as PglD from the C. jejuni pathway are also important for virulence. 34 PglD has recently been the subject of small molecule, non-nucleoside, inhibitor development efforts. 35 Each of the series of uridine analogs was designed to investigate multiple structure-activity relationships (SAR) (Figure 1). These include: 1) Variation of the distance between the uridine and an aryl group that was designed to occupy the sugar-binding site of the UDPsugar; 2) functionality, provided by amino acid side chains, that include metal ion-binding or metal ion-displacing groups; 3) a bifunctional squaramide moiety, which represents a wellstudied diphosphate mimetic; 4) variation of the aryl (sugar-binding site occupant) group including size, electron density and hydrogenbonding properties, which is introduced using copper-catalyzed azide-alkyne cycloaddition (CuAAC). By exploiting several points of diversification, the goal is to access different sets of inhibitors, which are active in distinct glycan-modifying enzymes. If the different sets of molecules are effective inhibitors of specific enzyme families, this would expand the utility and customizability of the SPS method towards given targets. If however, the same set of compounds are found to be effective towards different enzymes, such 'frequent hitters' would be avoided, as this promiscuity would suggest non-specific activity, and would not be valuable as leads for optimization. 36 The SPS methodology was also applied to the synthesis of compounds containing pseudouridine, thus expanding the repertoire and potential scope of the approach. As the most abundant modification in RNA, 37 there is considerable interest in the properties of this nucleoside in therapeutics, and recently pseudouridimycin was found to inhibit bacterial RNA polymerase in otherwise drug-resistant pathogens. 2 Since its discovery in the 1950s, pseudouridinyl analogs have been relatively understudied, due to cost and poor synthetic accessibility.

Solid-phase synthesis of uridine analog library.
The synthesis is based on the approach developed by Epple et al. 30 The optimized route features resin immobilization via the ribose 2' and 3' hydroxyl groups as a stable aliphatic acetal and an efficient, resin-based route from the 5'-hydroxyl intermediate to the 5'-aminouridine, 10. In general, uracil protecting groups were avoided, to simplify the overall schemes; thus the initial steps were optimized to minimize side reactions at the N-3 position. This immobilized intermediate serves as the common starting point for diversification and has been prepared on a 3.0 mmol scale, which provides sufficient material for the preparation of ten nucleoside analogs that were aliquoted into 10 mM stock solutions sufficient for hundreds of assays. The purity of each resin-bound intermediate was assessed by LC-MS analysis. To prepare 10, the keto ester 7 was synthesized by O-alkylation of 4-(4hydroxyphenyl)-2-butanone 6 with 5-bromovaleric acid methyl ester (Scheme 1). The ketone was activated with trimethylorthoformate (TMOF) and p-toluenesulfonic acid (pTsOH), which underwent condensation with the ribose diol to yield the acetal 8. Ester hydrolysis provided the uridine-carboxylate 9. The carboxylate was immobilized through amide bond formation with H-Ala-Wang polystyrene resin using 2-(1H-benzotriazol-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and N,Ndiisopropylethylamine (DIPEA). The 5'-OH of the uridine was then converted to the corresponding 5'-NH 2 , by means of a Mitsunobu reaction with 3,4,5,6tetrachlorophthalimide (TCP), followed by treatment with ethylenediamine (EDA) in dimethylformamide (DMF) to afford the on-resin 5'-NH 2 uridine 10 (see Scheme 1).
All crude products were then precipitated from the cleavage cocktail with cold ether, resuspended in 20% acetonitrile in water, and purified by RP-HPLC to yield the final products 1-5. Overall compound yields were 4-25% based on the initial loading of carboxylate 9 on the resin.

Enzyme inhibition by uridine analog library.
Purified compounds were assayed for inhibition activity against the three target enzymes using convenient assays in a multiwell plate format. The assays included the UDP-Glo GT assay (Promega), which detects production of UDP, the UMP/CMP-Glo PGT assay (Promega) 38 which detects UMP release, and the amino-sugar acetyltransferase assay based on Ellman's reagent (DTNB), which is applied to monitor the release of coenzyme A (CoASH) upon acyl transfer. 39

Inhibition of PglC Measured by Luminescence Using the UMP-Glo Assay.-
Bacterial PGTs catalyze transfer of a phosphosugar from a UDP-sugar to undecaprenol phosphate (Und-P) with the release of UMP. 40 In C. jejuni, the monotopic PGT PglC catalyzes the first membrane-committed step in N-linked glycosylation, and involves transfer of di-N-acetylbacillosamine (diNAcBac) phosphate from UDP-diNAcBac to Und-P ( Figure  S1). Recently, the structure of a PglC homolog from C. concisus, which shares 72% sequence homology with the C. jejuni enzyme has been reported. 41 Therefore, in the interests of later applying structure-driven nucleoside analog inhibitor optimization, the C. concisus enzyme was pursued in these inhibition studies. The UMP-Glo assay is based on a previously-reported procedure, 23 and begins with pre-incubation of PglC in the reaction buffer with Und-P and 100 μM inhibitor for 10 minutes at room temperature (RT). Upon addition of UDP-diNAcBac, the reaction was allowed to proceed for 15 min before quenching with the detection reagent and was transferred into a 96-well plate and luminescence data was collected. Assay results were plotted as the percentage of remaining activity compared to the positive control (no inhibitor).
We note that the Promega Glo assays detect free UMP or UDP generated during the course of the PGT or GT reactions. However, due to the structural similarity between synthesized inhibitors and the UDP and UMP-detecting enzymes used in the Glo assays, we also controlled for off-target inhibition of the Glo reagent enzymes. Glo assay control experiments were first conducted in the presence and absence of inhibitors at 2 μM UMP (for PglC) or 1 μM UDP (for TcdB). These concentrations of UMP and UDP represent the amount of nucleotide released in a typical assay. 38 Using this information the percent of background inhibition was used to adjust the luminescence readout.  Figure S2). Additionally, Lineweaver-Burk analysis supported competitive binding of 4b ( Figure S3). This observation is consistent with previous work which showed that negatively-charged metal-coordinating groups provide greater inhibitory activity. 23 This informs us that further improvement of potency might be achieved through optimization of the metal-binding moiety. Along with the recently obtained crystal structure of PglC, 41 a more refined SAR around the sugar mimetic should also allow for the synthesis of more potent inhibitors, readily obtained from the SPS strategy. Finally, this information, could potentially provide access to small molecules that disrupt protein glycosylation in related pathogens.

Inhibition of PglD Measured
Using the Ellman's Reagent Assay.-PglD from C. jejuni is a prokaryote-specific UDP-amino sugar acetyltransferase ( Figure S1). 39 For inhibition studies, PglD was pre-incubated in the reaction buffer with 100 μM inhibitor for 20 minutes at RT, followed by addition of UDP-4-amino-NAcBac, acetyl-CoA, and DTNB. Initial rates were determined by monitoring absorbance at 412 nm in the 6-minute linear portion of the reaction curve, and compared against a positive control without inhibitor. Figure 3 shows the activity of PglD in the presence of the 24 inhibitors. In this case, compound 2b emerged as the best lead. Assays in the presence of a range of concentrations of 2b afforded an IC 50 of 35 ± 7 pM ( Figure S2). However, Lineweaver-Burk analysis in this case suggests that 2b is a non-competitive inhibitor of PglD ( Figure S4, Table S1). Co-crystallization of 2b with PglD will be valuable to understand the binding mode, which can then be used to refine the design of structurally-targeted inhibitors. The ability of compound 2b to disrupt enzymatic activity provides multiple points of variation for the next generation of inhibitors, which would readily accessible through the SPS method. Combined with the recent development of potent nM inhibitors of the acetyl-CoA binding pocket, 35 a new class of inhibitors bearing a nucleoside, which may exhibit privileged entry into target bacterial cells, may advance in vivo inhibition studies with C. jejuni. proteins, which belong to the family of multidomain clostridial glucosylating toxins that inactivate small GTPases, which are key players in eukaryotic signaling. 31 Inactivation is the direct consequence of threonine glycosylation in RhoA-C, RhoG, Rac1, and Cdc42 proteins by the N-terminal glucosyltransferase domain (GTD) of the toxins. 31 In the inhibition studies we used the catalytically-active TcdB-GTD domain with Cdc42-His 6 as the glycosyl acceptor and followed previously-reported assay conditions. 42 TcdB-GTD was preincubated with Cdc42-His 6 and 100 μM inhibitor for 10 minutes at RT. Upon addition of UDP-Glc, the reaction was allowed to proceed for 5 min before quenching with detection reagents and was transferred into a 96-well plate and luminescence data collected. Assay results were plotted as the percentage of remaining activity compared to the positive control (no inhibitor). Figure 4 shows the percent activity of each GT reaction in the presence of the 24 inhibitors. In this case, two of the squaric acid derivatives (5c and 5f) from series 5 showed the most significant inhibition. Additionally, a single member of series 2 (2e), was also active. The lowest IC 50 value was 174 ± 21 μM for squaric compound 5c ( Figure S2), for which a Lineweaver-Burk plot indicated competitive binding ( Figure S5). Analysis of the crystal structure of TcdB-GTD shows a very deep and structured binding site with little space for extra substitution near the 5'-substituent of the uridine, but with more open space near the glucose binding site of the UDP-glucose substrate. 43 From this we can reason that extended structures, as in series 3 and 4 and analogs with longer amino acid side chains, as in series 1, 3, and 4, might not compete efficiently with the native substrate. Conversely, analogs with unsubstituted amino acids (series 2 and 5) featured a few compounds with inhibitory potential, and would be a focus of further syntheses. TcdB-GTD contains the GT-A specific DXD motif, where two catalytically-essential aspartates bind Mn 2+ , which then binds to the diphosphate of UDP-Glc. 44 An inhibitor scaffold which binds divalent cations would then be highly desirable, making squaramide compounds promising, and informing on future directions. This approach opens the possibility of developing competitive inhibitors of the glycosyltransferase domain in C. difficile toxins, to complement other efforts that have focused on noncompetitive inhibitors. 42

Synthesis of Pseudouridine-containing Inhibitor Compounds.
Following screening of the 24-membered nucleoside analog library at a fixed concentration, three of the promising leads were resynthesized as the corresponding pseudouridinyl variants (Scheme S1). The SPS and purification followed the established protocols for the uridine analogs (Scheme S1). Overall, the three compounds, PU2e, PU4b and PU4e, showed similar inhibitory activity relative to the corresponding uridine analogs ( Figure 5). This indicates that the uracil binding sites of these enzymes may allow for this modification without a detrimental impact on binding, perhaps by binding to the face of the uracil that shares a similar hydrogen-bond "acceptor-donor-acceptor" pattern. This finding gives way to the opportunity to pursue pseudouridinyl variants of any potent hits identified in the future, which could provide a means of reducing off-target effects in inhibitor development for cellbased studies.

Mammalian Cell Viability Measured by ATP-Glo Assay in Presence of Inhibitors.
The cytotoxicity of the presented nucleoside analogs was measured in a mammalian cell model. As membrane permeation of nucleoside analogs can occur via active transport, this is a major advantage in bacterial target accessibility, but may also be a concern as inhibitors could affect functionally-related glycan-modifying enzymes containing structurally similar active sites. 16 One such example is the potent antibiotic tunicamycin which competitively inhibits WecA, but has not been used as an antibiotic in humans because of cytotoxicity in mammalian cells, due to off-target inhibition of the PGT at the inception of the dolichol pathway. 40,45 Nucleoside analogs can also act as antimetabolites that chain terminate DNA and RNA polymerization particularly with 3'-OH substitution with H, N 3 , F, or alkylation. 1 However, the latter is unlikely to be a concern as all of the analogs are modified at ribose C-5' site and therefore cannot be activated as a polymerase building block.
To probe for general toxicity, we examined the nucleoside analogs through the use of a wellestablished cell line, IMR-90 human lung fibroblasts. This cell line was chosen because it is a non-carcinoma line that grows well in culture, is often used for proof-of-principle experiments, and is a robust model with a clear cell rounding index for future toxicityrelated studies. 42,46 Cell toxicity was measured using Cell Titer Glo (Promega), which is a luciferin-based assay that reports on the number of viable cells based on quantification of ATP, thus indicating the number of metabolically active cells. For the assay a 96-well plate with 20,000 cells per well was incubated overnight to allow adherence, followed by incubation with 50 μM inhibitor in complete media for 24 or 48 hours. Control wells contained either media, untreated cells, cells treated with 0.5% DMSO (inhibitor vehicle), or cells treated with 1 μg mL −1 doxorubicin (a known DNA-damage agent). The Cell Titer Glo reagent was added to induce lysis, and ATP measured by luminescence. Data was plotted as percentage of cell death compared to cells treated with vehicle ( Figure S6). No significant cell death was observed after 48 h with the nucleoside analog library, indicating a lack of cytotoxicity in this cell model. In contrast, 50% of cells treated with doxorubicin were dead at 24 h, with near complete cell death at 48 h. Cells treated with 10% DMSO showed complete rounding and detachment from wells after 24 h. Cells treated with 0.5% DMSO were basically unaffected and used to normalize the data from cells treated with nucleoside analogs. Different sets of plates were used at the 24 h and 48 h time points to maintain sterility, with minimal plate variation observed in reference to controls.

Conclusions.
In summary, we have established a practical and versatile SPS method for the facile generation of uridine-based nucleoside analogs. The synthetic route will be highly accessible to a broad range of laboratories, owing to the limited number of simple steps, minimal purification of intermediates, and lack of protecting groups manipulations. The SPS approach has also been shown to be compatible with the synthesis of pseudouridine analogues, which have recently gained interest among researchers, due to promising effects against drug-resistant pathogens. The representative library of 27 uridine or pseudouridinebased compounds were assayed in plate-based formats against three different glycanmodifying enzymes: the phosphoglycosyl transferase PglC from C. concisus, the UDP-4amino-sugar acetyltransferase PglD from C. jejuni, and the glycosyltransferase domain of TcdB from C. difficile. These three enzymes were chosen based on available structural information, to help facilitate future efforts to optimize lead compounds via a structuredriven approach.
Individual compounds exhibited increased inhibitory activity for each enzyme; the more effective compounds for TcdB did not contain amino acid side chains, whereas glutamic acid-containing constructs inhibited PglC to a greater degree, and one 3-fluoro compound showed good inhibition of PglD, with an IC 50 of 35 ± 7 μM. The identification of distinct leads in each enzyme class tested is a good sign towards the minimization of class promiscuity that can plague many enzyme-targeted approaches. Each enzyme is also highly relevant to the pathogenicity of the organisms. Additional support towards minimized offtarget effects was given by the lack of compound cytotoxicity in human lung fibroblasts upon 48h exposure.
These studies now provide the springboard for further adaptation of specific analogs, and can guide understanding of structural features of glycan-processing enzymes. Together, the ease of access to a broad class of customizable nucleoside analogs, along with the identification of multiple leads for further inhibitor development, provides a platform for small molecule inhibitor discovery for sugar-nucleotide modifying enzymes that have not been commonly pursued.

UMP-Glo Assay.
PglC assays were performed using the Promega UMP-Glo assays, which detects UMP. Due to the structural similarity between the synthesized inhibitors and UMP, we also controlled for off-target inhibition of the Glo reagent enzymes by the nucleoside analogues and subtracted it from the total inhibition. The quenching solution was prepared as described by Promega. Assays contained 20 μM Und-P, 10 % DMSO, 0.1 % Triton X-100, 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 20 μM UDP-diNAcBac 39 and 0.2 nM PglC in a final volume of 20 uL. Inhibitors were added from a 2 mM stock in DMSO, at a final concentration of 100 μM. PglC was pre-incubated in the reaction mixture lacking UDP-diNAcBac for 10 minutes at RT. The reaction rate was predetermined to be linear over 20 minutes at the given concentrations. Upon addition of UDP-diNAcBac, the reaction was allowed to proceed for 15 min before addition of quenching solution. The reaction mixture was transferred to a 96-well plate (white, nonbinding surface, Corning). The plate was shaken at low speed for 16 min, incubated for 44 min at RT and luminescence was read using a Synergy H1 hybrid plate reader (Biotek). Data was plotted using GraphPad Prism, as percentage remaining activity compared to the positive control (no inhibitor).

Ellman's Reagent Assay.
PglD assays were performed by monitoring the production of CoASH over the course of the reaction using DTNB. Absorbance was measured in continuous mode at 412 nm. Using a 96-well clear bottom plate, the inhibitors were added from a DMSO stock solution at a final concentration of 100 μM, followed by PglD in 50 mM HEPES, pH 7.4, 1 mM MgCl 3 , 0.05 mg mL −1 BSA, 0.001% Triton X-100. Inhibitors and enzyme were preincubated 20 min at RT, followed by addition of substrate and Ellman's reagent cocktail to final concentrations of 6 nM PglD, 274 μM UDP-4-amino-NAcBac, 47 295 μM AcCoA, 2 mM DTNB, and 3% DMSO in 100 μL volume. Initial rates were measured in the linear portion of the reaction curve over a 6-min time period at RT, and absorbance was read at 412 nm on the plate reader.

UDP-Glo Assay.
TcdB assays were performed using the Promega UDP-Glo assay, which detects UDP generated over the course of the reaction. Due to the structural similarity between the inhibitors and UDP, we also corrected for off-target inhibition of the Glo reagent enzymes by the nucleoside analogues. The quenching solution was prepared as described by Promega. Assays contained 30 μM Cdc42, 5% DMSO, 0.1 % Triton X-100, 50 mM HEPES pH 7.5, 100 mM KCl, 4 mM MgCl 2 , 1 mM MnCl 2 , 20 μM UDP-Glc, and 0.5 nM TcdB-GTD in a final volume of 20 μL. Inhibitors were added from a 2 mM stock in DMSO, at a final concentration of 100 μM. TcdB-GTD was pre-incubated in the reaction mixture lacking UDP-Glc for 10 minutes at RT. The reaction rate was pre-determined to be linear over 15 minutes at the given concentrations. Upon addition of UDP-Glc, the reaction was allowed to proceed for 5 min before addition of quenching solution. The reaction mixture was transferred to a 96-well plate (white, nonbinding surface, Corning). The plate was shaken at low speed for 30 s, and incubated for 1 h at RT, and luminescence was read on the plate reader. Data were plotted as in the UMP-Glo assay.

Expression and Purification of Enzymes.
PglC from C. concisus was expressed and purified as described previously. 38 PglD from C. jejuni was expressed and purified following the reported procedure. 35 For cloning, expression, and purification of TcdB-GTD from Clostridium difficile, see Supporting Information.

Mammalian Cell Viability.
Mammalian Cell Viability. IMR-90 human lung fibroblasts were obtained from the American Tissue Culture Collection (ATCC) and maintained in Eagle's Modified Essential Medium (VWR) supplemented with 10% (v/v) fetal bovine serum (FBS) and Pen-Strep (P/S), in a humidified incubator with 5% CO 2 and 37 °C. Proliferating cells were subcultured at a ratio of 1:3 when confluent, and plated in 96-well TC-treated white clearbottom plates with lids at 20,000 cells per well, as determined by hemocytomer counting. Overnight incubation with 200 μL complete media allowed adherence of cells to the plate, which were fully confluent by microscope inspection. Growth media was removed from cells by aspiration, followed by the addition of 50 μM nucleoside analogs 1-5 in growth media with 0.5% DMSO. Additional wells instead received either 1.7 μM doxorubicin hydrochloride, 10% DMSO, or 0.5% DMSO in complete media to serve as controls for cell toxicity. Plates were then incubated for 24 h and 48 h time points. Media was then removed by aspiration, 100 μL complete media was added to each well, and 100 μL Cell Titer Glo reagent (Promega) was added to induce lysis. One row of media without cells was used as a background measurement.  PglC activity was measured by UMP-Glo monitoring luminescence after 10 min preincubation with inhibitor and a 15-min reaction with UDP-diNAcBac, in reference to control with no inhibitor. Solid bars indicate compounds with the highest inhibitory activity. Error bars indicate mean ± SD; n=3. PglD activity measured by CoASH release using DTNB. Percent inhibition was determined after 20 min pre-incubation with inhibitor and a 6-min reaction with UDP-4-amino sugar, in reference to control with no inhibitor. Solid bars indicate compounds with the highest inhibitory activity. Error bars indicate mean ± SD; n=3. TcdB-GTD activity was measured by UDP-Glo monitoring luminescence. Substrate conversion was measured after 10 min pre-incubation with inhibitor followed by a 5-min reaction time with UDP-Glc. Conversions are normalized to control reaction without inhibitor. Solid bars indicate compounds with the highest inhibitory activity. Error bars indicate mean ± SD; n=3. A. Structures of pseudouridinyl inhibitors. B. Comparison of uridinyl vs pseudouridinyl inhibitors for three selected analogs referenced to control with no inhibitor. Error bars indicate mean ± SD; n=3.


extfieldsofstudy:
Materials Science
Medicine

text:
From the Synthesis of Nanovehicles to Participation in the First Nanocar Race—View from the French Team

This review article presents our accomplished work on the synthesis of molecular triptycene wheels and their introduction into nanovehicles such as wheelbarrows and nanocars, equipped with two and four wheels, respectively. The architecture of nanovehicles is based on polycyclic aromatic hydrocarbons, which provide a potential cargo zone. Our strategy allowed us to obtain planar or curved nanocars, exhibiting different mobilities on metallic surfaces. Our curved nanocar participated in the first nanocar race organized in Toulouse (France) on 28 and 29 April 2017.

Introduction
In 2013, in a perspective article on "molecule concept nanocars" [1], Christian Joachim and Gwénaël Rapenne proposed "a yearly competition whereby all nanocar designers would meet together for a few days, similar to the microrobotics contests held around the world". It took almost four years to find a way to organize such a race, find sponsors, leave the time necessary for competitors to design and synthesize their prototypes, and develop a four-tip scanning tunneling microscope (STM) suitable to host the race. In 2017, six competitors from Austria, Germany, Japan, Switzerland, the United States, and France finally met in Toulouse (France) to compare their designs and strategies to speed up the movement of a single nanocar on a metallic surface. On the 28 April 2017, the starting signal was sounded at exactly 11 a.m. by Dr. Jean-Pierre Launay, emeritus Professor at the University Paul Sabatier (Toulouse) and race commissioner.
The Nanocar Race [2] was seen as a major communication event by the general public. In this review, we will describe why it was a highly challenging scientific event, which was selected to be a part of "Research of the Year 2017" by Chemical Engineering News [3], the scientific journal of the American Chemical Society, as well as the leading French newspaper Le Monde [4]. The last 15 years of research in our group will be detailed, from the design and synthesis of the first molecular wheelbarrow (a platform with two wheels), to the different prototypes of nanocars developed for the nanocompetition.
Nanovehicles are molecular machines able to move efficiently and with some kind of control and directionality at the nanoscale. The first prototypes were technomimetic [5], with a chassis connected to wheels. As pioneers in the field, we proposed using triptycenes [6] to act as rolling elements [7] and synthesized the first nanovehicle with two wheels-a wheelbarrow [8]. Two years after us, Tour's group reported a family of nanovehicles consisting of a molecular-scale chassis with four [60]-fullerene wheels [9]. Various nanocars were subsequently synthesized by the same group using carboranes [10],

The First Two-Wheeled and Two-Legged Vehicle: The Molecular Wheelbarrow
The Toulouse team published the design for the first molecule-vehicle in 2002 [7], and it was synthesized in 2003. It was equipped with a chassis, two wheels at the front, two legs, and two handles at the rear, structurally similar to a wheelbarrow [8]. In the case of a macroscopic wheelbarrow, pushing results in the rotation of the wheels. The purpose of this first vehicle was thus to observe the rotational movement of the two front wheels while pushing on the handles and keeping the rear part of its chassis far from the surface, thanks to the two rear molecular legs.

Design
Wheelbarrow 1 consists of two legs (3,5-di-tert-butylphenyl groups, shown in green in Figure 1) and two three-cogged wheels (triptycene groups, shown in red in Figure 1), which can freely rotate around the axle thanks to the presence of ethynyl spacers [20]. As shown in Figure 1, the skeleton consists of polycyclic aromatic hydrocarbons (PAHs), which due to their rigidity make manipulation by the STM tip easy. As anticipated, the two 3,5-di-tert-butylphenyl legs were shown to be held in a conformation in which the phenyl groups are nearly perpendicular to the main aromatic board. This was exploited to lift the chassis, and thus to minimize its interaction with the surface. Moreover, tert-butyl groups connected to PAHs are used to increase organic solubility and are easily observed by STM techniques, inducing good contrast in the image. The two 4-tert-butylphenyl groups (in blue) play the role of handles for subsequent manipulation using the tip of the microscope.

Synthesis
The synthesis of the molecular wheelbarrow 1 was achieved in twelve steps, with an overall yield of 2% [21]. As shown in Scheme 1, our strategy used the repetition of a "double Knoevenagel condensation-Diels-Alder reaction" sequence on an α-diketo fragment. The first sequence allowed the connection of the two 3,5-di-tert-butylphenyl legs (Step a) and the handles (Step b), while the second one provided the precursor for the connection of the wheels (Steps d and e). Thus, 1,3-di(4-iodophenyl)propan-2-one was selected as partner in this last double Knoevenagel reaction to introduce the iodine centers necessary for the final double Sonogashira coupling, which yielded the molecular wheelbarrow (Step f).
The starting cyclopentadienone 4 was obtained first via a double Knoevenagel reaction of 1,3bis(3,5-di-tert-butylphenyl)propan-2-one 2 with diketopyracene 3 (Step a). The Diels-Alder reaction of cyclopentadienone 4 with di-(4-tert-butylphenyl)acetylene (Step b) provided the ethane-bridged precursor 5 in 97% yield. The 1 H-NMR spectrum clearly showed the expected 2:1 ratio between the different types of tert-butyl groups, belonging to the legs and to the handles, respectively. Oxidation of the ethane bridge of the pyracene fragment with benzeneseleninic anhydride (Step c) yielded the α-diketo fragment 6 necessary for the connection of the second axle. This is the key step in our strategy. Aryl halides are introduced at this stage for subsequent cross-couplings, in order to connect the triptycene wheels. The double Knoevenagel condensation with 1,3-di(4-iodophenyl)propan-2-one (Step d) followed by another Diels-Alder reaction with di(4-tolyl)acetylene provided the diiodo intermediate 8 in 30% yield (Step e). The two wheels were then simultaneously covalently attached to the axle via a double coupling of 9-ethynyltriptycene under classical Sonogashira conditions (Step f), affording the molecular wheelbarrow 1 in 55% yield.

Single Molecule Scanning Tunneling Microscopy Images and Manipulation
Such a molecular-scale machine inevitably displays a high level of complexity due to the incorporation of several functionalities within the same molecule. This complexity was reflected in the relatively large molecular weight of the wheelbarrow (1802 g·mol −1 ), which can often lead to problems in the deposition due to the possibility of thermal fragmentations during the sublimation step [22]. In the present case, the required linear wheel axle included two thermally-sensitive triple bonds. However, a few intact molecular wheelbarrows were successfully imaged on a Cu(100) surface, as shown in Figure 2. It leads to a rather complicated STM image, dominated by three intense maxima separated by various weaker patterns, as expected for such a complex chemical structure. The characteristic dimensions, the equivalency of different molecules in the STM images, and comparison with images calculated by electron scattering quantum chemistry (ESQC) method [23] indicated that intact molecular wheelbarrows had been imaged. Manipulation with the STM tip at 5 K resulted in a conformational change in the molecule, which unambiguously confirmed the molecularity of the observed object: the spots obtained were not an assembly of fragments. to the axle via a double coupling of 9-ethynyltriptycene under classical Sonogashira conditions (Step f), affording the molecular wheelbarrow 1 in 55% yield.

Single Molecule Scanning Tunneling Microscopy Images and Manipulation
Such a molecular-scale machine inevitably displays a high level of complexity due to the incorporation of several functionalities within the same molecule. This complexity was reflected in the relatively large molecular weight of the wheelbarrow (1802 g·mol −1 ), which can often lead to problems in the deposition due to the possibility of thermal fragmentations during the sublimation step [22]. In the present case, the required linear wheel axle included two thermally-sensitive triple bonds. However, a few intact molecular wheelbarrows were successfully imaged on a Cu(100) surface, as shown in Figure 2. It leads to a rather complicated STM image, dominated by three intense maxima separated by various weaker patterns, as expected for such a complex chemical structure. The characteristic dimensions, the equivalency of different molecules in the STM images, and comparison with images calculated by electron scattering quantum chemistry (ESQC) method [23] indicated that intact molecular wheelbarrows had been imaged. Manipulation with the STM tip at 5 K resulted in a conformational change in the molecule, which unambiguously confirmed the molecularity of the observed object: the spots obtained were not an assembly of fragments. Unfortunately, lateral motion of the wheelbarrow could not be achieved. This could be explained by the fact that two wheels may not be enough to allow the motion on the surface of such a large molecule, due to strong coupling. Increasing the number of wheels was proposed to enhance the molecular mobility of nanovehicles, and as a result the design of nanovehicles with four wheels was envisioned.

The First Rotation of Wheels on a Surface
Wheels are of course one of the key mechanical elements of a vehicle, even on a molecular chassis. At the macroscopic scale, the molecular wheels move the frame slightly away from the supporting surface, in order to lower the lateral diffusion barrier of the molecule-vehicle by reducing the frame-surface electronic interactions. Such decoupling might be achieved using simple molecular legs, although the movement of the molecule on the surface would then cost more energy, as it would require a tilting movement of each molecular leg. As in the macroscopic world, rotation around an axis is thus more energetically favorable.
It was shown that the mechanical motion of a single molecule on a surface can be triggered by the tip apex of the STM [24]. Pushing a molecule with an STM tip was a convenient way to probe the triptycene fragment to act as a wheel at the nanoscale. For that purpose, a prototype composed of two wheels linked to an axle was designed. Unfortunately, lateral motion of the wheelbarrow could not be achieved. This could be explained by the fact that two wheels may not be enough to allow the motion on the surface of such a large molecule, due to strong coupling. Increasing the number of wheels was proposed to enhance the molecular mobility of nanovehicles, and as a result the design of nanovehicles with four wheels was envisioned.

The First Rotation of Wheels on a Surface
Wheels are of course one of the key mechanical elements of a vehicle, even on a molecular chassis. At the macroscopic scale, the molecular wheels move the frame slightly away from the supporting surface, in order to lower the lateral diffusion barrier of the molecule-vehicle by reducing the frame-surface electronic interactions. Such decoupling might be achieved using simple molecular legs, although the movement of the molecule on the surface would then cost more energy, as it would require a tilting movement of each molecular leg. As in the macroscopic world, rotation around an axis is thus more energetically favorable.
It was shown that the mechanical motion of a single molecule on a surface can be triggered by the tip apex of the STM [24]. Pushing a molecule with an STM tip was a convenient way to probe the triptycene fragment to act as a wheel at the nanoscale. For that purpose, a prototype composed of two wheels linked to an axle was designed.

Synthesis and Deposition of a Bis(ethynyltriptycene) as Prototype of a Wheel Dimer
Copper(II)-mediated Glaser homocoupling of 9-ethynyltriptycene fragments-the wheels already used in the molecular wheelbarrow-gave the corresponding dimer of wheels linked via a butadiyne spacer. The latter was a good candidate, since it allowed almost free rotation of the attached fragments [20] in combination with a linear geometry. The ethynyltriptycene dimer has subsequently been studied as a prototype wheel dimer.
Its deposition on a Cu(110) surface was achieved very cleanly by sublimation under high vacuum [25]. Figure 3 shows the image obtained; each bright lobe corresponds to one triptycene unit, and the observed pair of lobes constitutes the image of a dimer, as confirmed by calculations. No fragmentation or decomposition was observed.

Synthesis and Deposition of a Bis(ethynyltriptycene) as Prototype of a Wheel Dimer
Copper(II)-mediated Glaser homocoupling of 9-ethynyltriptycene fragments-the wheels already used in the molecular wheelbarrow-gave the corresponding dimer of wheels linked via a butadiyne spacer. The latter was a good candidate, since it allowed almost free rotation of the attached fragments [20] in combination with a linear geometry. The ethynyltriptycene dimer has subsequently been studied as a prototype wheel dimer.
Its deposition on a Cu(110) surface was achieved very cleanly by sublimation under high vacuum [25]. Figure 3 shows the image obtained; each bright lobe corresponds to one triptycene unit, and the observed pair of lobes constitutes the image of a dimer, as confirmed by calculations. No fragmentation or decomposition was observed. Adapted from [22], copyright 2006, with permission from Elsevier.

Unidirectional Rotation of the Wheel
For the first time, it was possible to observe the rotation of a wheel on a surface, by working at 5 K under ultra-high vacuum. This was achieved by inducing the translation of the molecule upon pushing it with the STM tip [25]. In order to understand the motion of this molecule in detail, it was particularly important to study the manipulation signal (i.e., the tunneling current recorded at constant tip height during the motion), because the STM images before and after manipulation did not give information about the type of movement between measurements (translational or rotational).
The tip was moved across the molecular axle at a constant height, while the tunneling current was recorded. In most cases, it was possible to rotate one wheel, and in a few cases, the rolling of both wheels was observed (as shown in Figure 4). This behavior seems to depend on the precise shape of the tip apex, which in some cases allowed us to address both wheels during the manipulation. This triptycene dimer was the first molecule designed to yield a controlled rotation upon pushing the wheels using the STM tip. With this rolling motion proven, four triptycene units were then integrated into the next generation of nanovehicles, with the aim of increasing mobility on the surface. Step 1: the STM tip approaches the molecule; Step 2: the rotation of the wheel starts; Step 3: the STM tip lies on the other side of the wheel after a 120° rotation. Adapted from [25], copyright 2007, with permission from Springer Nature.

A Planar Nanocar
For the preparation of nanovehicles containing four wheels (i.e., nanocars), it was necessary to develop the synthesis of a larger chassis based on a perylene unit. This was to sterically accommodate

Unidirectional Rotation of the Wheel
For the first time, it was possible to observe the rotation of a wheel on a surface, by working at 5 K under ultra-high vacuum. This was achieved by inducing the translation of the molecule upon pushing it with the STM tip [25]. In order to understand the motion of this molecule in detail, it was particularly important to study the manipulation signal (i.e., the tunneling current recorded at constant tip height during the motion), because the STM images before and after manipulation did not give information about the type of movement between measurements (translational or rotational).
The tip was moved across the molecular axle at a constant height, while the tunneling current was recorded. In most cases, it was possible to rotate one wheel, and in a few cases, the rolling of both wheels was observed (as shown in Figure 4). This behavior seems to depend on the precise shape of the tip apex, which in some cases allowed us to address both wheels during the manipulation. This triptycene dimer was the first molecule designed to yield a controlled rotation upon pushing the wheels using the STM tip. With this rolling motion proven, four triptycene units were then integrated into the next generation of nanovehicles, with the aim of increasing mobility on the surface.

Synthesis and Deposition of a Bis(ethynyltriptycene) as Prototype of a Wheel Dimer
Copper(II)-mediated Glaser homocoupling of 9-ethynyltriptycene fragments-the wheels already used in the molecular wheelbarrow-gave the corresponding dimer of wheels linked via a butadiyne spacer. The latter was a good candidate, since it allowed almost free rotation of the attached fragments [20] in combination with a linear geometry. The ethynyltriptycene dimer has subsequently been studied as a prototype wheel dimer.
Its deposition on a Cu(110) surface was achieved very cleanly by sublimation under high vacuum [25]. Figure 3 shows the image obtained; each bright lobe corresponds to one triptycene unit, and the observed pair of lobes constitutes the image of a dimer, as confirmed by calculations. No fragmentation or decomposition was observed. Adapted from [22], copyright 2006, with permission from Elsevier.

Unidirectional Rotation of the Wheel
For the first time, it was possible to observe the rotation of a wheel on a surface, by working at 5 K under ultra-high vacuum. This was achieved by inducing the translation of the molecule upon pushing it with the STM tip [25]. In order to understand the motion of this molecule in detail, it was particularly important to study the manipulation signal (i.e., the tunneling current recorded at constant tip height during the motion), because the STM images before and after manipulation did not give information about the type of movement between measurements (translational or rotational).
The tip was moved across the molecular axle at a constant height, while the tunneling current was recorded. In most cases, it was possible to rotate one wheel, and in a few cases, the rolling of both wheels was observed (as shown in Figure 4). This behavior seems to depend on the precise shape of the tip apex, which in some cases allowed us to address both wheels during the manipulation. This triptycene dimer was the first molecule designed to yield a controlled rotation upon pushing the wheels using the STM tip. With this rolling motion proven, four triptycene units were then integrated into the next generation of nanovehicles, with the aim of increasing mobility on the surface. Step 1: the STM tip approaches the molecule; Step 2: the rotation of the wheel starts; Step 3: the STM tip lies on the other side of the wheel after a 120° rotation. Adapted from [25], copyright 2007, with permission from Springer Nature.

A Planar Nanocar
For the preparation of nanovehicles containing four wheels (i.e., nanocars), it was necessary to develop the synthesis of a larger chassis based on a perylene unit. This was to sterically accommodate Step 1: the STM tip approaches the molecule; Step 2: the rotation of the wheel starts; Step 3: the STM tip lies on the other side of the wheel after a 120 • rotation. Adapted from [25], copyright 2007, with permission from Springer Nature.

A Planar Nanocar
For the preparation of nanovehicles containing four wheels (i.e., nanocars), it was necessary to develop the synthesis of a larger chassis based on a perylene unit. This was to sterically accommodate all the wheels on the same molecular structure [26], as the chassis used for molecular wheelbarrow 1 was too short. Therefore, as shown in Scheme 2, our strategy started with the synthesis of di(4iodophenyl)cyclopentadienone 11 via a double Knoevenagel reaction between acenaphthenequinone 9 and 1,3-di(4-iodophenyl)propan-2-one 10 under basic conditions (Step a). Diels-Alder reaction with 1,2-di(3,5-di-tert-butylphenyl)ethyne gave-after aromatization-the half-chassis 12 along with evolution of carbon monoxide (Step b). The half-chassis 12 was subsequently dimerized by using a Scholl-type oxidative coupling with FeCl 3 as oxidant (Step c). In the last step, the resulting planar chassis 13 was functionalized with four ethynyltriptycenyl wheels via Sonogashira cross-couplings to yield our first prototype nanocar 14 in 9% overall yield. all the wheels on the same molecular structure [26], as the chassis used for molecular wheelbarrow 1 was too short. Therefore, as shown in Scheme 2, our strategy started with the synthesis of di(4-iodophenyl)cyclopentadienone 11 via a double Knoevenagel reaction between acenaphthenequinone 9 and 1,3-di(4-iodophenyl)propan-2-one 10 under basic conditions (Step a). Diels-Alder reaction with 1,2-di(3,5-di-tert-butylphenyl)ethyne gave-after aromatization-the halfchassis 12 along with evolution of carbon monoxide (Step b). The half-chassis 12 was subsequently dimerized by using a Scholl-type oxidative coupling with FeCl3 as oxidant (Step c). In the last step, the resulting planar chassis 13 was functionalized with four ethynyltriptycenyl wheels via Sonogashira cross-couplings to yield our first prototype nanocar 14 in 9% overall yield. This nanovehicle was deposited on Au(111). However, due to its planarity and the resulting high interaction of the polyaromatic chassis with the surface, it was not possible to move the vehicle. Thus, we envisioned the design of an alternative molecule-car with a modified chassis, which would undergo less surface coupling.

A Curved Nanocar
To decrease the interaction of the polyaromatic chassis with the surface, a highly curved chassis was prepared. The synthetic strategy remained identical, but the 3,5-di-tert-butylphenyl fragments positioned at both ends were substituted with 4-tert-butylphenyl groups [26]. In this case, planarity of contiguous phenyl rings was more easily achieved and dimerization of the half-chassis 16 under Scholl oxidative conditions led to an overcyclized perylene platform bearing six additional C-C bonds, as shown in blue (highlighted in bold) in Scheme 3. This nanovehicle was deposited on Au(111). However, due to its planarity and the resulting high interaction of the polyaromatic chassis with the surface, it was not possible to move the vehicle. Thus, we envisioned the design of an alternative molecule-car with a modified chassis, which would undergo less surface coupling.

A Curved Nanocar
To decrease the interaction of the polyaromatic chassis with the surface, a highly curved chassis was prepared. The synthetic strategy remained identical, but the 3,5-di-tert-butylphenyl fragments positioned at both ends were substituted with 4-tert-butylphenyl groups [26]. In this case, planarity of contiguous phenyl rings was more easily achieved and dimerization of the half-chassis 16 under Scholl oxidative conditions led to an overcyclized perylene platform bearing six additional C-C bonds, as shown in blue (highlighted in bold) in Scheme 3. The last step consisted of connecting the four triptycene wheels via a quadruple Sonogashira coupling to give the corresponding nanovehicle 17 in 34% yield. Geometric optimization of this secondgeneration nanovehicle showed that this overcyclized chassis has a curved shape at both ends ( Figure  5b). As with fullerenes, this geometry can be explained by the presence of alternating five-and sixmembered rings in the polycyclic aromatic hydrocarbon platform. The two five-membered rings are shown in red in Scheme 3. This specificity is of great interest, since it provides an appropriate shape to act as an efficient cargo zone. The planar and curved nanovehicles have very large differences in their solubilities and their spectroscopic properties. For instance, the color of the planar molecule is pink, while the curved one is dark green; hence, it was called "the green buggy".

Participation in the First Nanocar Race: The View from the French Team
On 28 and 29 April 2017, six teams from three continents met in Toulouse to compete with their molecular racers. Both families of nanocars-the technomimetic models incorporating wheels (from Austria, France and the USA) and the smaller and lighter models lacking wheels (from Germany, Japan and Switzerland)-entered the competition [27]. Since the ultra-high-vacuum-low temperature (UHV-LT) STM instrument at Centre d'Elaboration de Matériaux et d'Etudes Structurales (CEMES-CNRS) in Toulouse employs only four tips, four nanocars competed using this microscope. Thus, the The last step consisted of connecting the four triptycene wheels via a quadruple Sonogashira coupling to give the corresponding nanovehicle 17 in 34% yield. Geometric optimization of this second-generation nanovehicle showed that this overcyclized chassis has a curved shape at both ends ( Figure 5b). As with fullerenes, this geometry can be explained by the presence of alternating fiveand six-membered rings in the polycyclic aromatic hydrocarbon platform. The two five-membered rings are shown in red in Scheme 3. This specificity is of great interest, since it provides an appropriate shape to act as an efficient cargo zone. The last step consisted of connecting the four triptycene wheels via a quadruple Sonogashira coupling to give the corresponding nanovehicle 17 in 34% yield. Geometric optimization of this secondgeneration nanovehicle showed that this overcyclized chassis has a curved shape at both ends ( Figure  5b). As with fullerenes, this geometry can be explained by the presence of alternating five-and sixmembered rings in the polycyclic aromatic hydrocarbon platform. The two five-membered rings are shown in red in Scheme 3. This specificity is of great interest, since it provides an appropriate shape to act as an efficient cargo zone. The planar and curved nanovehicles have very large differences in their solubilities and their spectroscopic properties. For instance, the color of the planar molecule is pink, while the curved one is dark green; hence, it was called "the green buggy".

Participation in the First Nanocar Race: The View from the French Team
On 28 and 29 April 2017, six teams from three continents met in Toulouse to compete with their molecular racers. Both families of nanocars-the technomimetic models incorporating wheels (from Austria, France and the USA) and the smaller and lighter models lacking wheels (from Germany, Japan and Switzerland)-entered the competition [27]. Since the ultra-high-vacuum-low temperature (UHV-LT) STM instrument at Centre d'Elaboration de Matériaux et d'Etudes Structurales (CEMES-CNRS) in Toulouse employs only four tips, four nanocars competed using this microscope. Thus, the The planar and curved nanovehicles have very large differences in their solubilities and their spectroscopic properties. For instance, the color of the planar molecule is pink, while the curved one is dark green; hence, it was called "the green buggy".

Participation in the First Nanocar Race: The View from the French Team
On 28 and 29 April 2017, six teams from three continents met in Toulouse to compete with their molecular racers. Both families of nanocars-the technomimetic models incorporating wheels (from Austria, France and the USA) and the smaller and lighter models lacking wheels (from Germany, Japan and Switzerland)-entered the competition [27]. Since the ultra-high-vacuum-low

temperature (UHV-LT) STM instrument at Centre d'Elaboration de Matériaux et d'Etudes Structurales
(CEMES-CNRS) in Toulouse employs only four tips, four nanocars competed using this microscope. Thus, the Ohio team and the Rice-Graz team were sitting in the same room as the other teams but were remotely piloting their own nanocar, deposited in the STM located at their home university. This presented a further challenge to control a nanocar at the atomic scale from half the world away.
Except for the Rice-Graz team who competed on a silver track, the other nanocars were on gold. The latter surface was originally designated as the common surface for the race, and this was why the official ranking contains two winners, one on gold and one on silver. Each track was 100 nm long and included at least two turns.

Deposition and Imaging of the French Nanocar
The French nanocar had the advantage of being very robust. It was deposited by sublimation, as with the other nanocars, and was identified on the surface as a long 3 nm molecule. As shown in Figure 6, the triptycene wheels appear as very bright spots when compared with the central polyaromatic core. Ohio team and the Rice-Graz team were sitting in the same room as the other teams but were remotely piloting their own nanocar, deposited in the STM located at their home university. This presented a further challenge to control a nanocar at the atomic scale from half the world away. Except for the Rice-Graz team who competed on a silver track, the other nanocars were on gold. The latter surface was originally designated as the common surface for the race, and this was why the official ranking contains two winners, one on gold and one on silver. Each track was 100 nm long and included at least two turns.

Deposition and Imaging of the French Nanocar
The French nanocar had the advantage of being very robust. It was deposited by sublimation, as with the other nanocars, and was identified on the surface as a long 3 nm molecule. As shown in Figure 6, the triptycene wheels appear as very bright spots when compared with the central polyaromatic core.

The Race and the Official Ranking
On 29 April at 5:00 p.m., after two days and one night of intense efforts, the first ever international nanocar race ended. As mentioned, two teams were ranked first: the Swiss team, racing on Au(111), as stated in the original rules [28] and the Rice-Graz team, racing on Ag(111), since their nanocar appeared to move uncontrollably fast on gold. On Au(111) with the lightest nanocar of the competition (only 42 atoms), the Swiss team arrived first, with a distance of 100 nm covered in six hours [29]. The US team (Ohio) arrived second, with a distance of 43 nm covered. Their nanocar-the Bobcat Nano-wagon-was the largest to participate, with about 650 atoms. Finally, the Nano-Windmill piloted by the German team (Dresden) covered a distance of 11 nm. On Ag(111), the dipolar racer (which consists of one axle with two wheels) was very fast, with a distance of 1000 nm covered in only 29 h [30] but without competitors, since the others competed on Au(111). The characteristics of each team are given in Table 1.
We targeted two strategies to move our nanocar directionally; first, we tried pulling the molecule on the surface, as pushing was forbidden in the rules. The second strategy was to find a way for an electronic excitation to induce the movement. Unfortunately, due to the limited time available both in training and during the race, this propulsion mode failed to drive our nanocar. Only our first strategy was effective, and pulling the molecule with the STM tip allowed us to cover an impressive distance of 25 nm in 3 s. However, we were disqualified from the race as the jury decided not to recognize movement by pulling. We are very satisfied to have participated in the first nanocar race; remembering the motto from the creator of the modern Olympic games, Pierre de Coubertin, which emphasizes that "the importance is to participate". Moreover, for the first time, we were able to climb a step edge.

The Race and the Official Ranking
On 29 April at 5:00 p.m., after two days and one night of intense efforts, the first ever international nanocar race ended. As mentioned, two teams were ranked first: the Swiss team, racing on Au(111), as stated in the original rules [28] and the Rice-Graz team, racing on Ag(111), since their nanocar appeared to move uncontrollably fast on gold. On Au(111) with the lightest nanocar of the competition (only 42 atoms), the Swiss team arrived first, with a distance of 100 nm covered in six hours [29]. The US team (Ohio) arrived second, with a distance of 43 nm covered. Their nanocar-the Bobcat Nano-wagon-was the largest to participate, with about 650 atoms. Finally, the Nano-Windmill piloted by the German team (Dresden) covered a distance of 11 nm. On Ag(111), the dipolar racer (which consists of one axle with two wheels) was very fast, with a distance of 1000 nm covered in only 29 h [30] but without competitors, since the others competed on Au(111). The characteristics of each team are given in Table 1.
We targeted two strategies to move our nanocar directionally; first, we tried pulling the molecule on the surface, as pushing was forbidden in the rules. The second strategy was to find a way for an electronic excitation to induce the movement. Unfortunately, due to the limited time available both in training and during the race, this propulsion mode failed to drive our nanocar. Only our first strategy was effective, and pulling the molecule with the STM tip allowed us to cover an impressive distance of 25 nm in 3 s. However, we were disqualified from the race as the jury decided not to recognize movement by pulling. We are very satisfied to have participated in the first nanocar race; remembering the motto from the creator of the modern Olympic games, Pierre de Coubertin, which emphasizes that "the importance is to participate". Moreover, for the first time, we were able to climb a step edge. Table 1. The six registered participants and their respective nanocars, with their characteristic parameters: country, team leader, landing surface, and number of atoms of their nanovehicle (with the corresponding chemical formula). In red, the parameters outside of the original rules [28] but accepted by the jury. Table 1. The six registered participants and their respective nanocars, with their characteristic parameters: country, team leader, landing surface, and number of atoms of their nanovehicle (with the corresponding chemical formula). In red, the parameters outside of the original rules [28] but accepted by the jury. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. Table 1. The six registered participants and their respective nanocars, with their characteristic parameters: country, team leader, landing surface, and number of atoms of their nanovehicle (with the corresponding chemical formula). In red, the parameters outside of the original rules [28] but accepted by the jury. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. Table 1. The six registered participants and their respective nanocars, with their characteristic parameters: country, team leader, landing surface, and number of atoms of their nanovehicle (with the corresponding chemical formula). In red, the parameters outside of the original rules [28] but accepted by the jury. We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusion and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss moleculecar had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semiconductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34]. Au We received the "prize of elegance" due to the very smart STM images of our Green Buggy recorded during the race. The Japanese team was not ranked either, as their nanovehicle was unable to recover from a software crash, just one hour into the race. This team was awarded the "fair play" prize.

Conclusions and Perspectives
In summary, we have presented the design and synthesis of polyaromatic hydrocarbons conceived by analogy with macroscopic wheels, wheelbarrows, and nanocars. Concerning the nanocar race, we had confirmation that the smallest molecules completed the track distance first (the Swiss molecule-car had only 42 atoms and is commercially available). This is partly because smaller was better for easily and rapidly moving on the surface.
The first nanocar race was a real success, with a worldwide audience following the event on the internet, broadcasted live, night and day, with more than 100,000 viewers at peak times. We now have experience in organizing such an event, which may have a second edition around 2021. All the competitors went home with new ideas that will make them more competitive next time, and we can assume that new participants will enter. One could imagine organizing semi-finals, test selections to qualify for the final or to run the race at a higher temperature, in order to have a faster event. It has already been shown that control of motion is possible at higher temperatures on some semi-conductive surfaces, such as SmSi [31,32] or SiB [33]. Furthermore, the question of whether we need to create different categories will be addressed, in order to give everybody the same chance (e.g., dividing the nanovehicles by the number of atoms or by the molecular mass). We could also organize a different race, for example, moving the nanocar with cargo on its backbone. This would be similar to the "waiter's race" ("course de garçons de café" in French) created in Paris (France), which tests the speed of a waiter carrying a loaded tray without tipping it over [34].


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Chemistry Biology

text:
Modular Enzymatic Cascade Synthesis of Vitamin B5 and Its Derivatives

Abstract Access to vitamin B5 [(R)‐pantothenic acid] and both diastereoisomers of α‐methyl‐substituted vitamin B5 [(R)‐ and (S)‐3‐((R)‐2,4‐dihydroxy‐3,3‐dimethylbutanamido)‐2‐methylpropanoic acid] was achieved using a modular three‐step biocatalytic cascade involving 3‐methylaspartate ammonia lyase (MAL), aspartate‐α‐decarboxylase (ADC), β‐methylaspartate‐α‐decarboxylase (CrpG) or glutamate decarboxylase (GAD), and pantothenate synthetase (PS) enzymes. Starting from simple non‐chiral dicarboxylic acids (either fumaric acid or mesaconic acid), vitamin B5 and both diastereoisomers of α‐methyl‐substituted vitamin B5, which are valuable precursors for promising antimicrobials against Plasmodium falciparum and multidrug‐resistant Staphylococcus aureus, can be generated in good yields (up to 70 %) and excellent enantiopurity (>99 % ee). This newly developed cascade process may be tailored and used for the biocatalytic production of various vitamin B5 derivatives by modifying the pantoyl or β‐alanine moiety.

Coenzyme A( CoA) is an essential enzyme cofactor in all organisms, and its biosynthetic pathway enzymes have been identified as attractive targets for new antimicrobial drugs. [1,2] An interesting class of new antimicrobials that target CoA biosynthesis is constitutedb yp antothenamides (PanAms), which are secondary or tertiarya mides of pantothenic acid (vitaminB 5 , 5a,F igure 1), the biosynthetic precursor of CoA. Various PanAms have been shown to possess potent antimicrobial activity against several organisms, including the pathogenic bac-terium Staphylococcus aureus [3] as well as the malaria parasite Plasmodium falciparum. [4] However, pantetheinase enzymes that normally hydrolyze pantetheinei nh uman serum also act on the PanAms,thereby reducing their efficacy. [5,6] Interestingly, pantetheinase-mediated hydrolysis of PanAms could be preventedb ym odifying the b-alaninem oiety of the compounds. [7,8] Indeed, aP anAm with an added a-methyl group was shown to have superior antiplasmodial activity compared to its parent molecule. [9] However,s uch modificationsi ntroduce stereochemical complexity to the molecules, recent results of which have indicated the strong relevance to the antimalariala ctivity of PanAm analogues. [10] However,t he particularly challenging chemical synthesis of these compounds poses as ignificant barriert ot he discoveryo ft heir clinical potential. Therefore, it is of high interest to developa na symmetricb iocatalytic synthetic strategy that provides efficient andstep-economical access to pantothenic acid (5a)a nd both diastereoisomers of its a-methyl substituted derivative (5b,F igure 1), avoiding( de-)protecting steps and intermediate purifications.
The desired PanAms can be easily prepared from the correspondingp antothenic acids by transforming the carboxylic acid group to an amide. [11] We envisioned that pantothenic acid (5a)a nd its a-methyl substituted derivative( 5b)c ould be prepared from fumaric acid (1a)a nd mesaconic acid (1b), respectively,t hrough a modulart hree-step enzymaticc ascade (Scheme 1) involving 3methylaspartate ammonia lyase (MAL), an appropriate decarboxylases uch as aspartate-a-decarboxylase (ADC), b-methylaspartate-a-decarboxylase (CrpG) or glutamate decarboxylase (GAD),a nd pantothenate synthetase (PS). The expected chemoselectivity of each biocatalyst could allow for ao ne-pot reaction sequence due to the orthogonal reactivity of each enzyme.I nt his process, the new stereogenic centeri np roduct 5b can be established by either regio-and diastereoselective amination( as catalyzed by MAL), or diastereospecific decarboxylation by one of the decarboxylase enzymes.
MAL of Clostridium tetanomorphum is part of ac atabolic pathway for l-glutamate, in which it catalyzes the conversion of l-threo-3-methylaspartate to ammonia and mesaconate. [12] Using al arge molar excesso fa mmonia, the enzymea lso efficiently catalyzes the amination of mesaconate (1b)t og ive both (2S,3S)-3-methylaspartate (l-threo-2b)a nd (2S,3R)-3-methylaspartate (l-erythro-2b)a sp roducts (Scheme 1). It was found that l-threo-2b forms at am uch faster rate than that of l-erythro-2b,b ut at equilibrium (using a6 5-foldm olare xcesso f ammonia over 1b at pH 9) the molar ratio of these diastereoisomersi sa pproximately 1. [13] In addition, MAL accepts fumarate (1a)a sasubstrate, whichi sc onvertedt ol-aspartate (2a). The mechanism-inspired engineering of aM AL mutant( H194A) with strongly enhanced diastereoselectivity in the amination of 1b,g iving exclusively l-threo-2b,h as been previously reported. [13] Moreover,t he substrate scope of MAL has been expanded by structure-guided site-saturation mutagenesis, allowing for the biocatalytic production of ab road range of valuable 3substituted derivatives of l-aspartic acid. [14] ADC of E. coli is part of the biosynthetic pathway for pantothenate, in which it catalyzest he decarboxylation of 2a to give b-alanine (3a). [15] This enzyme has al imited substrate scope and showed no decarboxylation activity towards 2b. [16] The enzyme b-methylaspartate-a-decarboxylase( CrpG) of Nostoc sp. ATCC 53789 is involved in ab iosynthetic pathway for cryptophycin, in which it catalyzes the decarboxylation of l-erythro-2b to yield (R)-3-amino-2-methylpropanoic acid (3b). [17] CrpG is the only enzyme knownt oc atalyze the decarboxylation of 2b,w ith significant activity towardst he l-erythro isomer only.G AD of the hyperthermophilic archaeon Thermococcusk odakarensis has been reported to function as an ADC and is mostl ikelyr esponsible for the production of b-alanine necessary for pantothenate biosynthesis. [18] In this study,w e demonstrate that this enzyme exhibits decarboxylase activity towards 2b,b ut with the highest activity towards the l-threo isomer.N otably,A DC and CrpG must undergo self-processing leadingt of ormation of the catalytic pyruvoyl group, whereas GAD is aP LP-dependentd ecarboxylase that does not require autocatalytic self-processing.
Pantothenate synthetase (PS) of E. coli is involved in the last step of pantothenateb iosynthesis and catalyzes the adenosine triphosphate (ATP)-dependent condensation of (R)-pantoate (4) and b-alanine (3a)t of orm (R)-pantothenic acid (vitaminB 5 , 5a). PS enzymest ypically accept av ariety of b-alanine analogues in the condensation reaction,a lbeit with reducedc atalytic efficiency compared to that with the natural substrate. [15,19] Initially,w eset out to combine MAL and ADC in one pot to preparep roduct 3a.A ccordingly,s ubstrate 1a and NH 4 Cl were incubated with MAL and ADC, and the reaction was monitored by TLC ( Figure S3, Supporting Information). After 24 h, 1a was completely converted to product 3a,a sc onfirmed by 1 HNMR spectroscopy.T hese initial results showed that the two enzymes MAL and ADC are compatible for cascade synthesis in one pot. To further demonstrate the preparative usefulness of this two-step cascade system, a1 00 mg-scale synthesis was performed. Accordingly,s ubstrate 1a (25 mm)a nd NH 4 Cl (500 mm)w ere incubated with MAL (0.02 mol %) and ADC (0.6 mol %) in one pot (25 mL of buffer,p H8). Under these conditions, excellent conversion (> 99 %a fter 24 h) andg ood isolated yield of product 3a (85 %) were achieved ( Table 1 Unfortunately,A DC showed no decarboxylase activity towards either l-threo-2b or l-erythro-2b.T herefore, we cloned, expressed, and purified the decarboxylase CrpG, and then incubated it with a1 :1 mixture of l-threo-2b and l-erythro-2b. Undert hese conditions, the l-erythro isomer was fully decarboxylated, whereas the l-threo isomer was not converted, not even after prolongedi ncubation for 7d ( Figure S11, Supporting Information). This indicates that CrpG is highly diastereose-Scheme1.Proposed enzymatic cascade synthesis of vitamin B 5 and its derivatives. Table 1. Two-step enzymaticc ascade synthesis of b-alanine (3a)and both enantiomers of 3-amino-2-methylpropanoic acid (3b). [ Having established the preference of CrpG for l-erythro-2b, at wo-step enzymatic cascade reactionw as performeda tt he analytical scale by incubation of 1b and NH 4 Cl with MAL and CrpG in one pot. Interestingly,f ull conversion of startings ubstrate 1b was observed( Figure S4, Supporting Information), yieldings olely the (R)-enantiomer of product 3b,a sc onfirmed by 1 HNMR spectroscopy and chiral HPLC analysis. This is explained by ad ynamic kinetic asymmetric transformation [20,21] of the diastereoisomeric mixture of 2b (Scheme 2). In the first cascade step, MAL produced both l-erythro-a nd l-threo-2b as intermediate products. Subsequently,i nt he second step CrpG only decarboxylated l-erythro-2b to give exclusively( R)-3b. The remaining l-threo-2b was also convertedi nto l-erythro-2b by MAL, leading to full conversion of the startingm aterial (1b) to the desired product (R)-3b.
Several control experiments were also performed. Firstly, when 1b and NH 4 Cl were incubated with MAL alone, an approximately 1:1m ixture of l-threo-2b and l-erythro-2b was obtained ( Figure S12 A, Supporting Information). After removal of MAL from the reactionm ixture by heat inactivation and filtration,C rpG was added. After prolonged incubation (7 d), a mixture of unreacted l-threo-2b and product 3b was obtained (Figure S12 B, Supporting Information).S econdly, incubation of 1b and NH 4 Cl with the diastereospecific mutant of MAL (MAL-H194A)a nd CrpG in one pot resultedi nt he formation of lthreo-2b but did not yield product 3b,w hichi sc onsistent with the inability of CrpG to decarboxylate l-threo-2b (Figure S9, Supporting Information). These results confirm that MAL is responsible for both the synthesis and epimerization of l-threo-a nd l-erythro-2b,a nd that CrpG displays activityt owards l-erythro-2b only,a llowing for the selective synthesis of (R)-3b starting from the simplen on-chirald icarboxylic acid 1b.
CrpG displays activity towards l-erythro-2b,e nabling the enzymatics ynthesis of (R)-3b.T os ynthesize the opposite enantiomer of 3b,adecarboxylase with activity towards l-threo-2b was required. Our attempts to obtain CrpG variants by directed evolution through screening of single-site saturation mutagenesis libraries did not yield any mutants with detectablea ctivity towards l-threo-2b.T herefore, we cloned andp roduced two pyridoxal phosphate (PLP)-dependent GAD enzymes, and tested their ability to decarboxylate l-threo-2b.I nitially,w e workedo nt he GAD from Pyrococcus furiosus (PfGAD), which was reported to accept l-aspartate, l-glutamate and l-tyrosine as substrates. [22] Although many different expression conditions were tested, we weren ot able to produce PfGAD in as oluble form in an E. coli host. In an attempt to produce the soluble protein, different constructs were made as fusions with three solubility enhancers:m altose-binding protein (MBP), small ubiquitin-like modifier protein (SUMO) and Fh8, as mall protein secreted by the parasite Fasciola hepatica. However,i nefficient solubilization of PfGAD limited the effectiveness of this approach.T herefore, we selected the GAD from the hyperthermophilic archaeon Thermococcus kodakarensis (TkGAD), which has 71 %sequence similarityw ith PfGAD ( Figure S2, Supporting Information). TkGAD reportedly catalyzes the decarboxylation of l-glutamate and l-aspartate. [23] The gene encoding TkGAD was cloned and expressed, and the corresponding enzyme purified, yielding the soluble and active protein ( Figure S1, Supporting Information). Initially, TkGAD activity was tested towards l-aspartate (2a)a nd l-erythro-a nd l-threo-2b (Figure S10, Supporting Information). Thee nzyme completely converted 2a to 3a,w hereas the reaction with l-erythro-2b showed < 10 %c onversion. To our delight, l-threo-2b was also accepted as substrate by TkGAD, yieldingt he desired (S)-3b with more than 70 %c onversion.
Considering that TkGAD displays activity towards both diastereoisomers of 2b,adynamic kinetic asymmetric transformation approach, in which MAL and TkGAD are combined in one pot, would not yield the enantiopure (S)-3b product. Hence, for the one-pot,t wo-step, enzymatic cascade synthesis of (S)-3b, TkGAD was used in combination with the diastereospecific MAL-H194A mutant, which produces exclusively l-threo-2b upon aminationo f1b (Scheme 3a nd Figure S5 in the Supporting Information). Accordingly,s ubstrate 1b (10 mm)a nd NH 4 Cl (500 mm)w ere incubated with MAL-H194A (0.02 mol %) Scheme2.One-pot, two-step,enzymatic cascade reaction,involving MAL and CrpG, that fully converts mesaconate (1b)too nly (R)-3b.This is due to CrpG only acting on l-erythro-2b and to the MAL-mediated dynamickinetic asymmetric transformation of the l-threo-2b to the desired diastereomer. Having developed one-pot,t wo-step, enzymatic cascader eactions for the productiono f3a,( R)-3b and (S)-3b,w en ext verifiedw hether the PS enzyme is able to accept these compounds as substrates in condensation with (R)-pantoate (4) using small-scale (1 mL) reactions. We were pleased to find that PS accepted 3a,( R)-3b and (S)-3b as substrates in the condensation reaction, yielding the corresponding pantothenic acid products 5a and 5b,a sc onfirmed by 1 HNMR spectroscopy ( Figure S13, SupportingI nformation).
Having established that PS can be used to set up at hreestep enzymatic cascade reaction, the enzymesM AL, ADC, and PS werec ombined in one pot. Initially,t he reaction was tested by adding the three enzymes simultaneously and using an equimolar ratio of 1a, 4 and ATP. Although full conversion of the startings ubstrate 1a was observed after 24 h, the desired product (R)-5a was obtained in ac rude yield of only 50 % along with accumulationo f3a.T his indicated that furthero ptimization of the reaction conditions was necessary to improve the yield of product (R)-5a.A fter testing different ratios of 4 and ATP, am olar ratio of 2:3w as found to be best, which resulted in excellent conversion of the starting substrate into the desired product 5a (crude yield > 99 %; Figures S6 and S14, Supporting Information). Hence, under these conditions, no significant accumulation of intermediate product 3a was observed.
To assess the performance of this one-potm ulti-enzymatic cascade, a3 2mg-scale synthesis was performed.T he threestep cascade reaction wasperformed by addition of all componentss imultaneously including the three enzymes. After 24 h, the starting material 1a was completely consumed( conversion > 99 %, Figure S15 A, Supporting Information) andt he desired product (R)-5a was obtained in good isolated yield (70 %) and with excellent ee (> 99 %) ( Table 2, Figures S19 and S20, Supporting Information). Importantly,t he modularity of this enzymatic cascadea pproacha lso allows for the facile synthesis of both diastereoisomers of a-methyl-substituted vitamin B 5 ,t hat is (2R,2'R)-5b and (2S,2'R)-5b ( Figures S7 and S8, Supporting Information), with one stereogenic centerb eing set by the selected combination of enzymes and the other by the substrate (R)-pantoate (4). Under suitable reaction conditions (for details, see section 8.2 in the Supporting Information), and using the appropriate combination of enzymes in one-pot,t he desired products (2R,2'R)-5b and (2S,2'R)-5b were obtained with excellent de and ee values (> 99 %) andi n4 6-49 %i solated yield (Table 2; Figures S15, S21-S24, and S26 in the Supporting Information).
In conclusion, we have successfully developed ao ne-pot cascade process for the synthesis of enantiomerically pure vitamin B 5 startingf rom fumarate and utilizing MAL, ADC, and PS enzymes.S tarting from mesaconic acid,t he stereoselective synthesis of both diastereoisomers of a-methyl-substituted vitamin B 5 ,a ni mportanta ntibiotic precursor,w as achieved by using either the CrpG or GAD enzymei nsteado fA DC, with one stereogenic center being set by the selected combination of MAL/CrpG or MAL-H194A/GAD and the other derived from one of the starting substrates. Given the availability of engi-Scheme3.The one-pot, two-step,enzymatic cascade reaction that converts mesaconate (1b)too nly (S)-3b and relies on adiastereospecific mutant MAL (MAL-H194A) and the newly discovered stereoselectivity of TkGAD towards l-threo-2b.   neered MAL mutantsa nd natural PS enzymes with ab road substrate scope, work is in progress to expand the substrate scope of CrpG and GAD by protein engineering. Althought he decarboxylation step is actually stereochemically deconstructive with the loss of one chiral center, the cascade approachs trongly benefits from the use of stereo-divergent decarboxylases. These enzymes not only allow fort he synthesis of both diastereoisomerso fa-methyl-substituted vitamin B 5 ,b ut they also provide as trong drivingf orce to pull the equilibrium of the MAL-catalyzed reaction towards product formation.T he use of an irreversible decarboxylation step, with stereochemical kineticd istinction, is an important strategy in biocatalytic cascade synthesis to overcome thermodynamic limitations and maximize product yield. [24,25] Ap ossible constraint on the use of the developed cascade for large-scale transformations would be the dependence of the PS enzyme on the expensive cofactor ATP. This could be addressed by the incorporation of an auxiliary enzyme-catalyzeds tep fore fficient ATPr ecycling. SeveralA TP-recyclinge nzyme systems are availablea nd af ew have already been successfully implemented in preparative biocatalysis. [26][27][28] However,c onsidering that the starting materials (fumarate and mesaconate) can be efficiently produced in high yields by large-scale fermentation using metabolicallye ngineered E. coli strains, [29,30] we envision to co-express the enzymes employed for the cascade in such a fermentation host to directly obtain vitaminB 5 and its amethyl-substitutedd erivatives from cheap carbon and nitrogen sources. Indeed, such ac ell-baseda pproach would eliminate the need for ATPr ecycling.