The use of biocatalysis in the manufacture of small molecule active pharmaceutical ingredients has seen a marked increase over the past decade. Driven by academic and industrial interest in the application of enzymes as catalysts for transforming chemical routes, the biocatalytic toolbox available to a chemist has continued to expand. Despite this, the application of biocatalysis in early discovery chemistry has trailed in comparison to its use in manufacturing routes. The authors offer their perspective on the adoption of biocatalysis in the early discovery space: highlighting challenges including enzyme supply and the biocatalysis business model, as well as recent trends that could spur more collaboration and access to enzymes for early discovery R&D activities.

Several biocatalytic approaches for the preparation of optically pure methyl 3-oxocyclohexanecarboxylates (S)-, (R)-1 and 3-oxocyclohexanecarbonitriles (S)-, (R)-2 have been successfully demonstrated. Screening of reaction-focused enzyme collections was used to identify initial hits using three enzymatic strategies. Reaction optimization and scale-up enabled the production of chiral intermediates for route scouting efforts on scales of up to 100 g. The enzymes applied in these processes (lipases, enoate reductases, and nitrilases) have been shown to be robust catalysts for drug manufacturing and represent a green alternative to conventional methods to access these chiral cyclohexanone building blocks.

The glycoproteins of selected microbial pathogens often include highly modified carbohydrates such as 2,4-diacetamidobacillosamine (diNAcBac). These glycoconjugates are involved in host-cell interactions and may be associated with the virulence of medically significant Gram-negative bacteria. In light of genetic studies demonstrating the attenuated virulence of bacterial strains in which modified carbohydrate biosynthesis enzymes have been knocked out, we are developing small molecule inhibitors of selected enzymes as tools to evaluate whether such compounds modulate virulence. We performed fragment-based and high-throughput screens against an amino-sugar acetyltransferase enzyme, PglD, involved in biosynthesis of UDP-diNAcBac in Campylobacter jejuni. Herein we report optimization of the hits into potent small molecule inhibitors (IC50 < 300 nM). Biophysical characterization shows that the best inhibitors are competitive with acetyl coenzyme A and an X-ray cocrystal structure reveals that binding is biased toward occupation of the adenine subpocket of the AcCoA binding site by an aromatic ring.

As part of an ongoing study to elucidate the SAR of bisindole alkaloid inhibitors against the evolutionary conserved MRSA pyruvate kinase (PK), we present here the synthesis and biological activity of six dihalogenated analogues of the naturally occurring sponge metabolite deoxytopsentin, including the naturally occurring dibromodeoxytopsentin. The most active compounds displayed potent low nanomolar inhibitory activity against MRSA PK with concomitant significant selectivity for MRSA PK over human PK orthologues. Computational studies suggest that these potent MRSA PK inhibitors occupy a region of the small interface of the enzyme tetramer where amino acid sequence divergence from common human PK orthologues may contribute to the observed selectivity.

The public health care crisis caused by the emergence of drug resistant bacterial strains, e.g., methicillin resistant Staphylococcus aureus (MRSA) has underlined the urgent need to accelerate the discovery of new chemical entities active against antibiotic resistant bacteria. We report here the synthesis of a series thiazole containing deoxytopsentin analogues, which show moderate activity against a target MRSA pyruvate kinase enzyme: an evolutionary conserved hub protein critical for bacterial survival. A Hantzsch thiazole coupling between α-oxo-1H-indole-3-thioacetamides and 2-bromo-1-(1H-indol-3-yl)-ethanones provided facile access to the thiazole containing deoxytopsentin compounds.

1-Acyl-7-nitroindolines have been found to be useful photoactivated protecting groups for rapid release of carboxylates in aqueous solution. Mechanistic details of carboxylic acid photorelease from model compounds in solutions of varying H_2O-CH_3CN composition are now reported, using data from product studies, deuterium isotope effects, kinetic studies (UV-Vis), and nanosecond laser flash photolysis. Our data support a mechanism ( via T_1) that involves reaction of a critical photogenerated intermediate 21 (an acetic nitronic anhydride), obtained via photochemical transfer of the acetyl group from the amide nitrogen to one of the oxygen atoms of the nitro group. This mode of transfer has been reported in the photochemistry of a variety of N -acetyl- o -nitrodiphenylamines. Two competing pathways of reaction from 21 that differ in how the acyl group is cleaved are proposed to account for the products observed. In solutions of higher water content, the predominant reaction pathway of 21 is via an A_AL1-like cleavage that results in formal intramolecular redox reaction of the aromatic ring system, to give the released carboxylic acid and 7-nitrosoindole 3 (after tautomerisation of an initially formed nitroso-3 H -indole 9 ). In solutions of low water content, the major pathway for hydrolysis of 21 is via the standard addition–elimination mechanism (A_AC2) with water as the nucleophile, that releases the carboxylic acid and nitroindoline 4 . Laser flash photolysis studies in wholly aqueous medium gave a transient (within the 20 ns laser pulse) at λ _max 450 nm, assignable to 21 . Single exponential decay of this species in water ( k _obs = 5 × 10^6 s^−1) is assigned to the release of the carboxylic acid and formation of the nitroso-3 H -indole 9 , which is supported by time-resolved measurements of acid release using bromothymol blue. Therefore, 1-acyl-7-nitroindolines photorelease their protected functionalities at rates in the submicrosecond time scale.

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC, and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer of [³H]GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates.

The human pathogen Campylobacter jejuni possesses a general N-linked glycosylation system that is known to play a role in pathogenicity; however, a detailed understanding of this role remains elusive. A considerable hindrance to studying bacterial N-glycosylation in vivo is the absence of small molecule inhibitors to reversibly control the process. This report describes a pathway-screening assay that targets the early enzymes of C. jejuni N-glycan biosynthesis that would enable identification of inhibitors to the first four steps in the pathway. The assay includes PglF, PglE, PglD, PglC and PglA; the enzymes involved in the biosynthesis of an undecaprenyl diphosphate-linked disaccharide and monitors the transfer [ 3 H]GalNAc from the hydrophilic UDP-linked carrier to the lipophilic UndPP-diNAcBac (2,4-diacetamido-2,4,6-trideoxyglucose). The optimized assay has a Z'-factor calculated to be 0.77, indicating a robust assay suitable for screening. The diacylglycerol kinase from Streptococcus mutans, which provides a convenient method for phosphorylating undecaprenol, has been included in a modified version of the assay thereby allowing the screen to be conducted with entirely commercially available substrates. Abbreviations: Streptococcus mutans diacylglycerol kinase, DGK; tris(2carboxyethyl)phosphine, TCEP; UndOH, undecaprenol; UndP, undecaprenyl phosphate; diNAcBac, 2,4-diacetamido-2,4,6-trideoxyglucose; dimethyl sulfoxide, DMSO, disintegrations per minute, DPM; pure solvent upper phase, PSUP

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L-glycero-D-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6 '' position converting ADP-D-glycero-D-manno-heptose into ADP-L-glycero-D-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-L-glycero-D-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-beta-D-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.

UDP-N-acetylglucosamine 5-inverting 4,6-dehydratase (PseB) is a unique sugar nucleotide dehydratase that inverts the C-5'' stereocentre during conversion of UDP-N-acetylglucosamine to UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-hexos-4-ulose. PseB catalyzes the first step in the biosynthesis of pseudaminic acid, which is found as a post-translational modification on the flagellin of Campylobacter jejuni and Helicobacter pylori. PseB is proposed to use its tightly bound NADP+ to oxidize UDP-GlcNAc at C-4'', enabling dehydration. The alpha,beta unsaturated ketone intermediate is then reduced by delivery of the hydride to C-6'' and a proton to C-5''. Consistent with this, PseB from C. jejuni has been found to incorporate deuterium into the C-5'' position of product during catalysis in D2O. Likewise, PseB catalyzes solvent isotope exchange into the H-5'' position of product, and eliminates HF from the alternate substrate, UDP-6-deoxy-6-fluoro-GlcNAc. Mutants of the putative catalytic residues aspartate 126, lysine 127 and tyrosine 135 have severely compromised dehydratase, solvent isotope exchange, and HF elimination activities.

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the inversion of configuration at C-6' ' of the heptose moiety of ADP-d-glycero-d-manno-heptose and ADP-l-glycero-d-manno-heptose. The epimerase HldD operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. Previous studies support a mechanism in which HldD uses its tightly bound NADP+ cofactor to oxidize directly at C-6' ', generating a ketone intermediate. A reduction of the ketone from the opposite face then occurs, generating the epimeric product. How the epimerase is able access both faces of the ketone intermediate with correct alignment of the three required components, NADPH, the ketone carbonyl, and a catalytic acid/base residue, is addressed here. It is proposed that the epimerase active site contains two catalytic pockets, each of which bears a catalytic acid/base residue that facilitates reduction of the C-6' ' ketone but leads to a distinct epimeric product. The ketone carbonyl may access either pocket via rotation about the C-5' '-C-6' ' bond of the sugar nucleotide and in doing so presents opposing faces to the bound cofactor. Evidence in support of the two-base mechanism is found in studies of two single mutants of the Escherichia coli K-12 epimerase, Y140F and K178M, both of which have severely compromised epimerase activities that are more than 3 orders of magnitude lower than that of the wild type. The catalytic competency of these two mutants in promoting redox chemistry is demonstrated with an alternate catalytic activity that requires only one catalytic base: dismutation of a C-6' ' aldehyde substrate analogue (ADP-beta-d-manno-hexodialdose) to an acid and an alcohol (ADP-beta-d-mannuronic acid and ADP-beta-d-mannose). This study identifies the two catalytic bases as tyrosine 140 and lysine 178. A one-step enzymatic conversion of mannose into ADP-beta-mannose is also described and used to make C-6' '-substituted derivatives of this sugar nucleotide.

The first positive evidence for the utilization of a direct C-6' ' oxidation/reduction mechanism by ADP-l-glycero-d-manno-heptose 6-epimerase is reported here. The epimerase (HldD or AGME, formerly RfaD) operates in the biosynthetic pathway of l-glycero-d-manno-heptose, which is a conserved sugar in the core region of lipopolysaccharide (LPS) of Gram-negative bacteria. The stereochemical inversion catalyzed by the epimerase is interesting as it occurs at an "unactivated" stereocenter that lacks an acidic C-H bond, and therefore, a direct deprotonation/reprotonation mechanism cannot be employed. Instead, the epimerase employs a transient oxidation strategy involving a tightly bound NADP(+) cofactor. A recent study ruled out mechanisms involving transient oxidation at C-4' ' and C-7' ' and supported a mechanism that involves an initial oxidation directly at the C-6' ' position to generate a 6' '-keto intermediate (Read, J. A., Ahmed, R. A., Morrison, J. P., Coleman, W. G., Jr., Tanner, M. E. (2004) J. Am. Chem. Soc. 126, 8878-8879). A subsequent nonstereospecific reduction of the ketone intermediate can generate either epimer of the ADP-heptose. In this work, an intermediate analogue containing an aldehyde functionality at C-6' ', ADP-beta-d-manno-hexodialdose, is prepared in order to probe the ability of the enzyme to catalyze redox chemistry at this position. It is found that incubation of the aldehyde with a catalytic amount of the epimerase leads to a dismutation process in which one-half of the material is oxidized to ADP-beta-d-mannuronic acid and the other half is reduced to ADP-beta-d-mannose. Transient reduction of the enzyme-bound NADP(+) was monitored by UV spectroscopy and implicates the cofactor's involvement during catalysis.

We have previously shown that tamoxifen can reduce infarct sizes measured by 2,3,5,-triphenyltetrazolium chloride (TTC) staining at 72 h after 2 h of reversible middle cerebral artery occlusion (rMCAo) in rats. In this study, we tested whether improvement is found in both behavioral measures of protection and by histological measures of infarcted tissue at 7 and 14 days after 2 h rMCAo. Tamoxifen (10 mg/kg) was given once by intravenous injection 1 hr after reperfusion, i.e. 3 h after initiation of rMCAo. Neurobehavioral deficits were evaluated daily for 1 week or 2 weeks followed by infarct volumes measurements by hematoxylin-eosin (HE) staining. Tamoxifen-treated rats had significantly improved neurobehavioral deficit scores when evaluated daily throughout the 1 week or 2 week periods and showed 3 significantly reduced median infarct volumes measured after 1 week and 2 weeks. Median infarct values were 149 mm(3) (interquartile range, IQR: 92 to 258) and 124 mm(3) (IQR: 69 to 174) for the 1 and 2 week vehicle groups, respectively, compared with 5 mm(3) (IQR: 3 to 16) and 4 mm(3) (IQR: 0 to 48) for the comparable treated groups (both P < 0.05, Mann-Whitney test), giving a reduction of more than 90% in both cases. Thus, a single administration of tamoxifen given 3 h after initiation of rMCAo is extremely effective in producing long-term neuroprotection as assessed by neurobehavioral measures and histopathology in experimental stroke in rats. If these results are extrapolatable to human stroke, these data indicate that tamoxifen may be a useful neuroprotectant. (c) 2005 Elsevier Inc. All rights reserved.

ADP-l-glycero-d-manno-heptose 6-epimerase (AGME, RfaD) is a bacterial enzyme that is involved in lipopolysaccharide biosynthesis and interconverts ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep) with ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep). AGME is known to require a tightly bound NADP+ cofactor for activity and presumably employs a mechanism involving transient oxidation of the substrate. Four mechanistic possibilities are considered that involve transient oxidation at either C-7' ', C-6' ', or C-4' ' of the heptose nucleotide. In this contribution, the use of solvent isotope incorporation studies and alternate substrates provides strong evidence for a mechanism involving nonstereospecific oxidation/reduction directly at C-6' '. It was found that the epimerization proceeds without any detectable incorporation of solvent-derived deuterium or 18O-isotope into the product. This argues against mechanisms involving either proton transfers at carbon or dehydration/rehydration events. In addition, the deoxygenated analogues, 7' '-deoxy-ADP-l,d-Hep and 4' '-deoxy-ADP-l,d-Hep, were both found to serve as substrates for the enzyme, indicating that oxidation at either C-7' ' or C-4' ' is not required for catalysis.

The photochemistry of p-nitrobenzyl derivatives 6–10 has been studied in aqueous solution as a function of pH, using product analysis, UV–vis spectrophotometry, and laser flash photolysis (LFP). The compounds were chosen with the aim of further exploring the propensity of these systems to give rise to α-hydroxy-p-nitrobenzyl carbanions on photolysis, and to study their mechanisms of subsequent reaction. α-Hydroxy-substituted carbanions are anions that cannot be readily formed using thermal routes but which are believed to have some interesting chemistry. Three methods were employed for photogenerating these carbanions: (i) decarboxylation; (ii) retro-Aldol reaction; and (iii) carbon acid deprotonation. All three methods proved to be successful using the p-nitrobenzyl chromophore. Photogenerated α-hydroxy-p-nitrobenzyl carbanions react via disproportionation, giving rise to oxidized and reduced products; simple protonation of the anion was undetectable.Key words: photodecarboxylation, nitrobenzyl carbanions, photoredox, nitroaromatic compounds, excited-state carbon acids.

Several p -nitrobiphenyl derivatives show significantly higher photoreactivity compared to their p -nitrophenyl analogs, in addition to displaying a new acid-catalyzed photodecarboxylation mechanism in p -( p ′-nitrophenyl)phenylacetic acid.

The photochemistry of three p-nitrobiphenyl derivatives 9-11 has been investigated to explore the ability of photoexcited nitro groups to induce chemistry through the biphenyl ring system. Previous work has shown that the nitro group is highly electron withdrawing at both the meta and para positions (on the benzene ring) in the excited triplet state, inducing decarboxylations and retro-Aldol type reactions, as well as a novel intramolecular redox-type reaction. The mechanisms of all of these reactions are believed to involve photogenerated nitrobenzyl carbanion-type intermediates. Analogous reactions with enhanced quantum efficiencies were observed in the nitrobiphenyls studied in this work. This is further evidence that twisted ground state biaryls (and possibly higher order oligophenylenes) may be thought of as highly polarizable electronically conjugated π-systems in the excited state with the ability to induce efficient photochemistry. Moreover, a charge transfer triplet state is believed to be responsible for inducing highly efficient, novel acid-catalyzed pathways observed for the photodecarboxylation of 11 and the photoredox reaction of 9, neither of which has been observed in the corresponding nitrophenyl (parent) system.