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Two structurally related beta-lactams form different covalent complexes upon reaction with porcine elastase. The high resolution x-ray structures of these two complexes provide a clear insight into the mechanism of the reaction and suggest the design of a new class of serine protease inhibitors that resist enzyme reactivation by hydrolysis of the acyl intermediate. The presence of a hydroxyethyl substituent on the beta-lactam ring provides a new reaction pathway resulting in the elimination of the hydroxyethyl group and the formation of a stabilizing conjugated double bond system. In contrast, the presence of a diethyl substituent on the beta-lactam ring leads to addition of water. The two enzyme complexes show very different binding modes in the enzyme active site.

Deracemization of racemic chiral tertiary amines has been achieved by combination of an enantioselective amine oxidase, obtained through directed evolution, and ammonia borane in a one-pot process.

Cytochrome P450 RhF displays a high degree of substrate promiscuity, mediating a range of O-dealkylations, aromatic hydroxylations, epoxidations and asymmetric sulfoxidations. The self-sufficient nature of this CYP coupled with its ability to catalyse the oxidation of a wide range of functional groups highlights this enzyme as an excellent starting template for directed evolution and promising alternate to P450 BM3.

A series of diastereoisomers of beta-methyl-beta-phenylalanine analogues 1a-f have been prepared in enantiomerically pure form using a combination of chemo- and biocatalysis. Starting from l-threonine methyl ester 2, a range of beta,beta-disubstituted didehydroamino acids were obtained as their (Z)-isomers 6a-f. Asymmetric hydrogenation of these alkenes, using either the [Rh(R,R)-Et-DuPhos(COD)]BF4 or [Rh(S,S)-Et-DuPhos(COD)]BF4 catalyst, followed by hydrolysis yielded two of the four possible sets of diastereoisomers of the beta-branched amino acid. Subsequent stereoinversion, using a stereoselective amino acid oxidase in combination with a nonselective reducing agent, furnished the remaining two sets of diastereomers.

The lipase-catalyzed kinetic resolution of (R/S)-3-phenylbutyric acid 2 using solid-supported cyclohexane-1,3-dione (CHD) 6 is described. In each case the predominant enantiomer observed, after cleavage from the resin, was (R)-(−)-3-phenylbutyric acid (R)-2 (ee > 99%) rather than the expected (S)-enantiomer of 2. This observation is in contrast to the fact that Chromobacterium viscosum lipase shows high enantiospecificity for the (S)-enantiomer in the corresponding solution-phase hydrolysis reactions. The (R)-acyl group was subsequently released from the resin by NaOH hydrolysis, and the yield of the reaction could be improved by triple acylation of the resin.

Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain, lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-3H]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25% of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [3H]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.

A very short and efficient synthesis of the important drug candidate telaprevir, featuring a biocatalytic desymmetrization and two multicomponent reactions as the key steps, is presented. The classical issue of lack of stereoselectivity in Ugi- and Passerini-type reactions is circumvented. The atom economic and convergent nature of the synthetic strategy require only very limited use of protective groups.

The kinetic resolution of a chiral ester by enzyme catalysed hydrolysis is a well-known process and is illustrated by the preparation of a synthon for the natural product coriolic acid. The stereoselective hydrolysis of meso-diesters can give rise to useful optically active intermediates e.g. for the synthesis of carbocyclic ribonucleosides. The enantioselective esterification of a bicyclic secondary alcohol has been used to give access to homochiral prostaglandin precursors. Acylases have been employed to hydrolyse a γ-lactam in enantiospecific fashion and the optically active materials obtained have been converted into the anti-HIV agent carbovir and into an unnatural amino-acid which binds to the GABA receptor. The natural β-amino-acid cispentacin has been prepared by a similar strategy. The less well-developed areas involving enzyme-catalysed hydrolysis of phosphate esters, epoxides and nitriles are also discussed. Reduction of substituted bicyclo[3.2.0]heptanones by dehydrogenase enzymes (either as isolated enzymes or within whole cell systems) gives rise to useful materials for the construction of the pheromone eldanolide and the chemokinetic agent leukotriene B4. Oxidation of benzene and derivatives by dioxygenase enzymes furnishes substituted cyclohexadienediols that can be modified by employing [4+2] and [2+2] cycloaddition reactions. The utility of the enzymic Baeyer Villiger reaction is exemplified by its use in the preparation of δ-lactones that can be converted into anti-HIV agents. Soybean lipoxygenase is useful for the synthesis of 13-HODE and analogues while the opportunities offered by organisms such as Beauveria sulfurescens, that are capable of oxidising organic substrates at ostensibly non-activated positions, are noted. Enzymes that catalyse formation of carbon-carbon bonds are of importance. The uses of rabbit muscle aldolase to form trioses and lyases to form cyanohydrins provide good examples. Amongst future developments in the field of biotransformations, the use of more than one enzyme in the same reaction vessel in order to catalyse a cascade of sequential reactions is a particularly attractive proposition.

Incubation of penicillin N ( 3a ) with partially purified deacetoxy/deacetylcephalosporin C synthase (DAOC/DAC synthase) from Cephalosporium acremonium CO 728 gave in addition to the expected products, deacetoxycephalosporin C and deacetylcephalosporin C, a third β-lactam metabolite as a 3β-hydroxy-3α-methylcepham ( 9a ). Production of the 3β-hydroxycepham was promoted from [3- 2 H]penicillin N ( 3b ) which was rationalised by the operation of a kinetic isotope effect on a branched pathway in the enzymic process. The oxygen of the 3β-hydroxy group was shown to be derived in part from molecular oxygen. In addition, the 2β-methyl group of penicillin N was shown to be incorporated into C2 of the 3β-hydroxy-3α-methylcepham, a result in stereochemical accord with the equivalent transformation of the 2β-methyl group of penicillin N into C2 of deacetoxycephalosporin C 1 . A mechanistic interpretation, consistent with these observations, is offered.

Enantioselective oxidation of racemic O-methyl-N-hydroxycyclohexylethylamine, using a variant of monoamine oxidase N (MAO-N) from Aspergillus niger, yields unreacted (R)-enantiomer (e.e. = 99%) together with the oxime exclusively in the (E)-configuration.

The enantioselectivity and catalytic activity of Novozym 435® [Candidaantarctica lipase B (CALB)] in organic solvents was found to dramatically increase upon the addition of a non-reactive organic base, such as Et3N, to the reaction system.

Enantiomerically pure (R)-α-hydroxyaldehydes (>95% ee) are prepared from the corresponding α-hydroxyesters by silyl protection, reduction with diisobutylaluminium hydride, and finally deprotection under acidic conditions; subsequent coupling of these aldehydes with lithium hydroxypyruvate, catalysed by Escherichia coli transketolase, leads to novel optically pure triols.

Transketolase has been obtained in greater quantities from an over-expressed E. coli transformant carrying the transketolase gene. Crude extracts of this organism are suitable for use in small scale biotransformations to provide mmol quantities of product. Initial results indicate that the transketolase from E. coli is relatively non-specific for the aldehyde component of the reaction.

Perkin 1 Abstracts: Biocatalysis in Organic Synthesis aims to cover recent literature concerning the applications of enzymes and micro-organisms as catalysts in organic synthesis. The abstracts will emphasise the key synthetic step(s) that are mediated by the biocatalyst. Emerging technologies for biocatalyst design and optimisation will also be included.

Lipase-catalysed acetylation of 1 furnishes the ester 3 in 94% yield while, under the same reaction conditions, the isomer 2 affords the ester 5 in 86% yield.

Both β-glucosidase from almonds and β-xylosidase from Escherichia coli have been used to catalyse the synthesis of β-glucosides and β-xylosides of amino acid derivatives, respectively.

Shidasterone 1 is prepared from 2,3-isopropylidene-22-mesylate 20-hydroxyecdysone 4 by three different synthetic sequences; mechanistic considerations together with independent NOE studies reveal a (22R) configuration for Shidasterone 1.

A series of aromatic dinitriles have been examined as substrates for an immobilised whole cell Rhodococcus sp. that catalyses the hydrolysis of nitrites to amides and/or carboxylic acids. The fluorinated aromatic dinitriles 48 and 49 were regioselectively hydrolysed to the corresponding cyano amides 48a and 49a whereas the non-fluorinated analogues 41–44 were converted to cyano acids but with poorer regioselectivity.

The carboxylic acid 6 was converted into the amine 13en route to neplanocin-A 9. The same enantiomer of the amine 13 was made from D-ribose in a 13-step synthesis.

Both L-(>95% e.e.) and D-(60% e.e.)4-hydroxy-2-ketoglutarate 3 have been prepared on a mmol scale using 4-hydroxy-2-ketoglutarate (HKG) aldolase. Substrate specificity studies indicate that HKG aldolase isolated from different sources (Escherichia coli, bovine liver and rat liver) is highly specific for both pyruvate and glyoxylate.

Two novel analogues of the mannosyl transferase acceptor substrate (GlcNAc)2-Pi-Pi-dolichyl 2 have been prepared in which dolichyl is replaced by phytanyl, 4, and lauryl, 5; both compounds 4 and 5 were synthesized using readily available chitin as the disaccharide precursor. Compound 4 acts as an efficient acceptor substrate for mannosyl transferases from porcine liver and Saccharomyces cerevisiae.

Chiral esters of high optical purity were obtained by an enzyme-controlled inter-esterification procedure in which the constituent hydrolysis and esterification reactions were suitably matched.

The conversion of the tripeptide (L-α-amino-δ-adipoyl)-L-cysteinyl-D-valine (α-amino-δ-adipoyl = 5-amino-5-carboxypentanoyl) into isopenicillin N by the enzyme isopenicillin N synthetase has been shown to proceed with complete retention of stereochemistry; a similar result was observed during penicillin formation with isoleucinyl and allo-isoleucinyl tripeptides.

Isopenicillin N synthetase (IPS) cloned from Cephalosporium acremonium has been isolated from transformed Escherichia coli and purified to homogeneity. The resulting, abundant, recombinant protein, whilst undergoing slightly different N-terminal processing to that observed for the fungally-derived protein, has identical kinetics for the conversion of LLD-aminoadipoyl-cysteinyl-valine to isopenicillin N. Recombinant IPS converts analogue substrates into unusual beta-lactam antibiotics in exactly the same way as the fungal protein.

Two unsaturated peptide substrates were each cyclised by the enzyme isopenicillin N synthase (IPNS) from Cephalosporium acremonium CO 728 simultaneously to both desaturated and hydroxylated β-lactam products whose hydroxy groups have been shown to derive their oxygen atom from the cosubstrate dioxygen; a working hypothesis involving iron–oxo species is proposed to explain this phenomenon.

The application of biocatalysis in the pharmaceutical industry is rapidly growing as a result of increased access to enzymes that meet the demands of industrial processes. This expansion of activity has led to a corresponding increase in the demand for immobilised enzymes. EziG™ (EnginZyme AB, Sweden) is marketed as a general matrix for enzyme immobilisation on controlled porosity glass. In this work we identified criteria for a “general” enzyme immobilisation technology in the context of the requirements of the pharmaceutical industry. We subsequently evaluated EziG™ for generality in a series of case studies for the application of immobilised biocatalysts. In this study we have focussed on the challenges facing both academic and industrial applications such as enzyme stability, multistep reactions and reactions in continuous flow.

As the enzyme toolbox for biocatalysis has expanded, so has the potential for the construction of powerful enzymatic cascades for efficient and selective synthesis of target molecules. Additionally, recent advances in computer-aided synthesis planning are revolutionising synthesis design in both synthetic biology and organic chemistry. However, the potential for biocatalysis is not well captured by tools currently available in either field. Here we present RetroBioCat, an intuitive and accessible tool for computer-aided design of biocatalytic cascades, freely available at retrobiocat.com. Our approach uses a set of expertly encoded reaction rules encompassing the enzyme toolbox for biocatalysis, and a system for identifying literature precedent for enzymes with the correct substrate specificity where this is available. Applying these rules for automated biocatalytic retrosynthesis, we show our tool to be capable of identifying promising biocatalytic pathways to target molecules, validated using a test-set of recent cascades described in the literature.

The properties of the heme, flavin mononucleotide (FMN) and FeS domains of P450 RhF, from Rhodococcus sp. NCIMB 9784, expressed separately and in combination are analysed. The nucleotide preference, imidazole binding and reduction potentials of the heme and FMN domains are unaltered by their separation. The intact enzyme is monomeric and the flavin is confirmed to be FMN. The two one-electron reduction potentials of the FMN are −240 and −270mV. The spectroscopic and thermodynamic properties of the FeS domain, masked in the intact enzyme, are revealed for the first time, confirming it as a 2Fe–2S ferredoxin with a reduction potential of −214mV.

Transaminases have attracted considerable interest in their use as biocatalysts for the synthesis of compounds containing chiral amine units, which are widespread within the pharmaceutical, agrochemical, and fine chemical industry. Recent developments in enzyme- and process-engineering have expedited their use in asymmetric synthesis; however, industrial applications are still hindered by a number of factors, including equilibrium thermodynamics, product inhibition, and poor substrate tolerance. Detailed and comprehensive approaches to each of these challenges have been reported during the last two decades; the most representative enzyme discovery and screening strategies, protein and equilibrium engineering, and immobilization techniques are reviewed herein. Furthermore, we present a detailed look into the applications of transaminases for the synthesis of a variety of amine-containing compounds and the integration of transaminases into multienzymatic systems that allow access to a variety of highly complex products for the end user.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of