Gideon Grogan

This report reviews significant developments in applications of biological catalysis in synthetic organic chemistry for the year 2010. Hydrolases have been applied in kinetic resolution and quantitative DKR processes and have been evolved in vitro to give enzymes displaying improved enantioselectivity and solvent stability. Applications of reductases have included an unusual instance of the generation of optically active atropisomers and also the identification of stereoselective imine reductases in Streptomyces spp. Other biotransformations leading to optically active amines have focused on transaminases, and a wide-ranging genomics analysis has revealed nineteen new and previously rare (R)-selective transaminases. A further (R)-selective transaminase was extensively engineered for process suitability for application in the industrial synthesis of sitagliptin. Improved carbon–carbon bond formation by aldolases is being addressed through the identification and improvement of enzymes accepting non-phosphorylated dihydroxyacetone as a substrate donor for the synthesis of sugars. In addition, new enzymatic activities, including natural enzymes that catalyse the Stetter reaction, and also the Michael-type addition of water to cyclohexenone have been reported. Finally, advances in the in silico design of enzymes are proceeding rapidly, with the report of the design of the first enzyme known to catalyse an bimolecular Diels-Alder reaction.

The structure of the alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber reveals possible reasons for its remarkable tolerance to organic co-solvents and suggests new directions for structure-informed mutagenesis to produce enzymes of altered substrate specificity or improved selectivity.

Maleate isomerase (MI), a member of the Asp/Glu racemase superfamily, catalyzes cis−trans isomerization of the C2−C3 double bond in maleate to yield fumarate. Mutational studies, in conjunction with the structure of the C194A mutant of Nocardia farcinica MI cocrystallized with maleate, have revealed an unprecedented mode of catalysis for the superfamily in which the isomerization reaction is initiated by nucleophilic attack of cysteine at the double bond, yielding a covalent succinylcysteine-like intermediate.

This report reviews significant developments in the application of biological catalysis in synthetic organic chemistry for the year 2009. The continuing interest in lipases as catalysts for quantitative asymmetric transformation has resulted in their application to the synthesis of norsertraline and imidacloprid using dynamic kinetic resolution approaches. A lipase has also been used for the resolution of a complex dynamic compound library based on the reversible Strecker reaction. Esterases have been engineered for wider substrate specificity, and, in one case, to acquire epoxide hydrolase activity. A fluorophosphatase has been engineered to display activity toward the more toxic enantiomer of the nerve agent sarin. A new process has been formulated for the laccase-dependent regeneration of NAD(P)H for use in reductase-catalysed processes. Laccases themselves are attracting increasing interest as mild oxidation catalysts for aromatic coupling reactions leading to polycyclic biologically active compounds and dyes. An amino acid residue in enoate reductases has been highlighted as a major factor in determining enantioselectivity in those enzymes. In carbon–carbon bond formation, aldolases that are able to accept the non-phosphorylated C3 donor dihydroxyacetone have been applied to the synthesis of iminosugars, and the first application of the enzymatic Friedel-Crafts reaction has been realised. Last, aminomutases have found application in the synthesis of β-amino acids, in one case being combined with alanine racemase, furnishing an elegant process for the quantitative conversion of α-phenylalanine to its β-isomer.

The biocatalytic generation of high-value chemicals from abundant, cheap and renewable feedstocks is an area of great contemporary interest. A strain of Rhodococcus erythropolis designated MLT1 was isolated by selective enrichment from the soil surrounding hop plants, using the abundant triene β-myrcene from hops as a sole carbon source for growth. Resting cells of the organism were challenged with β-myrcene, and the major product of biotransformation was determined by mass spectrometric analysis to be the monoterpene alcohol geraniol. Controls demonstrated that the product was biogenic and that an aerobic environment was required. The ability to transform β-myrcene was shown to be restricted to cells that had been grown on this substrate as sole carbon source. Pre-incubation of cells with the cytochrome P450 inhibitors metyrapone or 1-aminobenzotriazole reduced geraniol production by 23% and 73% respectively, but reduction in activity was found not to correlate with the inhibitor concentration. A comparative analysis of insoluble and soluble cell extracts derived from cells of MLT1 grown on either β-myrcene or glucose revealed at least four proteins that were clearly overproduced in response to growth on β-myrcene. Mass spectrometric analysis of tryptic digests of three of these protein bands suggested their identities as an aldehyde dehydrogenase, an acyl-CoA dehydrogenase and a chaperone-like protein, each of which has a precedented role in hydrocarbon metabolism clusters in Rhodococcus sp. and which may therefore participate in a β-myrcene degradation pathway in this organism.

This report reviews significant developments in applications of biological catalysis in synthetic organic chemistry for the year 2008. Amongst the highlights, a carboxylesterase with specificity for tertiary alcohols has been engineered for inverted enantioselectivity, and the halohydrin dehalogenase HheC has been applied to the formation of chiral oxazolidinones using cyanate as a nucleophile. There have been notable advances in the screening and application of ketoreductases (KREDs) for both chiral reductions and the quantitative deracemisation of secondary alcohols. A KRED has also been used in combination with palladium catalysis to perform a two step Suzuki-coupling/carbonyl reduction in one-pot in aqueous medium. Studies on an unusual Baeyer-Villiger monooxygenase from Xanthobacter have revealed its ability to epoxidise a non-activated alkene. There has been renewed interest in transaminases for the production of chiral amines from prochiral ketones, including the imaginative use of both pyruvate decarboxylase and α-amino acid dehydrogenase to solve equilibrium issues that militate against quantitative conversion. In the area of carbohydrate-transforming enzymes, a new glycosynthase has been developed for the irreversible formation of glycoside links to glucuronic acid, and a glycosyltransferase from Helicobacter pylori has been described that catalyses the formation of S-glycosides. Finally, in silico enzyme design has been coupled with directed evolution techniques to deliver an enzyme which catalyses a reaction not known in Nature, the Kemp elimination, with far-reaching implications for the possible design of enzymatic catalysts in the future.

The Rhodococcus jostii RHA1 genome encodes a number of enzymes that can be exploited as biocatalysts. Study of the substrate spectrum and enantioselectivity of Baeyer–Villiger monooxygenases from R. jostii allowed the identification of short amino acid sequences specific to groups displaying certain catalytic characteristics. The gel illustrates the substrate acceptance spectra and selectivities of the different proteins. magnified image Microbial genome sequences are providing a wealth of information on new enzymes that have considerable potential as biocatalysts. The recently sequenced genome of Rhodococcus jostii RHA1, for example, has revealed an impressive array of catabolic enzymes, including many putative Baeyer–Villiger monooxygenases (BVMOs). We have cloned 23 target BVMO sequences from the genome of R. jostii RHA1 and heterologously expressed 13 of these as soluble proteins to unearth new substrate specificities and selectivities. Whole‐cell biocatalysts expressing the genes were screened against seven different test substrates. Each of these catalysts displayed activity toward at least three ketones. We observed a remarkable diversity of both regio‐ and enantioselectivity among the BVMOs from R. jostii RHA1 for the transformation of two chiral substrates, with some enzymes displaying high enantioselectivity for the isomers of 2‐methylcyclopentanone. With the notable exception of the product of gene ro03437, named MO14, the biocatalysts' sequences correlated well with their respective activities and selectivities. This correlation allowed the identification of sequence motifs specific to subgroups of the BVMOs from R. jostii and other organisms. Overall, the data improve predictive models of BVMO activity from sequence and suggest new avenues to pursue in engineering these enzymes.