A novel epoxide hydrolase (BMEH) with unusual (R)-enantioselectivity and very high activity was cloned from Bacillus megaterium ECU1001. Highest enantioselectivities (E > 200) were achieved in the bioresolution of ortho-substituted phenyl glycidyl ethers and para-nitrostyrene oxide. Worthy of note is that the substrate structure remarkably affected the enantioselectivities of the enzyme, as a reversed (S)-enantiopreference was unexpectedly observed for the ortho-nitrophenyl glycidyl ether. As a proof-of-concept, five enantiopure epoxides (> 99% ee) were obtained in high yields, and a gram-scale preparation of (S)-ortho-methylphenyl glycidyl ether was then successfully performed within a few hours, indicating that BMEH is an attractive biocatalyst for the efficient preparation of optically active epoxides.

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S)-N-Boc-3-hydroxypiperidine (S-NBHP) is a useful synthon for the synthesis of pharmaceutical intermediates including ibrutinib, the API of the newly approved drug Imbruvica, for the treatment of lymphoma. To our knowledge, there are no published biotransformation methods scalable to prepare S-NBHP. We report here the development of an efficient process catalyzed by recombinant ketoreductase (KRED) to reduce N-Boc-piperidin-3-one to obtain optically pure S-NBHP. The process has been optimized and demonstrated to have commercial potential with 100 g/L of substrate concentration, product of >99% ee with under 5% of enzyme loading (w/w). ■ INTRODUCTIONA large number of natural and unnatural bioactive molecules have one or more piperidine rings. 1 There are many active pharmaceutical ingredients (API) which also contain this moiety. 2 The hydroxyl group on the C3-position would introduce a chiral carbon atom and may significantly affect the bioactivity of the molecule. 3 As a result, chiral hydroxypiperidines and their derivatives are important synthons used in the pharmaceutical industry.Several methods have been reported for the synthesis of chiral piperidines including classic diastereomeric resolution, 4 asymmetric synthesis, 5 and asymmetric reduction, for example, of 4-oxo-piperidine-3-carboxylic acid esters followed by multistep conversions (Scheme 1). 6 All these methods suffered from low yields or lengthy synthesis. Lacheretz et al. reported the bioreduction of cyclic 3-oxo-amines using the tissue of Daucus carota, where six piperidin-3-one derivatives could be reduced to S-alcohols with varying enantioselectivity. 7 Among the substrates, the reduction of N-Boc-piperidin-3-one showed the highest enantioselectivity, giving (S)-N-Boc-3-hydroxypiperidine (S-NBHP) with 95% optical purity. Unfortunately the reaction is not practical due to low substrate concentration (3 mM), high catalyst concentration (23%, m/v), moderate chiral purity (95% ee) and low yield (73%).Ketoreductase (KRED)-mediated biotransformation has been applied more and more widely in pharmaceutical industry nowadays. 8 In general, there are two strategies to locate a KRED for practical application. On the one hand, the discovery of new natural KRED biocatalysts using activity screening and genome mining continues to enlarge the substrate spectrum. 9 On the other hand, enzyme engineering technologies including directed evolution and semirational and rational design can engineer KREDs with high efficiency and stability. 10 Directed evolution and rational design as the most important technique of protein engineering have emerged to be powerful tools for manipulating protein properties with numerous successful stories. 11 The availability of highly diversified KRED libraries makes it increasingly possible to discover biocatalysts to meet the metrics of industrial processes. Here we report such a work to screen and apply recombinant KREDs to catalyze the asymmetric preparation of S-NBHP on an industrial scale. ■ RESULTS AND DISCUSSIONThe target enz...

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Computational modeling approaches were successfully applied to a series of diarylpyridine derivatives as novel HIV-1 NNRTIs.