Abstract
Enantiopure styrene oxide (SO) and its derivatives are important building blocks for chiral synthesis. In this study, we developed an attractive "1-pot, 2-step" chemoenzymatic approach for producing enantiopure SO with 100% theoretical yield. This approach involved asymmetric reduction of α-chloroacetophenone by an alcohol dehydrogenase (ADH; step 1), followed by base-induced ring closure (epoxidation) of enantiopure 2-chloro-1-phenylethanol produced by the ADH (step 2). By-product formation during epoxidation was suppressed to <1% by adding methyl tert-butyl ether (MTBE) as the second phase. Therefore, with this optimized approach, ADH from Lactobacillus kefir (LkDH) successfully produced 1M (S)-SO, with 99% analytical yield and 97.8% enantiomeric excess (ee). In the preparation of (R)-SO, a semi-rational strategy of active pocket iterative saturation mutagenesis (ISM) was successfully used to inverse the enantioselectivity of LkDH (muDH2, F147L/Y190P/A202F/M206H/V196L/S96D/K97V), which produced the opposite enantiomer (R)-2-chloro-1-phenylethanol. Through the optimized chemoenzymatic approach, muDH2 was successfully used to prepare 1M (R)-SO, with 98.1% ee and 99.0% analytical yield. Our results indicated that this optimized chemoenzymatic approach could be used to produce both enantiomers of SO at concentrations as high as 120g/L within 14h, which is the highest concentration as far as we know. MuDH2 obtained through ISM also showed reversed enantioselectivity toward another 13 aromatic ketones, compared with wild-type (WT) LkDH. Furthermore, a molecular docking experiment demonstrated that muDH2 inverted the binding orientation of the substrate, which may be the reason for its inverse enantioselectivity.
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