title: Practical two-step synthesis of enantiopure styrene oxide through an optimized chemoenzymatic approach.
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 1 M (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 1 M (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 120 g/L within 14 h, 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.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Kai
authors.initials: K
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. hlwang@ecust.edu.cn.
authors.forename: Hualei
authors.initials: H
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Haiyang
authors.initials: H
authors.lastname: Fan
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China.
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, People's Republic of China. dzhwei@ecust.edu.cn.
authors.forename: Dongzhi
authors.initials: D
authors.lastname: Wei
mesh_terms: D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution; D004852:Epoxy Compounds / Q000378:metabolism*; D007778:Lactobacillus / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D009153:Mutagens / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004852:Epoxy Compounds; D009153:Mutagens; D050505:Mutant Proteins; C013690:styrene oxide; D000426:Alcohol Dehydrogenase
keywords: Alcohol dehydrogenase; Directed evolution; Enantioselectivity; Styrene oxide