Development of a new chemo-enzymatic catalytic route for synthesis of (S)- 2-chlorophenylglycine.

(S)-2-chlorophenylglycine ((S)-CPG) is a key chiral intermediate for the synthesis of clopidogrel. Herein, a novel, efficient and environmentally friendly chemo-enzymatic route for the preparation of optically pure (S)-CPG was developed. A straightforward chemical synthesis of the corresponding prochiral keto acid substrate (2-chlorophenyl)glyoxylic acid (CPGA) was developed with 91.7% yield, which was enantioselectively aminated by leucine dehydrogenase (LeuDH) to (S)-CPG. Moreover, protein engineering of LeuDH was performed via directed evolution and semi-rational design. A beneficial variant EsLeuDH-F362L with enlarged substrate-binding pocket and increased hydrogen bond between K77 and substrate CPGA was constructed, which exhibited 2.1-fold enhanced specific activity but decreased thermal stability. Coupled with a glucose dehydrogenase from Bacillus megaterium (BmGDH) for NADH regeneration, EsLeuDH-F362L completely converted up to 0.5 M CPGA to (S)-CPG in 8 h at 40 °C.