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 Es LeuDH-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 ( Bm GDH) for NADH regeneration, Es LeuDH-F362L completely converted up to 0.5 M CPGA to ( S )-CPG in 8 h at 40 °C.