Efficient Synthesis of Non-Natural L-2-Aryl-Amino Acids by a Chemoenzymatic Route

摘要

Enantiopure non-natural 2-aryl-amino acids are important intermediates for synthesizing pharmaceuticals. To develop an efficient chemoenzymatic process to produce nonnatural 2-aryl-amino acids, a penicillin G acylase (PGA) gene from Bacillus megaterium was cloned and expressed in Bacillus subtilis WB800. The recombinant PGA exhibited a high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-phenylacetyl derivatives of non-natural 2-aryl-amino acids. The L-2-aryl-amino acids were obtained in >99.9% enantiomeric excess (ee) and >49% conversion within 3 h. The position and type of the substituent in the substrate influence the recombinant PGA activity but do not affect the PGA enantioselectivity. The kinetic parameters of the recombinant PGA for different substrates were determined and compared. The mechanisms of enantioselectivity of PGA with respect to different substrates were elucidated. The chiral discrimination of PGA with respect to rac-2a-e was mainly because the Dsubstrates used in this study cannot interact with the active residues and bind to the active pocket as stably as the L-substrates. The unreacted N-phenylacetyl-D-2-aryl-amino acids can be completely racemized at 170 °C and then used as the substrate. A gram-scale production of L-2-aryl-amino acids was successfully achieved with approximately theoretical conversion, indicating that the chemoenzymatic approach appears to be promising for industrial applications.