Single-site modification of the P450-BM3 substrate-entrance facilitates the synthesis of optically pure pharmaceutically useful methyl trans-3-phenylglycidates

Abstract

Optically pure methyl trans-3-phenylglycidates are useful chiral blocks or intermediates in the pharmaceutic field. However, high efficiency and enantioselective synthetic methods remain to be developed. We report an enzymatic process, based on rationally modifying the site D68 at the entrance of the P450-BM3 monooxygenase, thereby creating a mutant D68V with high epoxidation activity and high stereoselectivity toward methyl cinnamate as a cheap substrate. About 9-fold increase in turnover number compared to the wild type was achieved. Furthermore, with molecular dynamics simulations, the reasons for increased activity and stereoselectivity were unveiled. The best mutant also presents high activity toward methyl cinnamate analogs with fluorinated benzyl group. All epoxy products were produced on a gram-scale by whole-cells catalysis. In addition, a series of (+)-prebalamide derivatives were chemically synthesized from these products. This work not only expands the biocatalytic toolbox for producing methyl trans-3-phenylglycidates, but also identifies a new hotspot for engineering P450-BM3.