Engineering a non-naturally enantioselective carbonyl reduction of diaryl α-keto amides by an “ene”-reductase from Bacillus subtilis and its mutant enzymes

Abstract

Optical diaryl α-hydroxyl amides are important chiral blocks for biologically active compounds. Given the less reactive keto carbonyl groups, diaryl α-keto amides are difficult to reduce; thus, the corresponding α-hydroxyl amides cannot be obtained. Here an “ene”-reductase (YqjM) from Bacillus subtilis was found to possess a promiscuous ability for enantioselective reduction of diaryl α-keto amide (1a) to obtain α-hydroxy amide 1b, but with poor activity and stereoselectivity. Structure-guided, iterative mutagenesis provided a YqjM variant Y28A/Y169I, which could achieve enantioselective conversion efficiently. The product (R)-1b with 84.8 ± 1.3 % yield and 95.6 ± 1.5 % e.e. was achieved within 4 h. With the Y28A/Y169I in hand, other diaryl α-hydroxy amides were also obtained. The non-natural carbonyl reduction mechanism catalyzed by Y28A/Y169I was given by a series of experimental evaluation and molecular dynamics (MD) simulation. This study provides a much simply, rapidly, environmentally friendly, and cost-effectively unconventional route to achieve optical diaryl α-hydroxy amides that are previously difficult to access. The simplified reaction operations not requiring a photocatalyst and blue light, and mild reaction conditions can greatly improve its application potential of the reaction.