Abstract:

Chiral N-substituted amino amides and esters are ubiquitous scaffolds in pesticides and pharmaceutical chemicals, but their asymmetric synthesis remains challenging especially for those with multiple chiral centers. In this study, IR104 from Streptomyces aureocirculatus was identified from 157 wild-type imine reductases for the synthesis of (S)-2-((R)-2-oxo-4-propylpyrrolidin-1-yl) butanamide (antiepileptic drug Brivaracetam) via dynamic kinetic resolution reductive amination from ethyl 3-formylhexanoate and (S)-2-aminobutylamide with high diastereoselectivity. To further improve the catalytic efficiency of IR104, its mutant D191E/L195I/E253S/M258A (M3) was identified by saturation mutagenesis and iterative combinatorial mutagenesis, which exhibited a 102-fold increase in the catalytic efficiency relative to that of wild-type enzyme and high diastereoselectivity (98:2 d.r.). Crystal structural analysis and molecular dynamics simulations provided some insights into the molecular basis for the improved activity of the mutant enzyme. The imine reductase identified in this study could accept chiral amino amides/esters as amino donors for the dynamic kinetic resolution reductive amination of racemic α-substituted aldehydo-esters, expanding the substrate scope of imine reductases in the dynamic kinetic resolution-reductive amination. Finally, IR104-M3 was successfully used for the preparation of Brivaracetam at gram scale. Using this mutant, various N-substituted amino amides/esters with two chiral centers were also synthesized with up to 99:1 d.r. and 96% yields and subsequently converted into γ- and δ-lactams, providing an efficient protocol for the synthesis of these important compounds via enzymatic dynamic kinetic resolution-reductive amination from simple building blocks.

Key words: Imine reductase, Dynamic kinetic resolution-reductive amination, Directed evolution, N-substituted amino amide, N-substituted amino ester