Rational design of imine reductase for asymmetric synthesis of α-methyl-benzylaminonitrogen heterocycles

Abstract

The potential applications of imine reductases (IREDs) in chiral amine synthesis have garnered significant attention. However, there is still limited research on IRED for reacting with bulky substrates. In this study, we addressed this gap by mutating engineered IRED M5 when confronted with a bulky amine substrate, α-methylphenylethylamine. Our investigation revealed the relatively low catalytic efficiency of engineered IRED M5 when tasked with α-methylphenylethylamine. To enhance the catalytic performance of IRED M5, the mutants M203V and F260A were obtained by rational design. F260A exhibited a substantial improvement in the conversion of tert‑butyl 3-(((R)-1-phenylethyl)amino)piperidine-1-carboxylate, exceeding 2-fold. This enhancement suggests that the F260A mutation likely led to a more conducive active site or increased substrate binding affinity. Furthermore, F260A demonstrated a significant enhancement in stereoselectivity when applied to tert‑butyl 4-(((S)-1-phenylethyl)amino)azepane-1-carboxylate, with the enantiomeric excess (e.e.) increasing from 9% to 86% (S). M203V increased conversions for 3-(((R/S)-1-phenylethyl)amino)piperidine-1-carboxylate. In addition, we revealed a possible the underlying molecular mechanism of the effect of F260A and M203V on catalytic performance. Our study provides a deeper understanding of the rational design according to the interaction between IRED cavity and substrate.