Blocking, Widening, Regulating, and Tailoring-Based Engineering of an Imine Reductase for Asymmetric Reductive Amination of Sterically Demanding Ketones with Ammonia

Abstract Chiral amines are essential building blocks for the synthesis of pharmaceuticals. Although imine reductases (IREDs) have emerged as powerful biocatalysts for chiral amine synthesis, their application in reductive amination using ammonia as an amino donor remains limited, especially for sterically demanding ketone substrates. Herein, we developed a BWRT (Blocking, Widening, Regulating, and Tailoring) strategy for engineering the active-site pocket of an IRED from Saccharopolyspora erythraea (SeIRED). The engineered variant, SeIRED-M4, containing 17 amino acid mutations, exhibited a 23,300-fold increase in specific activity along with a 19 °C increase in melting temperature (Tm) relative to the wild-type enzyme. SeIRED-M4 efficiently catalyzed the asymmetric reductive amination of a broad range of ketones with ammonia, affording the corresponding chiral amines with up to 98% conversion and >99% ee. The synthetic applicability of SeIRED-M4 was further demonstrated through the gram-scale synthesis of sitagliptin ((R)-1b), achieving 93% conversion and >99% ee at a substrate loading of up to 40 g/L. Collectively, this study demonstrates an active-site engineering strategy that improves catalytic performance by regulating substrate accessibility and productive binding orientation, highlighting the importance of controlling substrate-binding conformations in biocatalyst development for pharmaceutical applications.