Accessing all Four Stereoisomers of β ‐Fluoroamines by Engineered Imine Reductase‐Mediated Dynamic Kinetic Reductive Amination

Stereodivergent synthesis of chiral molecules featuring multiple stereocenters remains a fundamental challenge in synthetic chemistry. Chiral β ‑fluoroamines are favored structural motifs in pharmaceuticals and agrochemicals, and the precise stereochemical configurations of the C─F and C─N stereogenic centers directly influence bioactivity. Despite the high value of chiral β ‑fluoroamines, existing synthetic approaches lack the capacity to deliver all possible stereoisomers in a single operation. Here we report the stereodivergent engineering of an imine reductase (IRED) into a set of stereocomplementary variants that enable programmable access to all four stereoisomers of β ‐fluoroamines via dynamic kinetic reductive amination (DKRA) of α ‑fluoro‑ketones with amines. Leveraging the focused rational iterative site‐specific mutagenesis (FRISM) strategy, we precisely reprogrammed the enzyme active site by optimizing steric, electronic, and hydrogen‐bonding interactions with the key fluorinated imine intermediate. By generating and screening less than 100 variants, this engineering strategy yielded four stereoselective IRED variants, each selectively catalyzing the formation of a single stereoisomer of chiral β ‐fluoroamines with exceptional stereoselectivity (up to >20:1 dr and 99% ee ) across a broad substrate scope. Kinetic studies and molecular dynamics simulations further elucidated the source of divergent stereoselectivity. This work accelerates the development of stereodivergent enzyme families for complex chiral synthesis.