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.