Engineered ω-Transaminase Enables Remote Stereocontrol in the Synthesis of Chiral Pharmaceutical N-Heterocyclic Amines with Nonadjacent Stereocenters

Chiral N-heterocyclic amines containing nonadjacent stereocenters are essential and valuable synthons for the synthesis of various pharmaceuticals. ω-Transaminases (ω-TAs) are promising biocatalysts for the construction of chiral amine scaffolds. However, the remote stereocontrol in their synthesis remains challenging. Herein, we reported an enzymatic approach of kinetic resolution–asymmetric amination for synthesizing highly diastereoselective and enantioselective N-heterocyclic amines with remote stereocenters, catalyzed by only one ω-TA. An enzyme mining framework EnzSeer was developed to identify a ω-TA from Ancylobacter lacus (AlTA) that showed moderate activity and stereoselectivity toward 1-Boc-2-methyl-piperidin-5-one (1), a non-natural N-heterocyclic ketone. The structure-guided engineering of the active pocket of AlTA coupled with computational optimization of its peripheral regions significantly enhanced the catalytic performance of AlTA. The best mutant M3 exhibited a 17-fold improvement in catalytic efficiency (kcat/Km) and 118-fold increase in half-life at 45 °C. Employing the engineered enzyme, multiple chiral N-heterocyclic amines were synthesized with up to >99% ee, >95% de, and 99% conversion. The practical synthesis performance of M3 was confirmed through gram-scale synthesis of a key intermediate of ritlecitinib, achieving 38% yield, >99% ee, and 94% de. This work presents an EnzSeer framework for mining function-specific enzymes and provides engineered biocatalysts with high catalytic activity and thermostability, facilitating the development of an enzymatic pathway for producing diverse pharmaceutical chiral N-heterocyclic amines with nonadjacent stereocenters.