Unlocking the Substrate\nAcceptance of Phenylalanine\nAmine Dehydrogenase Enables the Asymmetric Synthesis of Pharmaceutical <i>N</i>‑Heterocyclic Primary Amines

<i>N</i>-Heterocyclic primary amines are highly\nvaluable\nand recurrent synthons in the pharmaceutical industry. Amine dehydrogenase\n(AmDH)-catalyzed direct asymmetric reductive amination of the readily\navailable <i>N</i>-heterocyclic ketones represents a promising\napproach for synthesizing <i>N</i>-heterocyclic primary\namines. However, the limited substrate acceptance of AmDHs restricts\ntheir application in reductive amination. In this endeavor, we unlocked\nthe substrate acceptance of phenylalanine amine dehydrogenase from <i>Bacillus badius</i> (F-<i>Bb</i>AmDH) to access a\npanel of <i>N</i>-Boc-substituted heterocyclic ketones with\na broad range of structural features. Based on the combination of\na reverse substrate design strategy and structural-guided steric hindrance\nmutagenesis, two active F-<i>Bb</i>AmDH mutants with expanded <i>N</i>-heterocyclic ketone specificity toward <i>N</i>-Boc-3-piperidone were generated, and two rounds of additional iterative\nsite mutagenesis further increased the catalytic activity by 143.4-fold.\nThe optimal triple mutant M3-2 displayed a significantly extended\nscope of <i>N</i>-Boc-substituted heterocyclic ketones,\nand its practical asymmetric synthesis performance was confirmed in\nthe gram-scale synthesis of the key intermediate of alogliptin, (<i>R</i>)-1-Boc-3-piperidinamine, achieving >99% ee, 86% conversion,\nand 80% (4.0 g) isolated yield. This work lays the foundation for\nthe biocatalytic synthesis of structurally diverse <i>N</i>-heterocyclic primary amines and gives referable guidance for engineering\noxidoreductases sharing an analogous pocket into versatile biocatalysts.