Engineering the substrate acceptance of l-amine dehydrogenase enables the collective biocatalytic synthesis of N-heterocyclic primary amines

Chiral N-heterocyclic primary amines are highly attractive synthons in the pharmaceutical industry. Amine dehydrogenases (AmDHs)-catalyzed direct asymmetric reductive amination of the readily available N-heterocyclic ketones represents a promising approach for synthesizing N-heterocyclic primary amines. However, the limited substrate acceptance of AmDHs restricts their application in biocatalytic synthesis. Here, we engineered the substrate acceptance of L-EsAmDH Es AmDH from Exiguobacterium sibiricum to access a panel of structurally diverse Nheterocyclic ketones. Through reverse substrate design and combining with substrate walking strategy, two active mutants with extended substrate specificity toward N-Boc-4-acetylpiperidine were identified, and two additional rounds of iterative site mutagenesis further increased the activity by 116.3-fold. The optimal mutant M4-2 (L49G/V303A/L307A/T143A) exhibited significantly expanded N-Boc, N-Bn, and N-Cbz-substituted heterocyclic ketones scope, and its practical biocatalytic synthesis performance was verified in the gram-scale synthesis of (R)-4-aminoethyl-1-Boc-piperidine, R )-4-aminoethyl-1-Boc-piperidine, achieving 95 % conversion, >99 % ee , and an overall isolated yield of 86 % (9.8 g). Our study lays the foundation for the collective biocatalytic synthesis of structurally diverse N-heterocyclic primary amines and gives referable guidance for engineering AmDH family members into versatile biocatalysts.