Intermolecular functionalization of tertiary C–H bonds to construct fully substituted stereogenic carbon centres represents a formidable challenge: without the assistance of directing groups, state-of-the-art catalysts struggle to introduce chirality to racemic tertiary sp³-carbon centres. Direct asymmetric functionalization of such centres is a worthy reactivity and selectivity goal for modern biocatalysis. Here we present an engineered nitrene transferase (P411-TEA-5274), derived from a bacterial cytochrome P450, that is capable of aminating tertiary C–H bonds to provide chiral α-tertiary primary amines with high efficiency (up to 2,300 total turnovers) and selectivity (up to >99% enantiomeric excess). The construction of fully substituted stereocentres with methyl and ethyl groups underscores the enzyme’s remarkable selectivity. A comprehensive substrate scope study demonstrates the biocatalyst’s compatibility with diverse functional groups and tertiary C–H bonds. Mechanistic studies explain how active-site residues distinguish between the enantiomers and enable the enzyme to perform this transformation with excellent enantioselectivity.

通过叔 C-H 键的分子间功能化来构建完全取代的立体碳中心是一项艰巨的挑战：在没有导向基团的帮助下，最先进的催化剂很难将手性引入外消旋叔 s p ³ -碳中心。这些中心的直接不对称功能化是现代生物催化的一个有价值的反应性和选择性目标。在这里，我们提出了一种源自细菌细胞色素 P450 的工程化氮宾转移酶 (P411-TEA-5274)，它能够胺化叔 C-H 键以高效提供手性 α-叔伯胺（总转换次数高达 2,300）和选择性（高达 >99% 对映体过量）。具有甲基和乙基的完全取代的立体中心的构建强调了该酶卓越的选择性。全面的底物范围研究证明了生物催化剂与不同官能团和叔 C-H 键的相容性。机理研究解释了活性位点残基如何区分对映体，并使酶能够以优异的对映选择性进行这种转化。