Biocatalytic Radical C(sp³)-N Coupling via Active Site Templating

Stereoselective nucleophilic substitution to access α-tertiary amines relies on copper-catalyzed radical approaches in which the substitution is mediated by metal-anilide coordination.¹ These systems, however, are constrained by competing arene radical alkylation pathways.² Here we report a distinct photoenzymatic mechanism for enantioconvergent nucleophilic substitution that operates without metal coordination to the nucleophile. Six rounds of protein engineering yielded a variant of a flavin-dependent oxidoreductase that promotes C(sp³)-N coupling between tertiary alkyl halides and simple anilines in good yields, with high chemoselectivity for N- over C-alkylation and high enantioselectivity across a broad substrate range. Multivariate statistical analysis, density functional theory, and mechanistic experiments show that the active site templates π-stacking, hydrogen-bonding, and water-bridged interactions between a tertiary radical and the aniline lone pair to generate an intermolecular n→SOMO hyperconjugative complex that is energetically disfavored in bulk solution, thereby simultaneously lowering the radical oxidation potential and suppressing arene addition.³ This work uncovers a previously inaccessible, copper-free manifold for nucleophilic substitution at sterically congested carbon centers and expands how enzymes can catalyze C(sp³)-N bond formation with control over both stereo- and chemoselectivity.