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.