Res Sq [Preprint]. 2026 Apr 3:rs.3.rs-9204910. doi: 10.21203/rs.3.rs-9204910/v1.

ABSTRACT

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.

PMID:41960347 | PMC:PMC13060504 | DOI:10.21203/rs.3.rs-9204910/v1