Emergence of a distinct mechanism of C-N bond formation in photoenzymes.

C-N bond formation is integral to modern chemical synthesis owing to the ubiquity of nitrogen heterocycles in small-molecule pharmaceuticals and agrochemicals. Alkene hydroamination with unactivated alkenes is an atom-economical strategy for constructing these bonds. However, these reactions are challenging to render asymmetric when preparing fully substituted carbon stereocentres. Here we report a photoenzymatic alkene hydroamination to prepare 2,2-disubstituted pyrrolidines by a Baeyer-Villiger mono-oxygenase. Five rounds of protein engineering afforded a mutant, providing excellent product yield and stereoselectivity. Unlike related photochemical hydroaminations, which rely on the oxidation of the amine or alkene for C-N bond formation, this work exploits a through-space interaction of a reductively generated benzylic radical and the nitrogen lone pair. This antibonding interaction lowers the oxidation potential of the radical, enabling electron transfer to the flavin cofactor. Experiments indicate that the enzyme microenvironment is essential in enabling a innovative C-N bond formation mechanism with no parallel in small-molecule catalysis. Molecular dynamics simulations were performed to investigate the substrate in the enzyme active site, which further support this hypothesis. This work is a rare example of an emerging mechanism in non-natural biocatalysis in which an enzyme has access to a mechanism that its individual components do not. Our study showcases the potential of enhancing emergent mechanisms using protein engineering to provide unique mechanistic solutions to unanswered challenges in chemical synthesis.