Enantiodivergent Carbon-Phosphorus Bond Formation via Enzymatic Carbene Insertion.

Phosphorus is one of the six elements of life, yet enzymatic carbon-phosphorus (C-P) bond formation is exceedingly rare in nature. This gap in the biosynthetic toolkit is particularly consequential, given the prominence of stereogenic-at-phosphorus P(V) scaffolds in pharmaceuticals and agrochemicals, where the absolute configuration at phosphorus often directly governs biological activity. Despite significant advances in the asymmetric synthesis of P-stereogenic compounds, a general enzymatic platform based on direct C-P bond formation has not been realized. Here, we describe the discovery and directed evolution of Aeropyrum pernix protoglobin (ApePgb) variants that catalyze carbene insertion into the P-H bonds of secondary phosphine oxides and H-phosphinates. Mechanistic investigations reveal that carbene P-H insertion proceeds through a stereoretentive kinetic resolution. Directed evolution of a single protein scaffold produced an enantiodivergent enzyme platform that selectively delivers either P-configuration with high enantioselectivity across a broad substrate scope. The evolution campaign further revealed latent activity for nitrene P-H insertion, opening a path toward P-stereogenic phosphinamides and phosphonamidates through future evolution.