Enantiodivergent Carbon–PhosphorusBond Formationvia 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.