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Chiral amines can be made by insertion of a carbene into an N–H bond using two-catalyst systems that combine a transition metal-based carbene-transfer catalyst and a chiral proton-transfer catalyst to enforce stereocontrol. Haem proteins can effect carbene N–H insertion, but asymmetric protonation in an active site replete with proton sources is challenging. Here we describe engineered cytochrome P450 enzymes that catalyse carbene N–H insertion to prepare biologically relevant α-amino lactones with high activity and enantioselectivity (up to 32,100 total turnovers, >99% yield and 98% e.e.). These enzymes serve as dual-function catalysts, inducing carbene transfer and promoting the subsequent proton transfer with excellent stereoselectivity in a single active site. Computational studies uncover the detailed mechanism of this new-to-nature enzymatic reaction and explain how active-site residues accelerate this transformation and provide stereocontrol. 

Abstract We report a computationally driven approach to access enantiodivergent enzymatic carbene N−H insertions catalyzed by P411 enzymes. Computational modeling was employed to rationally guide engineering efforts to control the accessible conformations of a key lactone‐carbene (LAC) intermediate in the enzyme active site by installing a new H‐bond anchoring point. This H‐bonding interaction controls the relative orientation of the reactive carbene intermediate, orienting it for an enantioselectiveN‐nucleophilic attack by the amine substrate. By combining MD simulations and site‐saturation mutagenesis and screening targeted to only two key residues, we were able to reverse the stereoselectivity of previously engineeredS‐selective P411 enzymes. The resulting variant,L5_FL‐B3, accepts a broad scope of amine substrates for N−H insertion with excellent yields (up to >99 %), high efficiency (up to 12 300 TTN), and good enantiocontrol (up to 7 : 93er).