Chiral amines can be made by insertion of a carbene into an N-H bond using two-catalyst systems that combine a transition metal 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 catalyze 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.Amines are ubiquitous in bioactive molecules and functional materials 1,2 , and the development of efficient and selective methods for C-N bond construction remains one of the central themes of modern organic chemistry and biochemistry 3-5 . Among the numerous ways to construct C-N bonds, carbene insertion into N-H bonds 6-10 benefits from the high reactivity of carbene species and excellent functional group compatibility to rapidly build complex nitrogen-containing molecules. In the last several years, empowered by directed evolution, metallo-haem-dependent enzymes (cytochromes P450, cytochromes c and globins, for example) have exhibited an impressive ability to catalyze non-natural carbene-and nitrene-transfer reactions with high efficiency and selectivity. Specifically, haem proteins have been engineered to perform carbene N-H insertion reactions with catalytic efficiency far exceeding their small-molecule counterparts (up to thousands of total turnover numbers (TTN)) [11][12][13][14] . However, compared to cyclopropanation 15 , C-H insertion 16 and many other carbene transfer reactions also catalyzed by haem proteins 17,18 , N-H insertion reactions are still underdeveloped, especially with respect to high stereocontrol.In small-molecule catalysis, a common strategy for asymmetric N-H insertion is to employ a transition-metal catalyst for carbene transfer along with a separate chiral proton-transfer catalyst (PTC) for stereoinduction (Fig. 1a) 19,20 . The carbene precursor first reacts to form a metal carbene species, which can be trapped by the amine substrate through nucleophilic attack, generating an ylide intermediate. The asymmetric protonation of the ylide is then guided by a chiral PTC, such as a chiral phosphoric acid 19 or amino-thiourea 20 ; other proton sources need to be strictly avoided to ensure high asymmetric induction. Computational studies by Shaik and coworkers 21 have revealed a similar mechanism for haem protein-catalyzed N-H insertion reactions. Thus, the challenge in achieving high enantioselectivity originates from the difficulty in precisely contr...

We report a biocatalytic platform of engineered cytochrome P411 enzymes (P450s with axial serine ligation) to carry out efficient lactone-carbene insertion into primary and secondary α-amino C-H bonds. Directed evolution of a P450 variant, P411-C10, yielded a lineage of enzyme variants capable of forming chiral lactone derivatives with high catalytic efficiency (up to 4000 TTN) and in a stereo-divergent manner. For carbene insertion into secondary C-H bonds, a single mutation was discovered to invert the two contiguous chiral centers and lead to the opposite enantiomers of the same major diastereomers. This work demonstrates the utility of engineered enzymes for asymmetric catalysis and highlights the remarkable tunability of these genetically-encoded biocatalysts for accessing desired selectivities.

Previous work has demonstrated that variants of a heme protein, Rhodothermus marinus cytochrome c (Rma cyt c), catalyze abiological carbene boron–hydrogen (B–H) bond insertion with high efficiency and selectivity. Here we investigated this carbon–boron bondforming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BORLAC that shows high selectivity and efficiency for B–H insertion of 5- and 6-membered lactone carbenes (up to 24,500 total turnovers and 97.1:2.9 enantiomeric ratio). The enzyme shows low activity with a 7-membered lactone carbene. Computational studies revealed a highly twisted geometry of the 7membered lactone carbene intermediate relative to 5- and 6-membered ones. Directed evolution of cytochrome c together with computational characterization of key iron-carbene intermediates has allowed us to expand the scope of enzymatic carbene B–H insertion to produce new lactone-based organoborons.

<p>Whereas enzymatic asymmetric carbene N–H insertion is a powerful method for preparation of chiral amines in principle, it has suffered from limited enantioselectivity in practice. In this work, we demonstrate that engineered cytochrome P450 enzymes can catalyze this abiological C–N bond-forming reaction with excellent activity and selectivity (up to 32,100 TTN, >99% yield and 98% e.e.) to prepare a series of bioactive <i>α</i>-amino lactones, which have not been accessed previously using a carbene insertion strategy. The enzymes are dual-function catalysts, effecting both carbene transfer and enantioselective proton-transfer catalysis, in a single active site. To gain insight into the mechanism of the enzymatic transformation, especially in the asymmetric protonation step, we performed extensive molecular dynamics simulations and density functional theory (DFT) calculations. Computational studies uncover the important roles of active-site residues that enable high activity and selectivity through interacting with the carbene intermediate and the amine substrate, and directing water molecules for selective proton transfer.<br></p><p></p>