Search for: All records

Engineered nonheme iron enzymes perform enantioselective radical azidation on aryl N -fluoroamide substrates. 

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. 

Propargyl amines are versatile synthetic intermediates with numerous applications in the pharmaceutical industry. An attractive strategy for efficient preparation of these compounds is nitrene propargylic C(sp3)−H insertion. However, achieving this reaction with good chemo-, regio-, and enantioselective control has proven to be challenging. Here, we report an enzymatic platform for the enantioselective propargylic amination of alkynes using a hydroxylamine derivative as the nitrene precursor. Cytochrome P450 variant PA-G8 catalyzing this transformation was identified after eight rounds of directed evolution. A variety of 1-aryl-2-alkyl alkynes are accepted by PA G8, including those bearing heteroaromatic rings. This biocatalytic process is efficient and selective (up to 2610 total turnover number (TTN) and 96% ee) and can be performed on preparative scale. 

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).

 

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).

 

Trifluoromethyl‐substituted cyclopropanes (CF₃‐CPAs) constitute an important class of compounds for drug discovery. While several methods have been developed for synthesis oftrans‐CF₃‐CPAs, stereoselective production of correspondingcis‐diastereomers remains a formidable challenge. We report a biocatalyst for diastereo‐ and enantio‐selective synthesis ofcis‐CF₃‐CPAs with activity on a variety of alkenes. We found that an engineered protoglobin fromAeropyrnum pernix(ApePgb) can catalyze this unusual reaction at preparative scale with low‐to‐excellent yield (6–55 %) and enantioselectivity (17–99 % ee), depending on the substrate. Computational studies revealed that the steric environment in the active site of the protoglobin forced iron‐carbenoid and substrates to adopt a pro‐cisnear‐attack conformation. This work demonstrates the capability of enzyme catalysts to tackle challenging chemistry problems and provides a powerful means to expand the structural diversity of CF₃‐CPAs for drug discovery.