Mutating a Highly Conserved Residue in Diverse Cytochrome P450s Facilitates Diastereoselective Olefin Cyclopropanation

Cytochrome P450s and other heme‐containing proteins have recently been shown to have promiscuous activity for the cyclopropanation of olefins using diazoacetate reagents. Despite the progress made thus far, engineering selective catalysts for all possible stereoisomers for the cyclopropanation reaction remains a considerable challenge. Previous investigations of a model P450 (P450BM3) revealed that mutation of a conserved active site threonine (Thr268) to alanine transformed the enzyme into a highly active and selective cyclopropanation catalyst. By incorporating this mutation into a diverse panel of P450 scaffolds, we were able to quickly identify enantioselective catalysts for all possible diastereomers in the model reaction of styrene with ethyl diazoacetate. Some alanine variants exhibited selectivities that were markedly different from the wild‐type enzyme, with a few possessing moderate to high diastereoselectivity and enantioselectivities up to 97 % for synthetically challenging cis‐cyclopropane diastereomers. Enzymatic cyclopropanation: Mutation of a highly conserved active site residue in a small panel of diverse cytochrome P450s allows for the rapid identification of trans‐ and cis‐selective enzymes for the carbenoid‐mediated cyclopropanation of styrenes.