Engineered Cytochrome c -Catalyzed Lactone-Carbene B-H Insertion

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 bond-forming chemistry with cyclic, lactone-based carbenes. Using directed evolution, we obtained a Rma cyt c variant BOR (LAC) 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 7-membered 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.