Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes

Abstract

Developing catalysts that produce each stereoisomer of a desired product selectively is a longstanding synthetic challenge. Biochemists have addressed this challenge by screening nature's diversity to discover enzymes that catalyze the formation of complementary stereoisomers. We show here that the same approach can be applied to a new-to-nature enzymatic reaction, alkene cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated "P411" variants of cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. We then enhanced their activities and stereoselectivities by directed evolution: just 1–3 rounds of site-saturation mutagenesis and screening generated enzymes that transform unactivated alkenes and electron-deficient alkenes into each of the four stereoisomeric cyclopropanes with up to 5,400 total turnovers and 98% enantiomeric excess. These fully genetically encoded biocatalysts function in whole Escherichia coli cells in mild, aqueous conditions and provide the first example of enantioselective, intermolecular iron-catalyzed cyclopropanation of unactivated alkenes.

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