Automated Flow Synthesis of Artificial Heme Enzymes for Enantioselective Biocatalysis

The remarkable efficiency with which enzymes catalyze small molecule reactions has driven their widespread application in organic chemistry. Here, we employ automated fast-flow solid-phase synthesis to access full-length enzymes without restrictions on the number and structure of non-canonical amino acids incorporated. We demonstrate the total syntheses of Fe-dependent Bacillus subtilis myoglobin (BsMb) and sperm whale myoglobin (SwMb), which displayed excellent enantioselectivity and yield in carbene transfer reactions. Absolute control over enantioselectivity in styrene cyclopropanation was achieved using L- and D-BsMb mutants which delivered each enantiomer of cyclopropane product in identical and opposite enantiomeric enrichment. BsMb mutants outfitted with non-canonical amino acids were used to facilitate detailed structure-activity relationship studies, revealing a previously unrecognized hydrogen-bonding interaction as the primary driver of enantioselectivity in styrene cyclopropanation.