Engineering leghemoglobin for highly enantioselective trans -(1 R ,2 R )-selective cyclopropanation of styrenes

Leghemoglobin (LegH), a plant-derived heme protein, remains largely underexplored in terms of its biocatalytic potential. This study reports the first instance of chiral catalysis using LegH in the asymmetric cyclopropanation of alkenes, successfully establishing it as a new biocatalyst. Wild-type LegH (WT-LegH) can catalyze the cyclopropanation of styrene with ethyl diazoacetate, but with low efficiency (20% yield) and weak enantioselectivity (3% ee), with a slight preference for the trans-(1R,2R)-configured product. Through semi-rational design and single-site saturation mutagenesis, the mutant LegHH62 T was obtained, which exhibited significantly improved catalytic performance (90% yield, 90% ee). After systematic optimization of reaction conditions, this mutant enables the achievement of a yield up to 91% with 93% ee and excellent diastereoselectivity, using a catalyst loading as low as 0.02 mol%. Substrate expansion studies revealed that LegHH62 T exhibited good catalytic activity and high R-enantioselectivity for various styrene derivatives. This research not only introduces an efficient new biocatalytic tool for the green synthesis of chiral cyclopropanes but also, due to its catalytic preference for the trans-(1R,2R) configuration, complements the trans-(1S,2S)-selective system previously developed by our group based on Vitreoscilla hemoglobin (VHb), thereby expanding the application potential of LegH in asymmetric synthesis.