Directed Evolution to Reverse Epoxide Hydrolase Enantioselectivity for meso-3,4-Epoxytetrahydrofuran

Chiral vicinal diols are important intermediates in the synthesis of pharmaceuticals. Epoxide hydrolases catalyze hydrolytic ring opening of epoxides to produce the corresponding vicinal diols, providing an attractive way to access these building blocks under mild conditions in a stereoselective and atom-efficient manner. In this study, an epoxide hydrolase is identified and engineered to form (3S,4S)-tetrahydrofurandiol in high optical purity via the desymmetrization of meso-3,4-epoxytetrahydrofuran. In nine rounds of directed evolution, the enzyme's native (3R,4R)-stereopreference was reversed and its activity was dramatically improved to achieve quantitative yield under remarkably high 500 g/L substrate concentration and low enzyme loading. Computational modelling provides insights on the changes in enzyme-substrate interaction that result in divergent enantioselectivities afforded by evolved variants.

Automated summary

Chiral vicinal diols are important intermediates in pharmaceutical synthesis. Epoxide hydrolases can catalyze the hydrolytic ring opening of epoxides, providing an attractive and efficient method to access these building blocks. In this study, an engineered epoxide hydrolase was able to produce (3S,4S)-tetrahydrofurandiol with high optical purity. Through directed evolution, the enzyme's stereopreference was reversed and its activity was significantly improved. Computational modelling provided insights into the changes in enzyme-substrate interaction resulting in divergent enantioselectivities.