Engineering Limonene Epoxide Hydrolases for the Enantiocomplementary Synthesis of Chiral 1,3-Diols and Oxetanes

Chiral 1,3-diols and oxetanes serve as essential building blocks in the synthesis of key pharmaceuticals. In this study, we report the identification and protein engineering of limonene epoxide hydrolases (LEHs) to enable the kinetic resolution of racemic oxetanes, facilitating the enantiocomplementary synthesis of 1,3-diols while simultaneously obtaining optically pure oxetanes. To minimize screening efforts, single-code and triple-code saturation mutagenesis strategies were implemented. The substrate scope was expanded to include 12 examples where both (R)- and (S)-selective ring openings of racemic oxetanes were achieved in up to 99% optical purity for the chiral 1,3-diol products and oxetanes. Structural and computational analyses provided insights into key active-site interactions contributing to stereoselectivity. Furthermore, a bienzymatic one-pot two-step cascade reaction was constructed by pairing variants of halohydrin dehalogenase and LEHs, which efficiently converted readily available racemic haloalcohols into chiral 1,3-diols with high isolated yields (up to 48%) and enantioselectivity (up to 98% ee). The scalability and practical applicability of these strategies were further demonstrated through preparative-scale reactions and subsequent derivatizations, yielding key pharmaceutical intermediates.