Direct Asymmetric Hydroalkoxylation of Styrenes Through Flavin Brønsted Acid Biocatalysis

Direct addition of free alcohols across alkenes would provide a concise route to chiral ethers, but controlling alkene activation, nucleophile capture, and stereochemistry remains difficult. We evolved an oleate hydratase, an enzyme that naturally adds water across alkenes, to favor alcohol addition instead. The resulting enzyme catalyzes intermolecular hydroalkoxylation of electron-rich styrenes in aqueous solution, reaching up to 80% yield and >99% enantiomeric excess. Evolution reshaped an alcohol-binding pocket around a conserved flavin-centered core, allowing alcohol to compete with water. Mechanistic studies support a redox-neutral cycle in which protein-bound neutral FADH 2 acts as a Brønsted acid to protonate the alkene. By placing direct asymmetric hydroalkoxylation within an engineerable enzyme scaffold, this work opens a route to expand both alkene and nucleophile scope through protein engineering.