Repurposing “Ene”-Reductase to Isomerase for Enantiodivergent Synthesis of Allenoates

The synthesis of chiral allenoates through enzymatic asymmetric isomerization of achiral alkynoates via proton transfer is a highly desirable yet unachieved transformation, primarily due to significant product inhibition encountered with natural isomerases. To circumvent this, we repurposed flavin-dependent "ene"-reductases (EREDs), traditionally oxidoreductases, for redox-neutral stereoselective 1,3-proton transfer. Directed evolution of an ERED from Galdieria sulphuraria (GsOYE) generated a panel of new-to-nature isomerases proficient in isomerizing both 3-butynoates and 2-butynoates, affording diverse chiral allenoates with excellent enantiodivergence and negligible product inhibition. The resulting allenoates were leveraged in chirality transfer [2 + 2], [3 + 2], and [4 + 2] cycloadditions to create complex polycycles with up to three stereogenic centers. Mechanistic studies revealed that directed evolution yielded GsOYE mutants capable of operating via either one- or two-base mechanisms, a functional divergence rarely seen with small-molecule catalysts. This study expanded the catalytic repertoire of EREDs, establishing a biocatalytic platform for proton transfer catalysis to set axial chirality.