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.