Engineering a Thiamine-Dependent Benzoylformate Decarboxylase for Stereodivergent Radical C(sp3)-C(sp3) Bond Formation.

The stereodivergent construction of C(sp3)-C(sp3) bonds between two prochiral radicals remains a formidable challenge. Here, we report the engineering of a thiamine-diphosphate-dependent radical enzyme to catalyze redox-neutral and stereodivergent cross-coupling of two electrophilic C(sp3) precursors. This new-to-nature enzyme reactivity was achieved via light-accelerated single-electron transfer from an unprecedented enzymatic vinyl Breslow intermediate, generating two distinct prochiral radicals whose stereoselective recombination forges the new C(sp3)-C(sp3) bond. Through systematic protein engineering, we developed four enzyme variants that collectively deliver all four stereoisomers with high efficiency (up to 82% yield, 99% enantiomeric excess (ee) and >20:1 diastereomeric ratio (d.r.)). This work establishes a platform for enzymatic, reductant-free radical cross-coupling with simultaneous control over two C(sp3) stereocenters.