Repurposing thiamine-dependent enzymes for radical biocatalysis.

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitous and versatile biocatalysts in living systems, catalyzing diverse C-C bond formation or cleavage reactions. Inspired by ThDP-dependent enzymes, chemists have developed biomimetic N-heterocyclic carbenes (NHCs) for organocatalysis, ligand design, as well as material synthesis. Inspired by the recent development in chemo-NHC-enabled radical catalysis, and based on the structural plasticity of ThDP enzymes-conserved cofactor-binding motifs coupled with highly evolvable active sites, our group has repurposed ThDP-dependent enzymes into efficient and stereoselective radical acyl transferases (RATs), three-component radical enzymes (3CREs), and C(sp3)-H bond radical acyl transferases (RATCH). Mechanistically, synergistic dual photo-/enzyme catalysis enabled the generation of an enzyme-bound ketyl radical and a prochiral carbon-centered radical. These two radicals then undergo stereocontrolled radical-radical cross-couplings within the active site, thus yielding a series of enantioenriched chiral ketones. This chapter outlines a detailed protocol for these photobiocatalytic reactions with engineered benzaldehyde lyases (PfBAL), catalogued by structure-guided semi-rational mutagenesis, protein expression and purification, photobiocatalytic reaction screening, and enantioselectivity determination. We hope this protocol can guide further work in expanding the catalytic repertoire of ThDP-dependent enzymes, particularly towards non-natural stereoselective radical transformations.