Photobiocatalytic enantioselective C(sp³)-H acylation using thiamine-dependent enzymes.

Thiamine diphosphate (ThDP)-dependent enzymes are a versatile class of enzymes traditionally applied in biocatalysis for carbon-carbon bond forming or cleaving reactions via two electron chemistry. Recent advances have revealed their potential to catalyze radical-mediated transformations when combined with chemical hydrogen atom transfer (HAT) strategies, enabling the enantioselective functionalization of C(sp³)-H bonds. This approach allows for the direct acylation of benzylic C(sp³)-H sites across a broad range of organic substrates, affording enantioenriched ketones with good to high levels of enantioselectivity, reaching up to 96 % enantiomeric excess. Mechanistic studies have shown the involvement of radical intermediates derived from both the Breslow intermediate and the C-H substrate, and have highlighted the critical roles of the photocatalyst and hydrogen atom abstraction reagent in enabling efficient catalysis. These findings have expanded the reaction scope of ThDP-dependent enzymes to 'new-to-nature' transformations, opening new avenues for asymmetric synthesis with enzymes. This chapter reviews the expression and purification of the thiamine-dependent enzyme benzaldehyde lyase (PfBAL) as well as the setup and execution of photobiocatalytic C(sp³)-H acylation reactions using this enzyme.