Repurposing TPP-dependent enzymes with anodic oxidation for the conversion of aldehydes to enantioenriched carboxylic acids.

Thiamine diphosphate (ThDP)-dependent enzymes are highly promiscuous and robust biocatalysts in nature, orchestrating precise C-C bond formation and cleavage reactions. Combining organophotoredox catalysis with protein engineering, our group has repurposed ThDP-dependent enzymes for stereoselective dual photo-/enzymatic radical transformations. Inspired by the carboxylic acid byproducts in photo/thiamine biocatalytic systems, we postulated that the input of external electricity could offer an alternative method to expand the catalytic repertoire of ThDP-dependent enzymes. Recently, we integrated ThDP-dependent enzymes with ferrocenemethanol-mediated anodic oxidation to achieve a new-to-nature dynamic kinetic oxidation of α-branched aldehydes to enantioenriched carboxylic acids. More recently, we extended this electroenzymatic platform to enable the desymmetrization of dialdehydes, affording products bearing axial chirality and silicon chirality. For dynamic kinetic oxidation, we propose a mechanism that the active site of ThDP-dependent enzymes specifically recognizes one enantiomer of the aldehyde, leading to the formation of a configuration-defined Breslow intermediate. This intermediate then undergoes two-step single-electron oxidation by oxidized ferrocene methanol to generate an acyl azolium cation. Subsequent nucleophilic attack by water yields the corresponding enantioenriched carboxylic acids. In this chapter, we present a comprehensive protocol for these electrobiocatalytic reactions. The workflow encompasses structure-guided site-saturation mutagenesis, protein expression and purification, reaction screening, enantioselectivity analysis and operational details of the electroenzymatic system. We anticipate that this framework will facilitate future efforts to expand the catalytic repertoire of ThDP-dependent enzymes, particularly for non-natural stereoselective electroenzymatic transformations.