Directed Evolution of Threonine Aldolases as Radical C–C Bond Forming Enzymes for Photobiocatalytic Synthesis of β-Branched-α-Tetrasubstituted Non-Canonical Amino Acids

The convergent, stereoselective, and protecting-group-free synthesis of non-canonical amino acids, particularly those bearing a tetrasubstituted α-stereogenic center, remains a challenging task. By leveraging pyridoxal radical biocatalysis, we developed photobiocatalytic oxidative coupling methods for the assembly of α-tetrasubstituted amino acids, including β-branched variants, in a diastereo- and enantioselective fashion. Through repurposing and directed evolution of Thermotoga maritima threonine aldolases, we transformed a traditionally two-electron pyridoxal phosphate (PLP) dependent enzyme into a highly active and stereoselective biocatalyst for single-electron C–C bond formation, enabling enantioconvergent conversion of a broad range of racemic organoboron substrates. Our evolved radical C–C bond forming PLP enzymes achieved a total turnover number (TTN) of 3,100 under dual photobiocatalytic conditions, the highest TTN reported to date for an unnatural photobiocatalytic transformation. Mechanistic studies using radical clock probes and electron paramagnetic resonance (EPR) spectroscopy uncovered an unexpected role of free PLP in oxidative radical generation under photochemical conditions. Molecular dynamics (MD) simulations further elucidated the origin of enantio- and diastereoselectivity in the key radical addition step between the enzymatic quinonoid and the carbon-centered radical. Collectively, these results underscore the power of pyridoxal radical biocatalysis to access a broad spectrum of valuable non-canonical amino acid products via intermolecular, enzyme-controlled asymmetric radical chemistry, a transformation that remains elusive to both state-of-the-art small-molecule catalysis and native enzymology.