Engaging Unstabilized Alkyl Radicals with Pyridoxal Radical Biocatalysis: Enantiodivergent Synthesis of Aliphatic Non-Canonical Amino Acids

ABSTRACT Harnessing transient, unstabilized alkyl radical intermediates for the enantioselective construction of valueadded chemical entities remains a fundamental challenge in biocatalysis. Through the repurposing and directed evolution of pyridoxal phosphate (PLP)-dependent tryptophan synthases, we advanced an open-shell enzyme platform capable of intercepting transient alkyl radicals for the efficient and enantioselective synthesis of aliphatic non-canonical amino acids. Engineering an orthogonal pair of radical PLP enzymes allowed unstabilized alkyl radicals, generated from diverse aliphatic organoboronates, to undergo dehydroxylative C(sp 3 )–C(sp 3 ) coupling with a common L-serine donor, affording either L- or D-amino acids with excellent enantiopurity in an enzyme-controlled fashion. Mechanistic and computational investigations employing radical clock substrates and unusual radical-mediated rearrangement processes revealed that the radical intermediates generated in this system exhibit unexpectedly long lifetimes, highlighting the power of this dual enzyme-photocatalyst platform to engage unactivated alkyl radicals. Collectively, these findings delineate a potentially general strategy for generating and utilizing unstabilized alkyl radicals and underscore the synthetic potential of radical pyridoxal biocatalysts for stereodivergent amino acid construction.