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(sp3)–C(sp3) 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. Competing Interest Statement Y.Y., L.C. and Z.B. has filed a provisional patent on the compositions, systems and methods for biocatalytic amino acid synthesis based on the results described in this manuscript. Other authors have no competing interest to declare.