Photobiocatalytic Diastereo- and Enantioselective Synthesis of Noncanonical Amino Acids Bearing Challenging Stereochemical Dyads and Triads

Abstract Catalyst-controlled stereodivergent synthesis of acyclic molecules bearing multiple stereogenic centers remains a longstanding challenge in asymmetric catalysis, particularly through intermolecular radical C–C coupling. Here, we report the directed evolution of pyridoxal 5′-phosphate (PLP)-dependent biocatalysts for the stereodivergent synthesis of noncanonical amino acids bearing either skipped α,γ-stereochemical dyads or contiguous α,β,γ-stereochemical triads. Starting from a thermophilic tryptophan synthase β-subunit, protein engineering generated complementary enzyme variants that selectively construct three of the four possible α,γ-stereoisomers through biocatalyst-controlled reversal of both remote γ-stereochemistry and proximal α-stereochemistry. These engineered enzymes catalyzed intermolecular radical C–C bond formation with a broad range of secondary alkyltrifluoroborates, affording α,γ-disubstituted amino acids in excellent yields and stereoselectivities. Furthermore, directed evolution enabled efficient β-dehydroxylative radical coupling of the non-native threonine-derived aminoacrylate intermediate, providing amino acids bearing contiguous α,β,γ-stereochemical triads with up to 96:4:<0.1:<0.1 d.r. and >99:1 e.r. To further demonstrate the synthetic utility of this platform, the stereochemically enriched amino acids were transformed into valuable γ-chiral amines through engineered PLP-dependent tryptophan decarboxylases, establishing a dual PLP enzyme-based biocatalytic cascade for the enantiodivergent synthesis of privileged amine scaffolds. Molecular dynamics simulations reveal that enzyme-controlled γ-stereoselectivity originates from active-site remodeling that modulates π–π interactions and steric contacts governing the facial selectivity of the radical addition step. This work expands the synthetic capabilities of radical biocatalysis by enabling catalyst-controlled stereodivergent assembly of complex acyclic stereochemical arrays and establishes engineered PLP enzymes as versatile catalysts for the synthesis of stereochemically complex amino acids and chiral amines.