Engineered Biocatalytic Synthesis of β‐N‐Substituted‐α‐Amino Acids

Abstract

Non‐canonical amino acids (ncAAs) are useful synthons for the development of new medicines, materials, and probes for bioactivity. Recently, enzyme engineering has been leveraged to produce a suite of highly active enzymes for synthesis of β‐substituted amino acids. However, there are few examples of biocatalytic N‐substitution reactions to make α,β‐diamino acids. Here, we use directed evolution to engineer the β‐subunit of tryptophan synthase, TrpB, for improved activity with diverse amine nucleophiles. Mechanistic analysis shows that high yields are hindered by product re‐entry into the catalytic cycle and subsequent decomposition. Additional equivalents of L‐serine can inhibit product reentry through kinetic competition, facilitating preparative scale synthesis. We show β‐substitution with a dozen aryl amine nucleophiles, including demonstration on a g‐scale. These transformations yield an underexplored class of amino acids that can serve as unique building blocks for chemical biology and medicinal chemistry.

Enzymatic Synthesis of Unprotected α,β-Diamino Acids via Direct Asymmetric Mannich Reactions

“…We have illustrated the synthetic utility of this LolT-based intermolecular Mannichase for gram-scale synthesis of L -tambroline. We expect this new Cα-C bond forming enzymatic platform will complement current Cβ-N bond forming PLP-dependent enzymes, which synthesize α,β-diamino acids through β-replacement reaction. , We also provided structural understanding for the engineered LolT variants, highlighting an unusual role of tryptophan-mediated π–π interaction in controlling stereoselectivity. These mechanistic insights gained here would inform future protein design and engineering work for related enzymatic transformations.…”