Non-canonical amino acids (ncAAs) are required for a range of applications in drug discovery, but these compounds are challenging targets for chemical synthesis, often requiring synthesis via long and inefficient routes. Biocatalysis can offer uniquely direct and selective methods to access ncAAs, either by using enzymes that naturally perform amino acid synthesis or by designing alternative enzymatic strategies. We have explored enzymatic methods for the synthesis of tryptophan analogs as well as hydroxylated amino acids. Using engineered tryptophan synthases, a range of unnatural tryptophan derivatives can be accessed in a single step from the condensation of an indole with serine. To create amino acids bearing functionality for cross-linking, we evolved a tryptophan synthase to act on N-allyl indoles, providing allyl-containing tryptophans that can be used to link peptides via olefin metathesis. To access hydroxyprolines, another desirable class of amino acids, we developed a ketoreductase-catalyzed dynamic kinetic resolution. This reaction delivers all four isomers of 3-hydroxyproline with high diastereo- and enantioselectivities, and can also be used to access 3-hydroxypipecolic acids. We have additionally explored the use of alpha-ketoglutarate- and Fe(II)-dependent oxidases for the C-H hydroxylation of amino acids. In particular, we evolved a proline hydroxylase for the highly selective and efficient preparation of a desired hydroxyproline isomer. These biocatalytic approaches to ncAAs are more efficient and greener than alternative chemical routes, and they have been enabling for the large-scale synthesis of clinical candidates as well as the early-stage evaluation of novel chemical space.

Evolution of new enzymes for the synthesis of non-canonical amino acids

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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