Cytochrome P450Blt Enables Versatile Peptide Cyclisation to Generate Histidine‐ and Tyrosine‐Containing Crosslinked Tripeptide Building Blocks

We report our investigation of the utility of peptide crosslinking cytochrome P450 enzymes from biarylitide biosynthesis to generate a range of cyclic tripeptides from simple synthons. The crosslinked tripeptides produced by this P450 include both tyrosine‐histidine (A−N−B) and tyrosine‐tryptophan (A−O−B) crosslinked tripeptides, the latter a rare example of a phenolic crosslink to an indole moiety. Tripeptides are easily isolated following proteolytic removal of the leader peptide and can incorporate a wide range of amino acids in the residue inside the crosslinked tripeptide. Given the utility of peptide crosslinks in important natural products and the synthetic challenge that these can represent, P450 enzymes have the potential to play roles as important tools in the generation of high‐value cyclic tripeptides for incorporation in synthesis, which can be yet further diversified using selective chemical techniques through specific handles contained within these tripeptides. Cytochrome P450Blt generates A−N−B and A−O−B crosslinked tripeptides within MRYxH and MRYxW peptides. Removal of the minimal leader peptide allows isolation of these cyclic tripeptides, with sequences further varied by incorporation of a range of residues at the position within the cyclic tripeptide. Biarylitide P450s therefore show potential as biocatalysts for the generation of a diverse range of cyclic tripeptide building blocks.