Chemoenzymatic Late-Stage Modifications Enable Downstream Click-Mediated Fluorescent Tagging of Peptides

Aromatic prenyltransferases from cyanobactin biosynthetic pathways catalyse the chemoselective and regioselective intramolecular transfer of prenyl/geranyl groups from isoprene donors to an electron-rich position in these macrocyclic and linear peptides. These enzymes often demonstrate relaxed substrate specificity and are considered useful biocatalysts for structural diversification of peptides. Here, we assess the isoprene donor specificity of the N1-tryptophan prenyltransferase AcyF from anacyclamide A8P pathway, using a library of 22 synthetic alkyl-pyrophosphate analogues, of which, many display reactive groups that are amenable to additional functionalisation. We further used AcyF to introduce a reactive moiety in a tryptophan-containing cyclic peptide and subsequently used click chemistry to fluorescently label the enzymatically modified peptide. This chemoenzymatic strategy allows late-stage modification of peptides and is highly useful for many applications.