Applying Promiscuous RiPP Enzymes to Peptide Backbone -Methylation Chemistry.

The methylation of peptide backbone amides is a hallmark of bioactive natural products, and it also greatly modifies the pharmacology of synthetic peptides. Usually, bioactive -methylated peptides are cyclic. However, there is very limited knowledge about how post-translational enzymes can be applied to the synthesis of designed -methylated peptides or peptide libraries. Here, driven by the established ability of some RiPP enzymes to process diverse substrates, we sought to define catalysts for the and macrocyclization of backbone-methylated peptides. We developed efficient methods in which short, synthetic -methylated peptides could be modified using side chain and mainchain macrocyclases, PsnB and PCY1 from plesiocin and orbitide biosynthetic pathways, respectively. Most significantly, a strategy for PsnB cyclase was designed enabling simple in vitro methods compatible with solid-phase peptide synthesis. We show that cyanobactin N-terminal protease PatA is a broadly useful catalyst that is also compatible with -methylation chemistry, but that cyanobactin macrocyclase PatG is strongly biased against -methylated substrates. Finally, we sought to marry these macrocyclase tools with an enzyme that -methylates its core peptide: OphMA from the omphalotin pathway. However, instead, we reveal some limitations of OphMA and demonstrate that it unexpectedly and extensively modified the enzyme itself . Together, these results demonstrate proof-of-concept for enzymatic synthesis of -methylated peptide macrocycles.