Bioconjugate Platform for Iterative Backbone N -Methylation of Peptides.

N -methylation of peptide backbones has often been utilized as a strategy towards the development of peptidic drugs. However, difficulties in the chemical synthesis, high cost of enantiopure N -methyl building blocks, and subsequent coupling inefficiencies have hampered larger-scale medicinal chemical efforts. Here, we present a chemoenzymatic strategy for backbone N -methylation by bioconjugation of peptides of interest to the catalytic scaffold of a borosin-type methyltransferase. Crystal structures of a substrate tolerant enzyme from Mycena rosella guided the design of a decoupled catalytic scaffold that can be linked via a heterobifunctional crosslinker to any peptide substrate of choice. Peptides linked to the scaffold, including those with non-proteinogenic residues, show robust backbone N -methylation. Various crosslinking strategies were tested to facilitate substrate disassembly, which enabled a reversible bioconjugation approach that efficiently released modified peptide. Our results provide general framework for the backbone N -methylation on any peptide of interest and may facilitate the production of large libraries of N -methylated peptides.