biostudies-literatureZheng YNational Institute of General Medical SciencesNIGMS NIH HHS14006-14014https://www.ebi.ac.uk/biostudies/studies/S-EPMC9928189biostudies-literatureUnknown12(22)<i>N</i>-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 <i>N</i>-methyl building blocks, and subsequent coupling inefficiencies have hampered larger-scale medicinal chemical efforts. Here, we present a chemoenzymatic strategy for backbone <i>N</i>-methylation by bioconjugation of peptides of interest to the catalytic scaffold of a borosin-type methyltransferase. Crystal structures of a substrate tolerant enzyme from <i>Mycena rosella</i> 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 rACS catalysisBioconjugate Platform for Iterative Backbone <i>N</i>-Methylation of Peptides.PMC9928189R01 GM131347R01 GM079038Zheng YOngpipattanakul CNair SKfalseBioconjugate Platform for Iterative Backbone <i>N</i>-Methylation of Peptides.<i>N</i>-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 <i>N</i>-methyl building blocks, and subsequent coupling inefficiencies have hampered larger-scale medicinal chemical efforts. Here, we present a chemoenzymatic strategy for backbone <i>N</i>-methylation by bioconjugation of peptides of interest to the catalytic scaffold of a borosin-type methyltransferase. Crystal structures of a substrate tolerant enzyme from <i>Mycena rosella</i> 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 r2022-01-01T00:00:00Z2022 Nov2026-05-28T11:29:15.83Z2025-02-19T03:27:05.857ZS-EPMC99281893679344810.1021/acscatal.2c04681