N-methylation of bioactive peptides as a conformational constraint tool to improve enzymatic stability

Bioactive peptides (BPs) are promising therapeutic agents due to their high selectivity and low toxicity. However, their clinical potential is often limited by rapid enzymatic degradation and poor pharmacokinetic profiles. N-methylation of the peptide backbone has emerged as an effective strategy to improve proteolytic stability, membrane permeability, and conformational control by limiting intramolecular hydrogen bonding and restricting structural flexibility. In this chapter, we present a simplified and cost-effective solid-phase peptide synthesis (SPPS) protocol for the preparation of N-methylated peptides and lipopeptides. The method employs Fmoc chemistry, DIC/HOBt coupling, and ChemMatrix Rink Amide resin under manual conditions, without requiring specialized instrumentation. This protocol enables the efficient incorporation of one or more N-methylated residues and is suitable for the development of protease-resistant analogs. It provides a practical tool for researchers aiming to enhance the metabolic stability and pharmacological potential of therapeutic peptide candidates.