Backbone N-Heteroatom Substitution as a Strategy to Enhance Peptide Proteolytic Stability

Abstract

Peptide-based ligands are well-suited to engage large biomolecular surfaces but are often limited by rapid proteolytic degradation in biological environments. Backbone modification offers a direct means to disrupt protease recognition while preserving side chain functionality; however, many established approaches impose conformational constraints that compromise biological activity. Here, we evaluate backbone N-amino and N-hydroxy substitution as a strategy for enhancing peptide proteolytic stability. Using a defined chymotrypsin substrate, we demonstrate that backbone N-amination confers pronounced, position-dependent protection when introduced at or adjacent to the scissile bond. Extending these findings to a β-sheet-forming antimicrobial peptide, we show that poly-N-amination dramatically enhances serum stability while preserving or enhancing conformation-dependent antibacterial activity. Together, these results expand the repertoire of peptide backbone modifications that mitigate proteolytic degradation while retaining the conformational and functional features required for the design of peptide- and protein-based biological probes.