N-Substituted Aza-Glycine Promotes C8 CO···HN Hydrogen Bonding: A Novel Motif to Design Constrained Peptidomimetics

Peptidomimetic modifications enhance the rigidity, cell permeability, and proteolytic stability of peptides, presenting significant promise for drug development. One such modification involves the isosteric replacement of the peptide backbone C_αH with a nitrogen (N) atom, resulting in azapeptides. In azapeptides, there are two backbone N atoms within a single residue, with the possibility of substituting either one or both simultaneously. Previous studies have effectively mimicked the side chain substitution patterns in peptides by focusing on substituents on the nitrogen at the C_α position of azapeptides. In this study, we explored the unconventional substitution at the N-terminus nitrogen by using N-substituted aza-glycine (azGly). We investigated peptide-azapeptide dimers featuring N-acetyl-proline at the N-terminus of an N-methyl azGly residue. These hybrid peptides produced turn structures stabilized by an unusual C8 CO···HN hydrogen bond, stabilizing the proline amide in its trans conformation. Computational studies, including Density Functional Theory (DFT), Natural Bond Orbital (NBO), and Atoms In Molecules (AIM) analyses, confirmed the stabilizing effect of this C8 hydrogen bond, which was further validated by NMR and CD spectroscopic studies. As the NH position is occupied by C_αHR in natural peptides and proteins, formation of such C8 hydrogen bond is unlikely. Overall, we provided a novel strategy to introduce non-natural C8 hydrogen bond into peptide backbone via the incorporation of N-substituted azGly, which could be used to design more rigid and stable peptides and peptidomimetics, with potential applications in drug development.