Proline-Free Local Turn via N-Oxidation: Crystallographic and Solution Evidence for a Six-Membered N–O⋯H–N Ring

N-oxides are emerging as versatile tools for modulating peptide conformation due to their strong proton-accepting ability and distinct electronic properties. In this study, we report the first crystallographic evidence that an N-oxidized peptide (NOP <b>5</b>) containing a proline residue forms an intramolecular six-membered hydrogen bond between the N-oxide oxygen and an adjacent amide proton. This conformational motif is not restricted to proline-containing sequences: NMR spectroscopic analyses (including DMSO-d₆ titration, VT-NMR, NOE, and concentration-dependent studies) reveal that NOPs <b>7</b> and <b>9</b>, in which proline is replaced by glycine, adopt the same hydrogen-bonded ring structure in aprotic solvents. Remarkably, this conformation persists even in protic solvent (CD₃OH), indicating the robustness of the N-oxide-induced hydrogen bond. DFT calculations further support the experimental findings and rationalize the conformational preferences of NOPs <b>5</b> and <b>7</b>. These results establish N-oxide as a potent and generalizable constraint for stabilizing peptide secondary structures, offering a new strategy for the design of peptidomimetics with tunable rigidity and solvent stability.