Nitrile-Imine Crosslinking in Peptide Ions with Aromatic Amino Acid Residues: Are Ring-Stacking Interactions Important?

Abstract

In this study, a series of diaryltetrazole–peptide conjugates containing aromatic amino acid residues were studied by ultraviolet photodissociation (UVPD) mass spectrometry. Upon irradiation at 213 nm and in the 250–290 nm range, the tetrazole unit underwent selective loss of molecular nitrogen (N2) to generate reactive nitrile-imine intermediates. These intermediates subsequently participated in intramolecular cross-linking reactions, primarily involving peptide backbone amide groups, leading to macrocyclic product ions. Product structures and dissociation pathways were proposed based on tandem mass spectrometry experiments, including UVPD-MS2 and collision-induced dissociation (CID-MS3). High-resolution ion mobility measurements were performed to determine experimental collision cross sections, providing structural information for both precursor and denitrogenated ions. Theoretical calculations were performed to support structural assignments and mechanistic interpretation. Conformational searches were conducted using Born–Oppenheimer molecular dynamics (BOMD), and density functional theory (DFT) computations were used to optimize geometries and evaluate relative energies of low-energy isomers. Time-dependent DFT calculations were further used to analyze electronic transitions and rationalize wavelength-dependent photodissociation behavior. Based on combined experimental and computational results, the mechanistic understanding of nitrile-imine cross-linking was refined. The results indicate that ground-state aromatic stacking interactions are not universally required for cross-link formation, while differences in excited-state electronic structures are more likely responsible for the observed wavelength-dependent photochemical behavior.