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

Covalent conjugates of peptides containing aromatic amino acid residues Phe, Tyr, 3-nitro-Tyr, Trp, and 5-hydroxy-Trp with diaryltetrazole groups carrying 4-nitro and 4-methoxy substituents and linked to the peptide lysine were synthesized and used to study photodissociation of their gas-phase ions at 213 and 250-290 nm (UVPD). UVPD resulted in a competitive loss of N₂ from the tetrazole ring and peptide backbone cleavage that was wavelength dependent, showing >90% specificity of tetrazole dissociation at 250-280 nm. This wavelength region corresponded to π-π* excitations within the diaryltetrazole moiety, as established by time-dependent density functional theory calculations. The intermediates from the N₂ loss were analyzed by collision-induced dissociation (CID-MS³) to reveal high (74-99%) yields of cross-linked macrocyclic products that were distinguished by internal residue losses. The 4-methoxyphenyltetrazole conjugate was exceptional in that it underwent facile elimination of 4-methoxyaniline. High-resolution cyclic ion mobility measurements were employed that pointed out near conformational homogeneity of the conjugate ions. The measured collision cross sections (CCS_exp) were matched, typically within 1%, by theoretical CCS_calc that were obtained by combined Born-Oppenheimer molecular dynamics, density functional theory, and ion trajectory calculations. Ion mobility measurements of the N₂-loss intermediates allowed us to separate mixtures of several products that in each case were dominated by a single component that was identified as a cyclic isomer. The calculated fully optimized ion structures showed no π-π stacking of the amino acid aromatic rings and the diaryltetrazole moieties for most conjugate combinations. The 3-nitrotyrosine-diaryltetrazole conjugate was an exception in that it preferred stacked structures in low-energy conformers. The electronic properties of the conjugates in the ground and multiple excited electronic states were addressed by time-dependent density functional theory calculations that provided vibronic absorption spectra at 300 K. Electron transitions that were near resonant with the laser excitation lines occurred within the diaryltetrazole system (as in the Phe conjugate) or comprised electron transfer with the aromatic amino acid residue for the 3-nitro-Tyr, and Trp residues. The Trp-diaryltetrazole interactions occurred dynamically as a result of the conformational motion in thermal ions. The electron-transfer excitation in the 3-nitro-Tyr and Trp side-chain groups was associated with internal energy distribution throughout the peptide chains, driving backbone dissociations while lowering the yields of tetrazole UVPD.