Proton-Driven Click Reactions of Nitrile Imines with Serine and Threonine in Gas-Phase Peptide Ions

Gas-phase ion conjugates containing a 4-(2-phenyl-2<i>H</i>-tetrazol-5-yl)benzoyl group that was tethered to the lysine side chain in peptides SAAAK and TAAAK, denoted here as (SAAA-<i>tet</i>-K + H)⁺ and (TAAA-<i>tet</i>-K + H)⁺, were found to undergo loss of N₂ and cyclization upon UV photodissociation (UVPD) at 213 and 250-300 nm, forming two distinct populations of photofragment ions. Ions of the first type underwent prompt dissociation following loss of N₂ that involved transfer of the N-terminal amide oxygen and two exchangeable hydrogen atoms onto the reactive nitrile-imine intermediate, followed by loss of an N-terminal HN = CH-R fragment, R = CH₂OH and CH(OH)CH₃ for Ser and Thr, respectively. This prompt dissociation was most abundant at 5.82 eV, depleting the population of the ions of the first type, but gradually decreased at lower excitation energies in the 4.96-4.13 eV range. The other types of photofragment ions were represented by stable denitrogenated intermediates that were formed competitively upon UVPD. These were probed by collision-induced dissociation, CID-MSⁿ, showing losses of internal alanine residues and indicating macrocyclic structures. Precursor ion structures were established by matching collision cross sections (CC<i>S</i>_exp), obtained by high-resolution cyclic ion mobility measurements, with theoretical CC<i>S</i>_calc that were obtained by density functional theory (DFT) calculations for low-Gibbs-energy ion conformers. The macrocyclic ring formation was found to involve the serine and threonine hydroxyl groups that attacked the nitrile-imine carbon atom, forming O-linked structures, as corroborated by ion mobility measurements and matching CC<i>S</i>_calc and CC<i>S</i>_exp. The cyclization energetics and kinetics was probed by Rice-Ramsperger-Kassel-Marcus (RRKM) calculations of rate constants and Born-Oppenheimer molecular dynamics at points along the C-O reaction coordinate that stressed the role of hydroxyl proton transfer onto the neighboring N-terminal amine group. Structures were also proposed for the products of UV-induced prompt dissociation for which matching CC<i>S</i>_calc and CC<i>S</i>_exp were obtained. The serine and threonine hydroxyl cyclization to nitrile imines upon UVPD represents a new reaction type in the formation of peptide macrocyclic structures.