Nitrile-Imine-Mediated Cross-Linking of Peptides to Oligonucleotides in Gas-Phase Ion Complexes

Gas-phase ion complexes of dinucleotides and trinucleotides with a diaryltetrazole-tagged peptide underwent covalent cross-linking upon UV photodissociation (UVPD) at 213 nm. The cross-linking reaction involved nitrile-imine intermediates produced by the loss of N₂ from the tetrazole, whereby cross-linking between the complex components competed with internal cross-linking within the peptide. The propensity for UVPD-induced cross-linking of DNA nucleobases was established for complexes of dinucleotides dAA, dCC, dGG, and dTT, that gave cross-link yields of 5%, 15%, 40%, and <1%, respectively. Analysis of UVPD-produced nitrile-imine intermediates by collision-induced dissociation (CID-MS³) gave cross-link yields of 71%, 75%, 94%, and 8% for dAA, dCC, dGG, and dTT, respectively. UVPD-CID-MS³ of isomeric trinucleotide-peptide complexes of dCGA, dAGC, dCAG, dACG, and dGCA showed nearly quantitative cross-linking that favored guanine regardless of its position in the sequence. Binding energies for the gas-phase ion complexes were obtained by Born-Oppenheimer molecular dynamics and density functional theory calculations at the M06-2X/def2qzvpp level. These calculations showed similar binding energies for the dAA, dCC, and dCAG complexes that were in the 195-221 kJ mol^-1 range, whereas binding to dGG and dTT was weaker. A mechanism for the novel cross-linking reaction between the nitrile imine and guanine was elucidated with a riboguanosine conjugate that was tagged with a diaryltetrazole group at 5'-O. Product analysis as well as the calculated structures and energies suggested that cross-links resulted from an attack on the aromatic ring of an imine intermediate by the guanine carbonyl oxygen, followed by proton migrations.