Revisiting the reactivity of uracil during collision induced dissociation: tautomerism and charge-directed processes

Abstract

In our recent work towards the nontarget identification of products of nucleic acid (NA) damage in urine, we have found previous work describing the dissociation of NA bases not adequate to fully explain their observed reactivity. Here we revisit the gas-phase chemistry of protonated uracil (U) during collision induced dissociation (CID) using two modern tandem mass spectrometry techniques; quadrupole ion trap (QIT) and quadrupole time of flight (Q-TOF). We present detailed mechanistic proposals that account for all observed products of our experiments and from previous isotope labeling data, and that are supported by previous ion spectroscopy results and theoretical work. The diverse product-ions of U cannot be explained adequately by only considering the lowest energy form of protonated U as a precursor. The tautomers adopted by U during collisional excitation make it possible to relate the complex reactivity observed to reasonable mechanistic proposals and feasible product-ion structures for this small highly conjugated heterocycle. These reactions proceed from four different stable tautomers, which are excited to a specific activated precursor from which dissociation can occur via a charge-directed process through a favorable transition state to give a stabilized product. Understanding the chemistry of uracil at this level will facilitate the identification of new modified uracil derivatives in biological samples based solely on their reactivity during CID. Our integrated approach to describing ion dissociation is widely applicable to other NA bases and similar classes of biomolecules.