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.

Selective quantitation of the neurotoxin BMAA by use of hydrophilic-interaction liquid chromatography–differential mobility spectrometry–tandem mass spectrometry (HILIC–DMS–MS/MS)

“…The absence of background in the product ion spectra of BMAA isomer standards collected by ESI-DMS-MS/MS (ESM Fig. S2) can also be attributed to DMS, as reported elsewhere [33]. As shown in Fig.…”

Elucidating collision induced dissociation products and reaction mechanisms of protonated uracil by coupling chemical dynamics simulations with tandem mass spectrometry experiments

“…The diketo form of uracil is reported to be the predominant isomer in condensed phase . In the gas phase, O7 and O8 proton affinities become close in energy, and so we find that tautomerization and PT are more plausible . The relative energies of the different tautomers studied in the present work are summarized in Table , where we also report the corresponding Boltzmann population at 300 K. It is worth noting that the global minimum, isomer 1 , cannot be generated directly from protonation of the diketo form of uracil.…”

“…Finally, small peaks at m / z 43 and 44 are also detected. Globally, the MS/MS spectrum is similar to that of protonated uracil generated in the gas phase by FAB or more recently by ESI …”

“…On the other hand, these authors suggested different mechanisms as responsible for reaction pathways leading to some of the observed fragment ions. For example, Nelson and McCloskey proposed that water loss from pyrimidine bases involves ring opening followed by proton transfer (PT) to an exocyclic oxygen, while Beach and Gabryelski discussed the possibility that this mechanism does not reflect experimental data . They also proposed that the retro Diels–Alder mechanism proposed by Nelson and McCloskey for a cyanic acid loss could not occur in a single step as a charge‐directed process .…”

“…[13] In this context, CID was used to study the gas-phase unimolecular reactivity of protonated uracil (m/z 113). [14][15][16] Those studies unanimously point out that the tandem mass spectrometry (MS/MS) spectrum of protonated uracil presents three main peaks: m/z 96 corresponding to ammonia loss, another peak at m/z 95 associated with elimination of water and finally an intense peak at m/z 70 tagged as cyanic or isocyanic acid loss.…”