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Uridine (Urd), a canonical nucleoside of RNA, is the most commonly modified nucleoside among those that occur naturally. Uridine has also been an important target for the development of modified nucleoside analogues for pharmaceutical applications. In this work, the effects of 5-halogenation of uracil on the structures and glycosidic bond stabilities of protonated uridine nucleoside analogues are examined using tandem mass spectrometry and computational methods. Infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and theoretical calculations are performed to probe the structural influences of these modifications. Energy-resolved collision-induced dissociation experiments along with survival yield analyses are performed to probe glycosidic bond stability. The measured IRMPD spectra are compared to linear IR spectra predicted for the stable low-energy conformations of these species computed at the B3LYP/6-311+G(d,p) level of theory to determine the conformations experimentally populated. Spectral signatures in the IR fingerprint and hydrogen-stretching regions allow the 2,4-dihydroxy protonated tautomers (T) and O4- and O2-protonated conformers to be readily differentiated. Comparisons between the measured and predicted spectra indicate that parallel to findings for uridine, both T and O4-protonated conformers of the 5-halouridine nucleoside analogues are populated, whereas O2-protonated conformers are not. Variations in yields of the spectral signatures characteristic of the T and O4-protonated conformers indicate that the extent of protonation-induced tautomerization is suppressed as the size of the halogen substituent increases. Trends in the energy-dependence of the survival yield curves find that 5-halogenation strengthens the glycosidic bond and that the enhancement in stability increases with the size of the halogen substituent.

The 2'-substituents distinguish DNA from RNA nucleosides. 2'-O-methylation occurs naturally in RNA and plays important roles in biological processes. Such 2'-modifications may alter the hydrogen-bonding interactions of the nucleoside and thus may affect the conformations of the nucleoside in an RNA chain. Structures of the protonated 2'-O-methylated pyrimidine nucleosides were examined by infrared multiple photon dissociation (IRMPD) action spectroscopy, assisted by electronic structure calculations. The glycosidic bond stabilities of the protonated 2'-O-methylated pyrimidine nucleosides, [Nuom+H] + , were also examined and compared to their DNA and RNA nucleoside analogues via energy-resolved collision-induced dissociation (ER-CID). The preferred sites of protonation of the 2'-O-methylated pyrimidine nucleosides parallel their canonical DNA and RNA nucleoside analogues, [dNuo+H] + and [Nuo+H] + , yet their nucleobase orientation and sugar puckering differ. The glycosidic bond stabilities of the protonated pyrimidine nucleosides follow the order: [dNuo+H] + < [Nuo+H] + < [Nuom+H] + . The slightly altered structures help explain the stabilization induced by 2'-O-methylation of the pyrimidine nucleosides.

Gas-phase conformations of the sodium-cationized forms of the 2'-deoxycytidine and cytidine mononucleotides, [pdCyd+Na] + and [pCyd+Na] + , are examined by infrared multiple photon dissociation action spectroscopy. Complimentary electronic structure calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory provide candidate conformations and their respective predicted IR spectra for comparison across the IR fingerprint and hydrogen-stretching regions. Comparisons of the predicted IR spectra and the measured infrared multiple photon dissociation action spectra provide insight into the impact of sodium cationization on intrinsic mononucleotide structure. Further, comparison of present results with those reported for the sodium-cationized cytidine nucleoside analogues elucidates the impact of the phosphate moiety on gas-phase structure. Across the neutral, protonated, and sodium-cationized cytidine mononucleotides, a preference for stabilization of the phosphate moiety and nucleobase orientation is observed, although the details of this stabilization differ with the state of cationization. Several low-energy conformations of [pdCyd+Na] + and [pCyd+Na] + involving several different orientations of the phosphate moiety and sugar puckering modes are observed experimentally.

The 2'-substituent is the primary distinguishing feature between DNA and RNA nucleosides. Modifications to this critical position, both naturally occurring and synthetic, can produce biologically valuable nucleoside analogues. The unique properties of fluorine make it particularly interesting and medically useful as a synthetic nucleoside modification. In this work, the effects of 2'-fluoro modification on the protonated gas-phase purine nucleosides are examined using complementary tandem mass spectrometry and computational methods. Direct comparisons are made with previous studies on related nucleosides. Infrared multiple photon dissociation action spectroscopy performed in both the fingerprint and hydrogen-stretching regions allows for the determination of the experimentally populated conformations. The populated conformers of protonated 2'-fluoro-2'-deoxyadenosine, [Adofl+H] + , and 2'-fluoro-2'-deoxyguanosine, [Guofl+H] + , are highly parallel to their respective canonical DNA and RNA counterparts. Both N3 and N1 protonation sites are accessed by [Adofl+H] + , stabilizing syn and anti nucleobase orientations, respectively. N7 protonation and anti nucleobase orientation dominates in [Guofl+H] + . Spectroscopically observable intramolecular hydrogen-bonding interactions with fluorine allow more definitive sugar puckering determinations than possible for the canonical systems. [Adofl+H] + adopts C2'-endo sugar puckering, whereas [Guofl+H] + adopts both C2'-endo and C3'-endo sugar puckering. Energy-resolved collision-induced dissociation experiments with survival yield analyses provide relative glycosidic bond stabilities. The N-glycosidic bond stabilities of the protonated 2'-fluoro-substituted purine nucleosides are found to exceed those of their canonical analogues. Further, the N-glycosidic bond stability is found to increase with increasing electronegativity of the 2'-substituent, i.e., H < OH < F. The N-glycosidic bond stability is also greater for the adenine nucleoside analogues than the guanine nucleoside analogues.

Modified nucleosides have been an important target for pharmaceutical development for the treatment of cancer, herpes simplex virus, and the human immunodeficiency virus (HIV). Amongst these nucleoside analogues, those based on 2',3'-dideoxyribose sugars are quite common, particularly in anti-HIV applications. The gas-phase structures of several protonated 2',3'-dideoxyribose nucleosides are examined in this work and compared with those of the analogous protonated DNA, RNA, and arabinose nucleosides to elucidate the influence of the 2'- and combined 2',3'-hydroxyl groups on intrinsic structure. Infrared multiple photon dissociation (IRMPD) action spectra are collected for the protonated 2',3'-dideoxy forms of adenosine, guanosine, cytidine, thymidine and uridine, [ddAdo+H] + , [ddGuo+H] + , [ddCyd+H] + , [ddThd+H] + , and [ddUrd+H] + , in the IR fingerprint and hydrogen-stretching regions. Molecular mechanics conformational searching followed by electronic structure calculations generates low-energy conformers of the protonated 2',3'-dideoxynucleosides and corresponding predicted linear IR spectra to facilitate interpretation of the measured IRMPD action spectra. These experimental IRMPD spectra and theoretical calculations indicate that the absence of the 2'- and 3'-hydroxyls largely preserves the protonation preferences of the canonical forms. The spectra and calculated structures indicate a slight preference for C3'-endo sugar puckering. The presence of the 3'- and further 2'-hydroxyl increases the available intramolecular hydrogen-bonding opportunities and shifts the sugar puckering modes for all nucleosides but the guanosine analogues to a slight preference for C2'-endo over C3'-endo. Graphical Abstract.

Modifications to a Paul-type quadrupole ion trap mass spectrometer providing optical access to the trapped ion cloud as well as hardware and software for coupling to a table-top IR optical parametric oscillator laser (OPO) are detailed. Critical experimental parameters for infrared multiple photon dissociation (IRMPD) on this instrument are characterized. IRMPD action spectra, collected in the hydrogen-stretching region with this instrument, complemented by spectra in the IR fingerprint region acquired at the FELIX facility, are employed to characterize the structures of the protonated forms of 2-thiouridine, [s 2 Urd+H] + , and 4-thiouridine, [s 4 Urd+H] + . The measured spectra are compared with predicted linear IR spectra calculated at the B3LYP/6-311+G(d,p) level of theory to determine the conformers populated in the experiments. This comparison indicates that thiation at the 2- or 4-positions shifts the protonation preference between the 2,4-H tautomer and 4-protonation in opposite directions versus canonical uridine, which displays a roughly equal preference for the 2,4-H tautomer and O4 protonation. As found for canonical uridine, protonation leads to a mixture of conformers exhibiting C2'-endo and C3'-endo sugar puckering with an anti nucleobase orientation being populated for both 2- and 4-thiated uridine. Graphical Abstract ᅟ.

The gas-phase conformations of the protonated forms of the DNA and RNA cytosine mononucleotides, [pdCyd+H] + and [pCyd+H] + , are examined by infrared multiple photon dissociation (IRMPD) action spectroscopy over the IR fingerprint and hydrogen-stretching regions complemented by electronic structure calculations. The low-energy conformations of [pdCyd+H] + and [pCyd+H] + and their relative stabilities are computed at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) and MP2(full)/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) levels of theory. Comparisons of the measured IRMPD action spectra and B3LYP/6-311+G(d,p) linear IR spectra computed for the low-energy conformers allow the conformers present in the experiments to be determined. Similar to that found in previous IRMPD action spectroscopy studies of the protonated forms of the cytosine nucleosides, [dCyd+H] + and [Cyd+H] + , both N3 and O2 protonated cytosine mononucleotides exhibiting an anti orientation of cytosine are found to coexist in the experimental population. The 2'-hydroxyl substituent does not significantly influence the most stable conformations of [pCyd+H] + versus those of [pdCyd+H] + , as the IRMPD spectral profiles of [pdCyd+H] + and [pCyd+H] + are similar. However, the presence of the 2'-hydroxyl substituent does influence the relative intensities of the measured IRMPD bands. Comparisons to IRMPD spectroscopy studies of the deprotonated forms of the cytosine mononucleotides, [pdCyd-H] - and [pCyd-H] - , provide insight into the effects of protonation versus deprotonation on the conformational features of the nucleobase and sugar moieties. Likewise, comparisons to results of IRMPD spectroscopy studies of the protonated cytosine nucleosides provide insight into the influence of the phosphate moiety on structure. Comparison with previous ion mobility results shows the superiority of IRMPD spectroscopy for distinguishing various protonation sites. Graphical Abstract ᅟ.