Open Access Dissertation
Date of Award
Mary T. Rodgers
TANDEM MASS SPECTROMETRY AND COMPUTATIONAL APPROACHES TO ELUCIDATE CONFORMATIONS AND N-GLYCOSIDIC BOND STABILITIES OF DNA AND RNA NUCLEOSIDES AND MONONUCLEOTIDES
Advisor: Dr. Mary T. Rodgers
Major: Analytical Chemistry
Degree: Doctor of Philosophy
The influence of noncovalent interactions with sodium cations on the conformations and energetics of ten DNA and RNA nucleosides as well as two adenine mononucleotides were elucidated via infrared multiple photon dissociation (IRMPD) action spectroscopy experiments and complementary electronic structure calculations. Energy-resolved collision-induced dissociation (ER-CID) experiments of protonated and metal cationized DNA and RNA nucleosides were performed using a Bruker amaZon ETD quadrupole ion trap mass spectrometer (QIT MS). By comparing the CID50% values of these nucleosides, which are the rf excitation amplitudes required for 50% dissociation of the precursor ion, the effects of local environment and modifications on the relative stabilities of the DNA and RNA nucleosides were elucidated. In particular, when the fragmentation pathways observed in the ER-CID experiments solely involve N-glycosidic bond cleavage, the survival yield analyses are directly correlated to the relative N-glycosidic bond stabilities of the DNA and RNA nucleosides.
For eight of the 10 sodium cationized DNA and RNA nucleosides examined in this thesis, the sodium cation preferentially binds to both the nucleobase and sugar moiety. In contrast, the guanine nucleosides bind the sodium cation solely via the guanine nucleobase. Theory suggests that the DNA and RNA forms of sodium cationized nucleosides exhibit highly parallel conformations. However, comparisons between the experimental IRMPD and calculated IR spectra demonstrate that for the pyrimidine nucleosides, stable low-energy bidentate conformers with an anti orientation of the nucleobase are dominantly populated in the experiments. This conclusion is confirmed by the relative Gibbs free energies calculated for water adducts of the ground tridentate syn oriented conformers and these bidentate anti oriented conformers of sodium cationized RNA pyrimidine nucleosides.
In general, the 2'-hydroxyl substituent of the RNA nucleoside stabilizes the N-glycosidic bond compared with their DNA analogues. Sodium cationization activates the N-glycosidic bond less effectively than protonation. The effects of methylation, 2'-fluoro substitution, and 2'-stereochemistry inversion on the N-glycosidic bond stabilities were also investigated via ER-CID experiments and survival yield analysis. The 2'-fluoro substituent significantly stabilizes the N-glycosidic bond compared with the analogous DNA and RNA nucleosides, whereas a 2'-O-methyl substituent produces a similar effect on the N-glycosidic bond stabilities as found for a 2'-hydroxyl substituent. Additionally, changing the stereochemistry of the 2'-hydroxyl substituent has only slight impact on the N-glycosidic bond stabilities. Methylation of different positions clearly impacts the relative N-glycosidic bond stabilities of these nucleosides. By comparing the CID50% values of protonated and metal cationized guanine nucleosides, the effects of local environment, i.e. pH and the presence of metal cations, on the relative N-glycosidic bond stabilities were also investigated. The order of relative stabilities of alkali metal cationized dGuo and Guo is consistent with the order of increasing size of the alkali metal. The binding between the metal cation and the nucleoside become weaker as the metal cation becomes larger.
The gas-phase conformations of the sodium cationized complexes of the neutral and deprotonated adenine mononucleotides have been examined in the present work via IRMPD action spectroscopy in both the IR fingerprint and hydrogen-stretching regions. Comparison of the measured IRMPD spectra with the calculated IR spectra of the stable low-energy conformations of these species calculated at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level of theory allows the structures populated in the experiments to be determined. Overall, the conformers of the sodium cationized forms of the neutral and deprotonated adenine mononucleotides that are dominantly populated in the experiments are highly parallel. Based on comparisons of the measured IRMPD and computed IR spectra, it is clear that in the dominant conformers of sodium cationized adenine mononucleotides populated, Na+ binds to the adenine nucleobase, sugar and phosphate moieties in a tridentate fashion, adenine exhibits a syn orientation, and the sugars prefer C3'-endo (3T2) puckering in both cases. In the dominant conformers of the disodium cationized deprotonated adenine mononucleotides populated, the first Na+ binds to the adenine nucleobase, sugar and phosphate moieties in a quadridentate fashion, the second Na+ binds solely to the phosphate moiety, adenine exhibits a syn orientation, and the sugars prefer O4'-endo (OT1) puckering in both cases. Present results are compared with those for the sodium cationized adenine nucleosides as well as the protonated and deprotonated forms of the adenine mononucleotides to elucidate the effects of the phosphate group and charge state on the gas-phase conformations of these nucleic acid building blocks.
Zhu, Yanlong, "Tandem Mass Spectrometry And Computational Approaches To Elucidate Conformations And N-Glycosidic Bond Stabilities Of Dna And Rna Nucleosides And Mononucleotides" (2018). Wayne State University Dissertations. 1978.