Access Type

Open Access Embargo

Date of Award

January 2016

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary T. Rodgers

Abstract

The gas-phase conformations of the protonated forms of the nucleic acid constituents: nucleosides and mononucleotides, were characterized by infrared multiple photon dissociation (IRMPD) action spectroscopy and theoretical calculations. The IRMPD spectroscopy experiments were performed using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) coupled to a free electron laser or a OPO/OPA laser system to measure the IRMPD action spectra over the IR fingerprint and hydrogen-stretching regions.

Theoretical calculations were performed using both mechanic dynamics and high level quantum chemical calculations to optimize structures, compute vibrational frequencies and IR intensities, and to obtain the relative stabilities of optimized structures. Comparisons of the measured IRMPD vs. computed linear IR spectra allow the stable conformers populated in the experiments to be determined. These conformers provide information regarding the preferred site of protonation, nucleobase orientation, and sugar puckering for each system investigated.

Comparisons of the measured IRMPD spectra and the stable gas-phase conformations of DNA vs. RNA species allow the effect of the 2'-hydroxyl substituent on the IRMPD spectra and conformations to be elucidated. The 2'-hydroxyl substituent does not significantly affect the measured IRMPD profiles and but greatly influence the IRMPD yields of DNA vs. RNA. The 2'-hydroxyl substituent does not significantly impact the stable gas-phase conformations of DNA vs. RNA. However, it provides an additional hydrogen-bond donor or acceptor, facilitating additional intramolecular hydrogen-bonding interactions to be formed. Therefore, a larger number of low-energy conformers are typically found to be populated in the experiments for the protonated RNA nucleosides and mononucleotides than their DNA analogues.

Comparisons of these results for the protonated nucleosides vs. mononucleotides elucidate the effect of the phosphate moiety on the conformations. The phosphate moiety serves as a stronger hydrogen-bond acceptor than the 5'-hydrxoyl substituent, and thus enhances the intramolecular hydrogen-bonding interactions formed between the phosphate, nucleobase and sugar moieties. Therefore, a very limited numbers of low-energy conformers are found to be populated in the experiments for the protonated mononucleotides. The phosphate moiety does not change the preferred protonation site of the adenine, guanine and cytosine mononucleotides as compared to their analogous nucleosides. However, the protonation preferences of the thymine and uracil mononucleotides are greatly affected by the phosphate moiety as compared to their analogous nucleosides.

The mechanisms and energetics for N-glycosidic bond cleavage of the protonated forms of 10 nucleosides were investigated by threshold collision-induced dissociation (TCID) techniques and theoretical calculations. The TCID experiments are performed using a custom-built guided ion beam tandem mass spectrometer to measure the CID cross sections as a function of collision energy. Stable gas-phase conformations of the protonated nucleosides determined from the IRMPD spectroscopy studies are taken as the reactants for the TCID experiments. The primary CID pathways observed in the experiments involve the cleavage of N-glycosidic bond, resulting in elimination of protonated or neutral nucleobase in competition.

Theoretical calculations were performed to map the potential energy surfaces (PESs) for N-glycosidic bond cleavage, and obtain important molecular parameters needed for threshold analyses. Except for protonated adenosine, [Ado+H]+, the calculated PESs for N-glycosidic bond cleavage of DNA vs. RNA indicate that the 2'-hydroxyl substituent does not affect the dissociation mechanisms, which typically involves two steps: N-glycosidic bond elongation and C2'-H transfer.

Threshold analysis allows the quantitative thermochemical energetics, activation energies (AEs) and reaction enthalpies (ΔHrxns), associated with the N-glycosidic bond cleavage processes to be determined. The measured AEs and ΔHrxns of the protonated DNA vs. RNA nucleosides indicate that the 2'-hydroxyl substituent typically increases these values by > 20 kJ/mol from DNA to RNA. Therefore, the 2'-hydroxyl substituent enhances the stability of N-glycosidic bond for the RNA species vs. their DNA analogues.

Comparisons of the measured AEs and ΔHrxns of the five protonated DNA and RNA nucleosides, respectively, allow the effects of different nucleobases on the stabilities of N-glycosidic bond to be determined. The trends found for the protonated DNA and RNA nucleosides indicate that the proton affinities (PAs) of the nucleobases only partially correlate with the leaving group propensities of the protonated nucleobase such that the trend in the measured AEs is not entirely consistent with the trend of the PAs. The trend suggests that the conformation of the reactant, site of protonation, tautomeric state, and orientation of the nucleobase relative to the sugar moiety all play a more significant role than the nucleobase PAs in determining the energetics associated with the N-glycosidic bond cleavage processes.

Included in

Chemistry Commons

Share

COinS