Access Type

Open Access Dissertation

Date of Award

January 2019

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry

First Advisor

Mary T. Rodgers

Abstract

Infrared ion spectroscopy has become an increasingly powerful tool for examining

the intrinsic structures of gas-phase ions. Infrared multiple photon dissociation (IRMPD)

action spectroscopy has been particularly successful. Several free electron laser (FEL)

facilities across the world have helped facilitate the growth of the IRMPD technique and

increasing interest has driven the development of more accessible IRMPD

instrumentation. The development of one such IRMPD instrumentation system is

described in this work, based around commercially available 3D quadrupole ion trap mass

spectrometers and Fourier-transform ion cyclotron resonance mass spectrometers.

The intrinsic gas-phase structures of nucleic acid monomers have been

extensively studied by IRMPD and complimentary theoretical approaches. These studies

have examined the common DNA and RNA nucleobases in several states of ionization.

The common DNA and RNA nucleosides have also been extensively examined in several

ionization states by several research groups. Studies of nucleotides have examined an

even more diverse set of ionization states. Several studies have also examined the impact of specific modifications on the intrinsic structures of specific nucleic acid monomers.

However, the incredible wealth of available synthetic and naturally occurring modifications

to these monomers still presents a substantial challenge in understanding the relationship

between structure and function for modified nucleic acid monomers.

Thiation of uridine at the 2- or 4- position are important modifications for tRNA.

4-thiouridine is a naturally occurring modification in tRNA that is thought to offer some

protection against near-UV exposure by cross-linking with a nearby cytidine. Whereas

2-thiouridine and 2-thiouridines further modified at the 5-position can be found at the

wobble position of the tRNA anticodon. The altered base-pairing of 2-thiouridine and the

modified 2-thiouridines is important to recognition of the codon on an mRNA. Previous

study of the protonated 2-thiouracil [s2Ura+H]+ and 4-thiouracil [s4Ura+H]+ indicate a

change in the protonation preference vs protonated uracil [Ura+H]+. IRMPD action

spectroscopy experiments in both the IR fingerprint region and hydrogen-stretching

region are performed on protonated 2-thiouridine [s2Urd+H]+ and 4-thiouridine [s4Urd+H]+.

Complimentary molecular dynamics simulations are used to explore the conformational

space available to the protonated thiouridines and generate candidate structures. Density

functional theory calculations at the B3LYP/6-311+G(2d,2p)//B3LYP/6-311+G(d,p) level

of theory further optimize the candidate structures, predict their IR spectra, and calculate

reasonably accurate energetics. Comparison of the measured IRMPD action spectra and

the predicted IR spectra reveal the preferred conformations of [s2Urd+H]+ and [s4Urd+H]+

and those populated in the experiments. Protonation of 2-thiouridine prefers formation of

the 2-sylfhydryl-4-hydroxyl, with some O4 protonated conformers present. Whereas

protonation of 4-thiourudine prefers protonation at S4, with a minor contribution from 2-hydroxyl-4-sulfhydryl tautomers. A mixture of C2′-endo and C3′-endo sugar puckering is

observed, with anti oriented nucleobases preferred.

Common targets for modification of pharmaceutically active nucleoside analogues

are the 2′- and 3′-hydroxy moieties on the sugar. Nucleosides with an arabinose sugar

moiety are some of the oldest nucleoside analogue drugs. The arabinose analogues of

cytidine, araCyd, and adenosine, araAdo, have both found use pharmaceutically. The

arabinose sugar moiety inverts the stereochemistry at the 2′-position vs ribose, and

previous studies by NMR, crystallography, and theoretical calculations are not consistent

on the impact of this modification on nucleoside structure. Nucleosides based upon the

2′,3′-dideoxyribose sugar moiety are also common pharmaceutically. Understanding the

impact of these two modifications on intrinsic nucleoside structure is important to

understanding the basis for their pharmaceutical activity. IRMPD action spectroscopy

experiments were performed for the protonated arabinose analogues of the common RNA

nucleosides adenosine, guanosine, cytidine, and uridine. IRMPD experiments were

performed for the protonated 2′,3′-dideoxyribose analogues of these RNA nucleosides as

well as the analogue of thymidine. Complimentary theoretical calculations explore the

conformational space of these ions to generate candidate structures and predict their IR

spectra and energetics. Comparison of these predicted IR spectra and the experimental

IRMPD spectra reveals the conformations that contribute to the experiments. An

intramolecular O2′H···O5′ hydrogen-bonding interaction unique to the arabinose

analogues is observed for each of the arabinose analogues alongside conformations

parallel to those observed previously for the DNA nucleosides. This unique intramolecular

hydrogen-bonding interaction strongly prefers C2′-endo sugar puckering, which does have some impact on the overall sugar puckering preferences, but C3′-endo sugar

puckering is also observed experimentally for the protonated arabinose analogues.

Comparison of the experimental IRMPD spectra of the 2′,3′-dideoxyribose analogues

reveals a stronger preference for C3′-endo sugar puckering. Otherwise, largely parallel

conformations are observed between the protonated 2′,3′-dideoxyribose nucleosides and

the previously studied protonated 2′-deoxyribose nucleosides.

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