Open Access Dissertation
Date of Award
Mary T. Rodgers
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.
Hamlow, Lucas Ash, "Modification Of Bruker Amazon Etd And Solarix Mass Spectrometers Towards Infrared Multiple Photon Dissociation: Structural Characterization Of Modified Nucleosides" (2019). Wayne State University Dissertations. 2322.