Access Type

Open Access Dissertation

Date of Award

January 2014

Degree Type


Degree Name




First Advisor

Mary T. Rodgers






December 2014

Advisor: Professor Mary T. Rodgers

Major: Analytical Chemistry

Degree: Doctor of Philosophy

Binding of metal cations to the nucleobases can lead to formation of rare tautomers of the nucleobases. The infrared multiple photon dissociation (IRMPD) action spectroscopy of five alkali metal cation-cytosine complexes, M+(cytosine), where M+ = Li+, Na+, K+, Rb+, and Cs+, are examined using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) coupled to a free electron laser. This work suggests that only the ground-state tautomeric conformations are accessed for all five M+(cytosine) complexes when electrospray ionization (ESI) is used as the ionization technique.

Based on the structural information obtained from the IRMPD studies, the binding affinities of four alkali metal cations to cytosine are measured using the threshold collision-induced dissociation (TCID) techniques in a guided ion beam tandem mass spectrometer to understand the effects of the size of the alkali metal cation on the strength of binding. The bond dissociation energies (BDEs) of the M+(cytosine) complexes are found to decrease as the size of the alkali metal cation increases from Na+(0.98 Å) to Cs+(1.67 Å) as a result of the electrostatic nature of the binding.

The base-pairing interactions of the proton-bound dimers of cytosine are likely the major factor that helps stabilize noncanonical DNA i-motif conformations, which are associated with Fragile X syndrome, the most widespread inherited cause of mental retardation in humans. Modifications of cytosine, such as methylation and halogenation, can influence the binding modes or the strength of the base-pairing interactions. The IRMPD action spectroscopy of four proton-bound homodimers, (5xC)H+(5xC), where x = H, F, Br, and Me, and three proton-bound heterodimers, (C)H+(5xC), where x = F, Br, and Me, are examined using a FT-ICR MS coupled to an optical parametric oscillator (OPO) laser. In the case of the proton-bound homodimers, it is clear that the only tautomeric conformation accessed in the experiments is the ground-state II+∙∙∙i_3a conformation. In the case of the heterodimers, the ground-state structures, II+∙∙∙i_3a, are accessed in the experiments. The first-excited conformers of the proton-bound heterodimers, i∙∙∙II+_3a, where the excess proton is now bound to the base with the lower PA, and which lie 2.4-7.4 kJ/mol higher in free energy, may also be accessed in the experiments, but are likely only present in low abundance.

Quantitative determination of the base-pairing energies (BPEs) of 20 proton-bound homo- and heterodimers is then performed using a guided ion beam tandem mass spectrometer to illustrate the effects of modifications on the strength of the base-pairing interactions in the proton-bound dimers. The modified cytosines included in this work are 5-methylcytosine (5MeC), 5-fluorocytosine (5FC), 5-bromocytosine (5BrC), 5-iodocytosine (5IC), 1-methylcytosine (1MeC), 5-fluoro-1-methylcytosine (1Me5FC), 5-bromo-1-methylcytosine (1Me5BrC), and 1,5-dimethylcytosine (15dMeC). Relative N3 proton affinities (PAs) of the modified cytosines are also extracted from the experimental data from competitive analyses of the two primary dissociation pathways that occur in parallel for the proton-bound heterodimers of cytosine and modified cytosines. Methylation clearly influences the BPE of the proton-bound dimers and N3 PA of cytosine. In the case of the homodimers, 5-hypermethylation is found to increase the BPE, whereas 1-hypermethylation is found to exert almost no effect on the BPE. Hence, 1,5-dimethylation of both cytosines results in an intermediate increase in the BPE. In the case of the heterodimers, methylation of a single cytosine at the N1, C5 or N1 and C5 positions weakens the BPE, and therefore would tend to destabilize DNA i-motif conformations. In contrast to its effects on the BPEs, methylation of cytosine increases the N3 PA regardless of the position of substitution. The N3 PAs of cytosine and the methylated cytosines follow the order: 15dMeC (979.9 ± 2.9 kJ/mol) > 1MeC (964.7 ± 2.9 kJ/mol) > 5MeC (963.2 ± 2.9 kJ/mol) > C (949.9 ± 2.8 kJ/mol), indicating that N1-methylation has a greater influence on the N3 PA than C5-methylation, and the effects of N1, C5-dimethylation on the N3 PA are roughly additive.

Halogen substituents exhibit different behavior from methylation due to their electron-withdrawing properties. 5-Halogenation is found to decrease the BPE, but exert almost no effect on the BPE in the presence of 1-methylation. Halogenation is found to decrease the N3 PA. The N3 PAs of cytosine and the halogenated cytosines follow the order: 1Me5BrC (959.9 ± 3.3 kJ/mol) > 1Me5FC (955.7 ± 3.3 kJ/mol) > C (949.9 ± 2.8 kJ/mol) > 5BrC (930.9 ± 3.6 kJ/mol) > 5FC (926.3 ± 3.5 kJ/mol), indicating that 1-methylation has a greater influence on the N3 PA than C5-halogenation.

This work is then extended to study the proton-bound dimers of 2′-deoxycytosine (dCyd) and 5-methyl-2′-deoxycytosine (5MedCyd). It is found that 5-permethylation of cytosine residues increases the base-pairing interactions in the presence of 2'-deoxyribose sugar, and thus should stabilize DNA i-motif conformations. Experimentally, the BPE of the (5MedCyd)H+(dCyd) proton-bound dimer is greater than that of the (dCyd)H+(dCyd) homodimer, whereas theory suggests that 5-methylation of a single cytosine residue exert almost no effect or slightly decrease in the BPE. Thus, single 5-methylation of cytosine residue should lead to minor destabilization of DNA i-motif. The N3 PAs of 5MedCyd is 994.4 ± 8.4 kJ/mol, and is 6.1 kJ/mol greater than that of dCyd, 988.3 ± 8.0 kJ/mol, suggesting that methylation increases the N3 PA of the nucleoside.

However, the BPEs of all proton-bound dimers examined here are still much greater than those of canonical Watson-Crick G±C and neutral C*C base pairs, suggesting that DNA i-motif conformations are still favored over conventional base pairing such that the DNA i-motif conformations should be stable upon modifications. In all cases, excellent agreement between TCID measured BPEs and N3 PAs and B3LYP calculated values is found, suggesting that B3LYP calculations can be employed to provide reliable energetic predictions for related systems.

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