Study of Reaction Dynamics of Protonated/Deprotonated and Radical Cations of Guanine in Nucleobases and Nucleosides: Singlet Oxygen Oxidation, C8-Water Addition, Cross-Linking with Lysine and Base-Pair Dissociation

Author

Yan Sun, The Graduate Center, City University of New YorkFollow

Date of Degree

2-2020

Document Type

Dissertation

Degree Name

Ph.D.

Program

Chemistry

Advisor

Jianbo Liu

Committee Members

Yolanda Small

Cherice Evans

Junyong Choi

Subject Categories

Analytical Chemistry | Physical Chemistry

Keywords

DNA, on-line mass spectrometry, gas phase, reaction kinetics, hydration, computational study

Abstract

Among the four DNA nucleobases, guanine (G) has the lowest oxidation potential and represents a preferential target for oxidation and ionization. This leads to the formation of guanine radical cation (G^•+) in various oxidative environments. Of the biologically relevant oxidants, electronically excited singlet oxygen (¹O₂) exclusively damages the guanine bases and gives rise to mutagenesis, DNA-protein cross-linking and cellular death. Combining our home-made electrospray ionization (ESI) guided-ion-beam tandem mass spectrometer, with reaction potential surface calculations and kinetics modeling, five projects have been accomplished as described below.

In project 1, the reactions of deuterated water (D₂O) with radical cations of guanine (9HG^•+), 9-methylguanine (9MG^•+), 2′-deoxyguanosine (dGuo^•+) and guanosine (Guo^•+) were studied in the gas phase, including measurements of reaction cross sections and computation of reaction potential energy surfaces. For each reactant ion, the energetically favorable product channel corresponds to the formation of water complexes, but it accounts for only 5% of the total reaction cross section. The dominant product channel is H/D exchange between the reactants. C8-hydroxylation of the guanine moiety, the most biologically important channel, was observed for 9HG^•+. The C8-hydroxylation mechanism was elucidated in the presence of single and double water molecules, of which the second water eliminates the reaction barrier for C8-water addition via a proton shuttle mechanism. All reactions show N9-substituent dependence, with overall reactivity being 9HG^•+ >> 9MG^•+ > dGuo^•+ ≈ Guo^•+.

In project 2, we report a kinetics and mechanistic study on the ¹O₂ oxidation of 9-methylguanine (9MG) and the cross-linking of the oxidized intermediate 2-amino-7,9-dihydro-purine-6,8-dione (9MOG^OX) with N^α-acetyl-L-lysine-methyl ester (abbreviated as LysNH₂) in aqueous solutions of different pH. This experiment revealed strong pH dependence of 9MG oxidation and of the addition of nucleophiles (water and LysNH₂₎ at the C5 position of 9MOG^ox. The oxidation rate constant of 9MG was determined to be 3.6 x 10⁷ M^-1s^-1 at pH 10 and 0.3 x 10⁷ M^-1s^-1 at pH 7, both of which were measured in the presence of 15 mM LysNH₂. The ωB97XD density functional theory coupled with various basis sets and the SMD implicit solvation model was used to explore the reaction potential energy surfaces for the oxidation of 9MG and the formation of C5-water and C5-LysNH₂ adducts of 9MOG^ox. The present work has confirmed that, regardless of cross-linking with LysNH₂, the initial ¹O₂ addition represents the rate-limiting step for the oxidative transformations of 9MG. All of the downstream steps are exothermic and proceed rapidly. The C5-cross linking of 9MOG^ox with LysNH₂ significantly suppresses the formation of spiroiminodihydantoin (9MSp) resulting from the C5-water addition. The latter becomes dominant only at the low concentration of the competing LysNH₂.

In project 3, collision-induced dissociation (CID) of 9-methylguanine–1-methylcytosine base pair radical cation ([9MG⋅1MC]^•+) and its monohydrate ([9MG⋅1MC_W]^•+, W = H₂O) with xenon and argon was carried out using guided-ion beam tandem mass spectrometry. [9MG⋅1MC]^•+ has two structures, a conventional structure 9MG^•+⋅1MC that consists of H-bonded 9-methylguanine radical cation (9MG^•+) and neutral 1-methylcytosine (1MC), and a proton-transferred structure [9MG – H]^•⋅[1MC + H]⁺ that is formed by intra-base pair proton transfer from the N1 of 9MG^•+ to the N3 of 1MC. 9MG^•+⋅1MC is slightly more stable than [9MG – H]^•⋅[1MC + H]⁺ and the two structures can be distinguished by CID in that 9MG^•+⋅1MC dissociates into 9MG^•+ and 1MC whereas [9MG – H]^•⋅[1MC + H]⁺ dissociates into neutral [9MG – H]^• radical and protonated [1MC + H]⁺. An interesting finding is that the CID mass spectra of [9MG⋅1MC]^•+ were overwhelmingly dominated by [1MC + H]⁺, which is contrary to what would be expected on the basis of statistical kinetics. The non-statistical phenomenon implies extensive intra-base pair proton transfer of [9MG⋅1MC]^•+. Monohydration of [9MG⋅1MC]^•+ reversed the order of stability of conventional and proton-transferred structures and changed their CID pathways. Major CID pathways of [9MG⋅1MC_W]^•+ include elimination of a water ligand and more interestingly, elimination of a methanol molecule. In addition, 9MG^•+, protonated [9MG + H]⁺ and [1MC + H]⁺ were detected in the CID of [9MG⋅1MC_W]^•+.

In project 4, we have extended the investigation to protonated [9MG⋅1MC + H]⁺ base pair ions and its monohydrate, aimed at determining whether non-statistical dissociation is a general feature of guanine–cytosine base pairs by comparing with CID of radical and deprotonated forms of base pairs. We report a guided-ion beam tandem mass spectrometric study on CID of protonated 9-Methylguanine–1-Methylcytosine base pair (abbreviated as [9MG⋅1MC + H]⁺) and its monohydrate [9MG⋅1MC + H_W]⁺ (W = H₂O) with Xe and Ar gases. Product ion mass spectra and cross section were measured as a function of center-of-mass collision energy, from which various dissociation pathways and dissociation threshold energies were determined. Electronic structure calculations at the DFT, RI-MP2, DLPNO-CCSD(T) levels of theory and Rice-Ramsperger-Kassel-Marcus modeling were used to map out reaction potential energy surfaces (PESs) and identify product structures. It was found that intra-base pair proton transfer within [9MG⋅1MC + H]⁺ results in two base pair conformers, [9MG + H]⁺⋅1MC and 9MG⋅[1MC + H]⁺, during base pair collisional activation. Both structures contain a Waston-Crick H-bonding motif but differ in the location of the central H. [9MG + H]⁺⋅1MC has the H bound to the N1 of 9MG while 9MG⋅[1MC + H]⁺ has the H shifted to the N4 of 1MC. [9MG + H]⁺⋅1MC lies in energy 0.17 eV lower than 9MG⋅[1MC + H]⁺, and its dissociation threshold is 0.85 eV lower than 9MG⋅[1MC + H]⁺. Surprisingly, the abundance of [1MC + H]⁺ in CID products is significantly higher than that of [9MG + H]⁺, contrast with what expected from a statistical dissociation product distribution. Hydration of [9MG⋅1MC + H]⁺ by an explicit water enriches base pair dissociation chemistry; particularly, it induces the reaction of water ligand with the methyl groups of collisionally activated nucleobases and results in formation of a methanol molecule.

In project 5, the reaction of ¹O₂ with radical cations of Guanine (9HG^•+), 9-Methylguanine (9MG^•+), 2′-Deoxyguanosine (dGuo^•+) and Guanosine (Guo^•+) were studied by a home-made electrospray ionization (ESI) guided-ion beam scattering tandem mass spectrometer in the gas phase. Reaction products and cross sections were measured over a center-of-mass collision energy (Ecol) range from 0.05 to 1.0 eV. No product was observed for the reactions of dry 9HG^•+ and 9MG^•+ with ¹O₂ due to the rapid decay of transient oxidation intermediates to starting reactants. But the reaction peroxide products of their monohydrates 9HG^•+_W and 9MG^•+_W with ¹O₂ were successfully captured due to the relaxation of reaction products via elimination of water ligand. Similar oxidation products were detected for dGuo^•+ and Guo^•+. In this case, no hydration was needed for the nucleoside reactants as the intramolecular vibrational redistribution can help stabilize the intermediates and products. 9MG was chosen as a model compound to map out reaction potential energy surfaces (PESs) because it has similar properties to dGuo and Guo. Calculations were carried out at various levels of theory including ωB97XD/6-31+G(d,p), RI-MP2/aug-cc-pVQZ, DLPNO-CCSD(T)/aug-cc-pVTZ, CASSCF(21,15)/6-31+G(d,p), RI-NEVPT2/6-31+G(d,p) and CASPT2(21,15)/6-31G(d,p) of which CASSCF, CASPT2 and NEVPT2 were used specifically to correct for the muti-referential characters of the ¹O₂ reaction with guanine radicals. Two possible reaction pathways were found for the ¹O₂ addition to 9MG^•+: C8-addition to the formation of a C8-peroxide and C5-addition to the formation of a C5-peroxide. C8-addition pathway is energetically more favorable than C5-addition.

Recommended Citation

Sun, Yan, "Study of Reaction Dynamics of Protonated/Deprotonated and Radical Cations of Guanine in Nucleobases and Nucleosides: Singlet Oxygen Oxidation, C8-Water Addition, Cross-Linking with Lysine and Base-Pair Dissociation" (2020). CUNY Academic Works.
https://academicworks.cuny.edu/gc_etds/3530