Reaction Kinetics, Product Branching, and Potential Energy Surfaces of ¹O₂-Induced 9-Methylguanine–Lysine Cross-Linking: A Combined Mass Spectrometry, Spectroscopy, and Computational Study

Abstract

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-9-methyl-9H-purine-6,8-dione (9MOGOX) with N α-acetyl-lysine-methyl ester (abbreviated as LysNH2) in aqueous solutions of different pH. Experimental measurements include the determination of product branching ratios and reaction kinetics using mass spectrometry and absorption spectroscopy, and the characterization of product structures by employing collision-induced dissociation. Strong pH dependence was revealed for both 9MG oxidation and the addition of nucleophiles (water and LysNH2) at the C5 position of 9MOGOX. The 1O2 oxidation rate constant of 9MG was determined to be 3.6 × 107 M–1·s–1 at pH 10.0 and 0.3 × 107 M–1·s–1 at pH 7.0, 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 1O2 oxidation of 9MG and the formation of C5-water and C5-LysNH2 adducts of 9MOGOX. Computational results have shed light on reaction pathways and product structures for the different ionization states of the reactants. The present work has confirmed that the initial 1O2 addition represents the rate-limiting step for the oxidative transformations of 9MG. All of the downstream steps are exothermic with respect to the starting reactants. The C5-cross-linking of 9MOGOX with LysNH2 significantly suppressed the formation of spiroiminodihydantoin (9MSp) resulting from the C5-water addition. The latter became dominant only at the low concentration (∼1 mM) of LysNH2.

Dynamics and Multiconfiguration Potential Energy Surface for the Singlet O2 Reactions with Radical Cations of Guanine, 9-Methylguanine, 2′-Deoxyguanosine, and Guanosine

“…The spin-restricted DFT is unable to describe the static correlation arising from the two degenerate π* antibonding orbitals and overestimates the 1 O 2 excitation energy, whereas the broken-symmetry, spin-unrestricted DFT brings about spin contamination from the ground-state 3 O 2 . This problem exists not only in the 1 O 2 reactant but also in the intermediates and TSs for 1 O 2 addition to guanine. ,− In our previous study of the 1 O 2 reactions with protonated and deprotonated guanine ions, − spin contamination in the PES was corrected using Yamaguchi’s approximate spin-projection method, , in which the spin-projected total electronic energy of an open-shell singlet state was calculated as a weighted combination of broken-symmetry, spin-unrestricted singlet and triplet states. However, the approximate spin-projection was validated only for systems containing singlet and triplet states. ,,,, The present system, on the other hand, is more complicated in that it mixes doublet and quartet states.…”

“…20−22,26−48 The consequential primary and secondary damage is implicated in DNA strand breaks, 2 DNA−protein cross-links, 43,48 mutation, 49 and apoptosis 50 well as in photodynamic therapy for cancer. 51 Most of the mechanistic insights on the 1 O 2 oxidation of guanine were obtained from the measurements of guanine residues in various contexts (including isolated nucleobases, nucleosides, oligonucleotides, and single-and double-stranded DNA) in solution [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]47,48 and their protonated and deprotonated ions in the gas phase, 44−46 augmented by reaction potential energy surface (PES) investigations [43][44][45][46][47][48]52,53 and molecular dynamics simulations. 41,42,44−46 A currently accepted mechanism is that guanine is attacked by 1 O 2 on its imidazole ring, forming a transient peroxide and/or endoperoxide that quickly converts to a hydroperoxide 8-OOH-G.…”

“…1 O 2 is generated in living systems through enzymatic and nonenzymatic reactions, type II photosensitization, chemical excitation, etc . , and is known to cause DNA damage. ,− The 1 O 2 damage to DNA initiates exclusively at the guanine residues. − ,− The consequential primary and secondary damage is implicated in DNA strand breaks, DNA–protein cross-links, , mutation, and apoptosis as well as in photodynamic therapy for cancer . Most of the mechanistic insights on the 1 O 2 oxidation of guanine were obtained from the measurements of guanine residues in various contexts (including isolated nucleobases, nucleosides, oligonucleotides, and single- and double-stranded DNA) in solution − ,, and their protonated and deprotonated ions in the gas phase, − augmented by reaction potential energy surface (PES) investigations − ,, and molecular dynamics simulations. ,,− A currently accepted mechanism is that guanine is attacked by 1 O 2 on its imidazole ring, forming a transient peroxide and/or endoperoxide that quickly converts to a hydroperoxide 8-OOH-G. The 8-OOH-G in isolated nucleosides and short oligonucleotides undergoes a series of reactions and ultimately forms spiroiminodihydantoin and guanidinohydantoin; whereas that within DNA is reduced to 8-OG .…”

Singlet O2 Oxidation of the Radical Cation versus the Dehydrogenated Neutral Radical of 9-Methylguanine in a Watson–Crick Base Pair. Consequences of Structural Context

“…Very recently, we investigated the reaction of singlet oxygen ( 1 O 2 ) with radical cations of guanine, 9-methylguanine, 2′-deoxyguanosine and guanosine . Singlet O 2 is one of the reactive oxygen species generated in living systems through enzymatic and nonenzymatic reactions, type II photosensitization, chemical excitation, and so forth. , Singlet O 2 causes DNA damage, ,− and lesions are initiated exclusively at the guanine residues. ,− Our work found that the 1 O 2 oxidation of the guanine radical cation leads to the formation of an 8-peroxide, from which a variety of products evolve. Note that under normal biological conditions, the encounter probability of 1 O 2 with guanine radical species is low due to their low local concentrations and short lifetimes.…”

Singlet O2 Reactions with Radical Cations of 8-Bromoguanine and 8-Bromoguanosine: Guided-Ion Beam Mass Spectrometric Measurements and Theoretical Treatments

“…The present work focuses on reactions of the radical cations of 8-bromoguanine (abbreviated as 8BrG •+ ) and 8-bromoguanosine (8BrGuo •+ ) with electronically excited singlet oxygen O 2 [a 1 Δ g ]. , 1 O 2 is a biologically relevant reactive oxygen species that reacts efficiently with cellular constituents including proteins, DNA, and lipids . Guanine represents the exclusive DNA target for 1 O 2 . − The resulting primary and secondary damage of guanine nucleobases and nucleosides is implicated in DNA strand breaks, DNA-protein cross-links, , mutation, and apoptosis as well as in photodynamic therapy for cancer . Interestingly, oxidized forms of guanine such as OG are even more susceptible to the 1 O 2 oxidation than guanine and guanosine. ,− However, no study has been reported for the 1 O 2 oxidation of neutral 8BrG, 8BrGuo, or their radical cations.…”