Density functional theory was used to study the thermodynamics and kinetics for the glycosidic bond cleavage in deoxyuridine. Two reaction pathways were characterized for the unimolecular decomposition in vacuo. However, these processes are associated with large reaction barriers and highly endothermic reaction energies, which is in agreement with experiments that suggest a (water) nucleophile is required for the nonenzymatic glycosidic bond cleavage. Two (S(N)1 and S(N)2) reaction pathways were characterized for direct hydrolysis of the glycosidic bond by a single water molecule; however, both pathways also involve very large barriers. Activation of the water nucleophile via partial proton abstraction steadily decreases the barrier and leads to a more exothermic reaction energy as the proton affinity of the molecule interacting with water increases. Indeed, our data suggests that the barrier heights and reaction energies range from that for hydrolysis by water to that for hydrolysis by the hydroxyl anion, which represents the extreme of (full) water activation (deprotonation). Hydrogen bonds between small molecules (hydrogen fluoride, water, or ammonia) and the nucleobase were found to further decrease the barrier and overall reaction energy but not to the extent that the same hydrogen-bonding interactions increase the acidity of the nucleobase. Our results suggest that the nature of the nucleophile plays a more important role in reducing the barrier to glycosidic bond cleavage than the nature of the small molecule bound, and models with more than one hydrogen fluoride molecule interacting with the nucleobase provide further support for this conclusion. Our results lead to a greater fundamental understanding of the effects of the nucleophile, activation of the nucleophile, and interactions with the nucleobase for this important biological reaction.

The structural and spectral properties of (ortho and para) C8-aryl-purine adducts formed from carbon attachment by phenolic toxins were investigated through DFT calculations and UV-vis absorbance and emission studies. The global minima of both the deoxyadenosine (dA) and deoxyguanosine (dG) adducts adopted a syn conformation about the glycosidic bond due to the presence of an O5'-H...N3 hydrogen bond, where the anti minima are 20-30 kJ mol-1 higher in energy. While the nucleobase adducts are planar, the presence of the deoxyribose sugar induces a twist about the carbon-carbon bond connecting the phenol and nucleobase rings. ortho-Phenolic adducts are less twisted than the corresponding para adducts due to stabilization provided by an intramolecular O-H...N7 bond. Solvation calculations, in combination with UV-vis studies, demonstrate that the structural preference is solvent dependent, where solvents with hydrogen-bonding abilities disrupt the intramolecular O-H...N7 hydrogen bond such that a greater degree of twist is observed, and less polar solvents stabilize the planar structure. Indeed, the ratio of twisted to planar conformers is estimated to be as large as 50:50 in some aprotic solvents. Thus, the combined experimental and computational approach has provided a greater understanding of the structure of the ortho- and para-dA and dG C-bonded phenoxyl adducts as the first step to understanding the biological consequences of this form of DNA damage.

Previous computational work suggests that isolated C8-phenoxyl-2'-deoxyguanosine nucleoside adducts preferentially adopt a syn orientation about the glycosidic bond, which is the first step in the mechanism by which many bulky C8 adducts exert their mutagenic effects. Since it is not clear whether these results can be directly extrapolated to the preferred conformation in DNA helices, approaches that more accurately reflect the physiological environment were used in the present study to understand the anti/syn preference of the ortho and para C8-phenoxyl-2'-deoxyguanosine adducts. Using nucleoside models and methods (B3LYP) similar to those previously implemented, we determine that the syn conformer is less stable than previously predicted when geometries relevant to B-DNA are considered. This indicates that the conformational energy trend is model dependent and stresses the importance of considering models that better mimic the DNA environment when determining the conformational preference of damaged bases. Therefore, we expanded our computational model to include the 5'-monophosphate group. Since the correct anti/syn energy trend for 2'-deoxyguanosine (dG) 5'-monophosphate has only been found using very specific computational models and prior knowledge of the biologically relevant nucleotide conformation, which is unavailable for most damaged systems, we initially benchmark our computational approach by studying the natural nucleotide. Despite the wide use of gas-phase optimizations in the current literature, only through the implementation of solvation-phase optimizations, as well as the use of a counterion model for the phosphate backbone, is the correct anti/syn energy trend predicted. Indeed, this is the first time in the literature that a biologically relevant syn structure is characterized for dG using methods suitable for studying bulky DNA adducts. Subsequently, our newly identified approach for DNA lesions was used to study C8-phenoxyl DNA adducts. In contrast to previously published results, we predict that the ortho and para adducts may adopt both the anti and syn conformations in DNA helices. These results have implications for the base-pairing properties and mutagenicity of these adducts, which must be further considered in future work.

Density functional theory was used to study the glycosidic bond cleavage in deoxynucleotides with the main goal to determine the effects of the nucleobase, hydrogen bonding with the nucleobase, and the (bulk) environment on the reaction energetics. Since direct glycosidic bond cleavage is a high-energy process, two nucleophile models were considered (HCOO–···H2O and HO–), which represent different stages of activation of a water nucleophile. The glycosidic bond cleavage barriers were found to decrease, while the reaction exothermicity increases, with an increase in the nucleobase acidity. The gas-phase barriers and reaction energies for bond cleavage in all deoxynucleotides were found to be significantly affected by hydrogen-bonding interactions with the nucleobase (by up to 30 kJ mol–1 depending on the nucleophile). Although the barriers increase and reaction energies become less exothermic in enzymatic and aqueous environments, the effects of the bulk environment are similar in the presence and absence of small molecules bound to the nucleobase. Therefore, the effects of hydrogen bonding with the bases are approximately the same in all environments. Our results suggest that hydrogen bonding with the nucleobase may play an important role in the glycosidic bond cleavage in both pyrimidine and purine nucleotides in a variety of environments.

Phenolic toxins and mutagenic diazoquinones generate C-linked adducts at the C8 site of 2'-deoxyguanosine (dG) through the intermediacy of radical species. We have previously reported the site-specific incorporation of these adducts into oligonucleotides using a postsynthetic palladium-catalyzed cross-coupling strategy [Omumi (2011 ) J. Am. Chem. Soc. 133 , 42 - 50 ]. We report here the structural impact of these lesions within two decanucleotide sequences containing either 5'- and 3'-flanking pyrimidines or purines. In the complementary strands, the base opposite (N) the C-linked adduct was varied to determine the possibility of mismatch stabilization by the modified nucleobases. The resulting adducted duplex structures were characterized using UV thermal denaturation studies, circular dichroism, fluorescence spectroscopy, and molecular dynamics (MD) simulations. The experimental data showed the C-linked adducts to destabilize the duplex when base paired with its normal partner C but to increase duplex stability within a G:G mismatch. The stabilization within the G:G mismatch was sequence dependent, with flanking purine bases playing a key role in the stabilizing influence of the adduct. MD simulations showed no large structural changes to the B form double helix, regardless of the (anti/syn) adduct preference. Consideration of H-bonding and stacking interactions derived from the MD simulations together with the thermal melting data and changes in fluorescent emission of the adducts upon hybridization to the complementary strands implied that the C-linked phenolic adducts preferentially adopt the syn-conformation within both duplexes regardless of the opposite base N. Given that biological outcome in terms of mutagenicity appears to be strongly correlated to the conformational preference of the corresponding N-linked C8-dG adducts, the potential biological implications of phenolic C-linked adducts are discussed.

Direct addition of aryl radical species to the C(8)-site of 2'-deoxyguanosine (dG) affords C(8)-aryl-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons (PAHs), and certain phenolic toxins. A common property of C(8)-arylpurine adduction is the accompaniment of abasic site formation. To determine how the C(8)-aryl moiety contributes to sugar loss, UV-vis spectroscopy has been employed to determine N(7) pK(a1) values and hydrolysis kinetics, while density functional theory (DFT) calculations have been utilized to probe the structural features and stability of the C(8)-aryl-dG adducts bearing different para and ortho substituents. In all cases, the C(8)-aryl-dG adducts adopt a syn conformation containing a strong O(5)'-H...N(3) hydrogen bond with the aryl ring twisted with respect to the nucleobase. The adducts undergo N(7)-protonation with ionization constants and calculated N(7) proton affinity (PA) values similar to those measured for dG. The hydrolysis kinetics shows that C(8)-aryl-dG nucleoside adducts are more prone than dG to acid-catalyzed hydrolysis, with those bearing para substituents having k(1) values that are ca. 90- to 200-fold larger than k(1) for dG, while the effects for the ortho adducts are only ca. 9- to 60-fold larger. Changes in the rate of hydrolysis are further explained by calculations showing that glycosidic bond cleavage in the syn orientation of both neutral and N(7)-protonated dG has a lower barrier than the anti orientation, and the bulky (phenyl) group further decreases the barrier. Despite adduct reactivity in acidic media, all adducts are relatively stable at physiological pH with t(1/2) approximately 25 days, suggesting that they are unlikely intermediates leading to abasic site formation at physiological pH. This information has allowed development of a new rationale for the tendency of abasic site formation to accompany C(8)-arylpurine adduction within duplex DNA at neutral pH.

Bulky DNA adducts are formed through the covalent attachment of aryl groups to the DNA nucleobases. Many of these adducts are known to possess conformational heterogeneity, which is responsible for the variety of mutagenic outcomes associated with these lesions. The present contribution reviews several conformational and mutagenic themes that are prevalent among the DNA adducts formed at the C8-site of the guanine nucleobase. The most important conclusions obtained (to date) from experiments are summarized including the anti/syn conformational preference of the adducts, their potential to inflict DNA mutations and mismatch stabilization, and their interactions with DNA polymerases and repair enzymes. Additionally, the unique role that computer calculations can play in understanding the structural properties of these adducts are highlighted.

Pyrrole- and indole-linked C(8)-deoxyguanosine nucleosides act as fluorescent reporters of H-bonding specificity. Their fluorescence is quenched upon Watson-Crick H-bonding to dC, while Hoogsteen H-bonding to G enhances emission intensity. The indole-linked probe is ∼ 10-fold brighter and shows promise as a fluorescent reporter of Hoogsteen base pairing.