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.
Contents.1. Experimental 2. NMR spectra of adducts 1-4 3. Table S1 4. Figure S1 5. Figure S2a-c Experimental General Methods: 1 H NMR spectra were recorded on a Bruker Avance 300 DPX or Bruker Avance 400 DPX operating at 300.1 MHz and 400.1 MHz, respectively, and 13 C NMR spectra were recorded on a Bruker Avance 300 DPX operating at 75.5 MHz.1 H NMR spectra were referenced to the residual proton solvent signal of the deuterated solvent and 13 C NMR spectra were referenced to the 13 C NMR resonance of the deuterated solvent. All 13 C NMR spectra were acquired using the J-modulated (JMOD) pulse sequence with C, CH 2 carbon atoms pointing up and CH, CH 3 carbon atoms pointing down.Unless otherwise noted, commercial compounds were used as received. Boronic acids were purchased from Sigma-Aldrich. Pd 3 (OAc) 6 was purchased from Strem Chemical and 2'-deoxyguanosine was from ChemGenes. Formic acid was obtained from Sigma-Aldrich. The synthesis of 8-Br-dG was performed according to literature methods. 1 High resolution mass spectrometry (HRMS) was conducted at the McMaster Regional Centre for Mass Spectrometry, Hamilton, Ontario.UV-Vis spectra were recorded on a Cary 300-Bio UV-Vis spectrophotometer equipped with a Peltier block-heating unit, stirrer and temperature controller.
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.
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