Time And Concentration Dependence Of Fenton-Induced Oxidation Of dG

The influence of incubation time and Fenton reagent concentrations was investigated on the oxidation of 2′-deoxyguanosine. The compounds identified and quantified, through use of an LC-MS/MS system, were 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) and 8,5′-cyclo-2′-deoxyguanosine (8,5′cyclodG) and the secondary oxidation products guanidinohydantoin and dehydro-guanidinohydantoin. 8-oxodG and 8,5′cyclodG formed very quickly, reaching a maximum rapidly, but with 8-oxodG a rapid decline occurred thereafter due to its further oxidation into the secondary products, which formed more slowly. Due to the better stability, 8,5′cyclodG correlated better with the general level of oxidation than 8-oxodG. The results emphasize the advantages of measuring other oxidation adducts than 8-oxodG alone.     

Oxidation of Guanine Nucleosides to 4-Amidinocarbamoyl- 5-hydroxyimidazoles by Dimethyldioxirane

Abstract

Final oxidation products generated from guanosine and 2′-deoxyguanosine by reaction with dimethyldioxirane have been identified as 4-amidinocarbamoyl-5-hydroxyimidazoles.     

Chemical and biological consequences of oxidatively damaged guanine in DNA

Abstract

Of the four native nucleosides, 2′-deoxyguanosine (dGuo) is most easily oxidized. Two lesions derived from dGuo are 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (Fapy)?dGuo. Furthermore, while steady-state levels of 8-oxodGuo can be detected in genomic DNA, it is also known that 8-oxodGuo is more easily oxidized than dGuo. Thus, 8-oxodGuo is susceptible to further oxidation to form several hyperoxidized dGuo products. This review addresses the structural impact, the mutagenic and genotoxic potential, and biological implications of oxidatively damaged DNA, in particular 8-oxodGuo, Fapy?dGuo, and the hyperoxidized dGuo products.     

Acetylation of Nucleosides by Acetylsalicylic Acid (Aspirin)1

Abstract

Acetylsalicylic acid (aspirin) reacted with adenosine, cytidine, guanosine and their 2′-deoxynucleosides to give acetylated nucleosides. Cytidine and 2′-deoxycytidine gave N⁴-acetylated nucleosides in nitromethane while in pyridine fully acetylated products were obtained. Adenosine and 2′-deoxyadenosine also gave fully acetylated products. However, guanosine and 2′-deoxyguanosine gave 2′,3′,5′-tri-O-acetylribosyl and 3′,5′-di-O-acetyl-2′-deoxyribosyl nucleosides, respectively. The corresponding aglycons also gave acetylated heterocycles under various conditions.     

Formation of spiroiminodihydantoin nucleoside from 8-oxo-7,8-dihydro-2′-deoxyguanosine by nitric oxide under aerobic conditions

When 8-oxo-7,8-dihydro-2′-deoxyguanosine in potassium phosphate buffer of pH 7.4 was bubbled by nitric oxide at room temperature under aerobic conditions, two major products were formed. They were identified as the diastereomers of spiroiminodihydantoin deoxyribonucleoside on the basis of their identical ESI-MS and UV spectra and HPLC retention times with those of the major products in reaction of 8-oxo-7,8-dihydro-2′-deoxyguanosine with hypochlorous acid. A 1000-fold excess of 2′-deoxyguanosine did not inhibit the reaction of 8-oxo-7,8-dihydro-2′-deoxyguanosine with nitric oxide. The results suggest that an 8-oxo-7,8-dihydroguanine moiety formed in DNA may react with nitric oxide in the presence of oxygen molecule generating spiroiminodihydantoin in humans.     

Structure of a neothramycin-2′-deoxyguanosine adduct

The interaction of neothramycin, a pyrrolo (1,4)benzodiazepine antibiotic with antitumor activity, with deoxyribonucleosides was studied as a model of the drug-DNA binding. The antibiotic was demonstrated to react with 2′-deoxyguanosine in aqueous dimethyl sulfoxide. Two reaction products were found on thin-layer plates. The major one was purified with a low yield, and the chemical structure was determined by PMR and mass spectrometry to be a one-to-one adduct of neothramycin and 2′-deoxyguanosine. A covalent bond was formed between C-3 of the antibiotic and N-2 of 2′-deoxyguanosine by dehydration.     

A One Step Synthesis of O6-Methyl-2′-deoxyguanosine

Abstract

A single step chemical synthesis of N⁷-methyl-2′-deoxyguanosine (m⁷dG), N¹-methyl-2′-deoxyguanosine (m¹dG) and O⁶-methyl-2′-deoxyguanosine (m⁶dG) is described. The products were separated on the silical gel plates and characterized by nuclear magnetic resonance and mass spectrometry.     

DNA strand breaks and base modifications induced by cholesterol hydroperoxides

Abstract

Cholesterol (Ch) can be oxidized by reactive oxygen species, forming oxidized products such as Ch hydroperoxides (ChOOH). These hydroperoxides can disseminate the peroxidative stress to other cell compartments. In this work, the ability of ChOOH to induce strand breaks and/or base modifications in a plasmid DNA model was evaluated. In addition, HPLC/MS/MS analyses were performed to investigate the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) after the incubation of 2′-deoxyguanosine (dGuo) with ChOOH and Cu²⁺. In the presence of copper ions, ChOOH induced DNA strand breaks in time and concentration-dependent manners. Purine and pyrimidine base modifications were also observed, as assessed respectively by the treatment with Fpg and Endo III repair enzymes. The detection of 8-oxodGuo by HPLC/MS/MS is in agreement with the dGuo oxidation in plasmid DNA. ChOOH-derived DNA damage adds further support to the role of lipid peroxidation in inducing DNA modifications and mutation.     

Quantitative determination of 8-hydroxy-2′-deoxyguanosine in human urine by isotope dilution mass spectrometry: normal levels in hemochromatosis

A previously developed method for quantitative determination of 8-hydroxyguanine by gas chromatography-mass spectrometry was modified to allow measurement of 8-hydroxy-2′-deoxyguanosine in human urine. [4,5,6,8-¹³C₄]8-Hydroxy-2′-deoxyguanosine was prepared by enzymatic coupling of [4,5,6,8-¹³C₄]8-hydroxyguanine to deoxyribose-1-phosphate. Samples of human urine (2 ml) were spiked with the labeled nucleoside (13 nmol) and subjected to solid phase extraction and reversed phase high performance liquid chromatography. The 8-hydroxy-2′-deoxyguanosine thus isolated was hydrolyzed by treatment with aqueous formic acid, and the resulting 8-hydroxyguanine was converted into its tetrakis-trimethylsilyl derivative and subjected to gas-liquid chromatographic-mass spectrometric analysis. Repeated determinations of 8-hydroxy-2′-deoxyguanosine in pools of urine showed coefficients of variation of 5 and 8% at concentrations of 8-hydroxy-2′-deoxyguanosine equal to 18 and 2 nM, respectively. Determination of 8-hydroxy-2′-deoxyguanosine in samples of urine spiked with different amounts of the unlabeled nucleoside showed a mean recovery of 102%. Application of the analytical method to a group of 11 apparently healthy subjects (mean age, 47 years) showed a mean level of endogenously produced 8-hydroxy-2′-deoxyguanosine equal to 1.33 ± 0.29 μmol/mol creatinine. The level recorded for another group of 15 younger subjects (mean age, 28 years) was somewhat higher, that is, 1.58 ± 0.84 μmol/mol creatinine, corresponding to a 24-h production rate of 8-hydroxy-2′-deoxyguanosine equal to 20.6 ± 11.6 nmol (288 ± 140 pmol/24 h · kg body weight). Hemochromatosis is a hereditary disease characterized by increased absorption of iron from the gastrointestinal tract and deposition of iron in organs. Application of the analytical method to a group of 12 patients with hereditary hemochromatosis who were under treatment with venesections showed a mean level of urinary 8-hydroxy-2′-deoxyguanosine equal to 1.39 ± 0.40 μmol/mol creatinine. This value was not significantly different from those of healthy subjects. The fact that these patients had only slight or moderate iron overload at the time of urinary sample collection may have influenced the urinary levels of 8-hydroxy-2′-deoxyguanosine in the present study.     

Deuteration and Tritiation of 2′-Deoxyribonucleosides in the 5′ and 4′ Positions

Abstract

The deuterations of 2′-deoxyguanosine in the 4′ and 5′ positions have been described elsewhere (1). The starting material is the 5′-aldehyde formed by mild oxidation with N,N-dicyclohexyl carbodiimide in dimethyl sulphoxide of the fully protected nucleoside with free 5′-alcoholic function. The 5′4euteration was achieved by reduction with deuterated sodium borohydride. Incorporation of deuterium in the 4′-position was achieved v i a an enhanced keto-enol tautomerim by heating the aldehyde in 50/50 D20/pyridine, with subsequent reduction of the aldehyde with NaBH4. The 6-furanoid form was isolated from the I-lyxo by-product by reverse phase HPLC. Applied to pyrimidine 2′-deoxyribonucleosides, this method was shown to give deuterated 2′-deoxycytidine and thymidine in good yield.     

Induction of 1,N(2)-etheno-2'-deoxyguanosine in DNA exposed to beta-carotene oxidation products

Epidemiological studies testing the effect of β-carotene in humans have found a relative risk for lung cancer in smokers supplemented with β-carotene. We investigated the reactions of retinal and β-apo-8′-carotenal, two β-carotene oxidation products, with 2′-deoxyguanosine to evaluate their DNA damaging potential. A known mutagenic adduct, 1,N²-etheno-2′-deoxyguanosine, was isolated and characterized on the basis of its spectroscopic features. After treatment of calf thymus DNA with β-carotene or β-carotene oxidation products, significantly increased levels of 1,N²-etheno-2′-deoxyguanosine and 8-oxo-7,8-dihydro-2′-deoxyguanosine were quantified in DNA. These lesions are believed to be important in the development of human cancers. The results reported here may contribute toward an understanding of the biological effects of β-carotene oxidation products.     

Formation of N-(1-Oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine by the Reaction of 2′-Deoxyguanosine with 3-Deoxyglucosone

3-Deoxyglucosone (3DG) has weaker mutagenicity than methylglyoxal by the Ames test. 3DG reacted readily with 2′-deoxyguanosine (dG) in nucleosides. Two major products (G-A and G-B) were isolated and purified from the reaction mixture of 50 mM 3DG and 50 mM dG at 50°C and pH 7.4 for 6d. G-A was identified as N-(1-oxo-2,4,5,6-hydroxyhexyl)-2′-deoxyguanosine. G-B was identified as a diastereomer of G-A.     

Impairment of the Viability of Transformed Chinese Hamster Cells in a Nonsubcultured Culture under the Influence of Exogenous Oxidized Guanoside is Manifested Only in the Stationary Phase of Growth

Despite the fact that oxidation products of nucleotides and nucleosides are markers of oxidative stress, reports of the paradoxical ability of these compounds to protect cells from the harmful effects of reactive oxygen species began to appear more often. Among all nitrogenous bases, guanine is most susceptible to the influence of oxidative stress; therefore, guanosine is oxidized more often than other bases. In the present work, the effect of exogenous 8-oxo-2′-deoxyguanosine on the growth and “stationary phase aging” (accumulation of “age-related” changes in cultured cells during cell proliferation slowing down within a single passage and subsequent “aging” in the stationary growth phase) of nonsubcultured transformed Chinese hamster cells was studied. We showed that the nucleoside is rapidly absorbed by the cells from the medium, but it does not affect the growth of the culture, and impairs the viability of the cells in the late stationary growth phase. Thus, no mitogenic or geroprotective effect of 8-oxo-2′-deoxyguanosine was found.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

Synthesis and Characterization of Oligodeoxynucleotides Containing 5′,8-Cyclopurine-2′-Deoxyribonucleosides

Abstract

Oligodeoxynucleotides containing the two 5′R and 5′S diastereoisomers of 5′,8-cyclo-2′-deoxyadenosine (CyclodAdo) and 5′,8-cyclo-2′-deoxyguanosine (CyclodGuo) have been synthesized using the phosphoramidite chemistry. The structural assignment and a few biochemical features of these modified DNA fragments are reported.     

Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO^?) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5′,8-cyclo-2′-deoxyguanosine (5′,8-cdG) and 5′,8-cyclo-2′-deoxyadenosine (5′,8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]₄ (TG4T), d[AGGG(TTAGGG)₃] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)₃A] (mutTel24) were exposed to HO^? radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L?>?OC???SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (～50% solution folded) and mutTel24 (～90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33?Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.     

Synthesis of Suitably-Protected Phosphoramidites of 2′-Fluoro-2′-Deoxyguanosine and 2′-Amino-2′-Deoxyguanosine for Incorporation Into Oligoribonucleotides

Abstract

A novel synthesis of 2′-fluoro-2′-deoxyguanosine employing DAST as the fluorinating agent is presented. The preparation of its phosphoramidite as well as that of 2′-amino-2′-deoxyguanosine is also described.     

链霉菌FIM-080014产生的核苷类代谢产物研究

本文采用大孔吸附树脂、硅胶柱色谱、反相柱色谱、凝胶sephadex LH-20及HPLC等方法对链霉菌FIM-080014发酵液及菌丝体中的代谢产物进行分离,得到7个核苷类化合物。通过NMR及MS等方法鉴定了上述化合物的结构,分别为尿嘧啶(1)、尿嘧啶核苷(2)、2'-脱氧尿嘧啶核苷(3)、5'-C-甲基尿嘧啶核苷(4)、2'-脱氧胸腺嘧啶核苷(5)、2'-脱氧鸟嘌呤核苷(6)和次黄嘌呤(7),其中化合物4首次从微生物代谢产物中分离得到。活性分析表明化合物1～7对神经氨酸酶具有一定的抑制活性,其IC50值分别为3.7、2.1、4.4、1.4、3.6、2.5和2.4 mM。     

Oxidation Chemistry of Adenosine-3′, 5′-Cyclic Monophosphate at Pyrolytic Graphite Eletrode

The voltammetric oxidation of adenosine-3′,5′-cyclic monophosphate (3′,5′-CAMP) has been studied in the pH range 2.13–10.07 using pyrolytic graphite electrode (PGE). Voltammetric, coulometric, spectral studies, and product characterization indicate that the oxidation of 3′,5′-CAMP occurs in an EC reaction involving a 6H⁺, 6e process at pH 7.24. Electrooxidized products were seperated by semipreparative high performance liquid chromatography (HPLC) and were characterized by mp, ¹HNMR, FTIR, and GC-mass as allantoin cyclic ribose monophosphate and 3 dimers as the major products. A detailed interpretation of the redox mechanism of 3′,5′-CAMP also has been presented to account for the formation of various products.