Effects of bromide upon reaction of nucleosides with hydrogen peroxide induced by ultraviolet light

When ultraviolet light was irradiated on a neutral solution of deoxynucleosides and hydrogen peroxide, concentrations of all the deoxynucleosides decreased greatly. Addition of bromide in the system suppressed the reactions of 2′-deoxycytidine, 2′-deoxythymidine, and 2′-deoxyadenosine, but not that of 2′-deoxyguanosine. Addition of hydroxyl radical scavengers suppressed the reaction. The effect of deuterium oxide, an enhancer of singlet oxygen, was small. It is reported that hydroxyl radical, generating from hydrogen peroxide by ultraviolet irradiation, can react with bromide forming bromine radical, and that bromine radical reacts with bromide forming dibromide radical anion. Our result of dose dependency of bromide suggests that dibromide radical anion is the reaction species to react only with 2′-deoxyguanosine.     

Effects of Halides on Reaction of Nucleosides with Ozone

GRAPHICAL ABSTRACT

Ozone (O₃), a major component of photochemical oxidants, is used recently as a deodorizer in living spaces. It has been reported that O₃ can directly react with DNA, causing mutagenesis in human cells and carcinogenesis in mice. However, little is known about the effects of coexistent ions in the reaction of O₃. In the present study, we analyzed the effects of halides on the reaction of O₃ with nucleosides using reversed-phase high-performance liquid chromatography with ultraviolet detection. When aqueous O₃ solution was added to a nucleoside mixture in potassium phosphate buffer (pH 7.3), the nucleosides were consumed with the following decreasing order of importance: dGuo > Thd > dCyd > dAdo. The effects of addition of fluoride and chloride in the system were slight. Bromide suppressed the reactions of dGuo, Thd, and dAdo but enhanced the reaction of dCyd. The major products were 5-hydroxy-2′-deoxycytidine, 5-bromo-2′-deoxycytidine, and 8-bromo-2′-deoxyguanosine. The time course and pH dependence of the product yield indicated formation of hypobromous acid as the reactive agent. Iodide suppressed all the reactions effectively. The results suggest that bromide may alter the mutation spectrum by O₃ in humans.     

Consequences of the salvage of purine compounds on the proliferation of rat T-lymphocytes with normal or inhibited purine de novo synthesis

We studied the ability of purine compounds to restore the proliferation of concanavalin-A-stimulated rat T-lymphocytes under conditions of purine de novo synthesis inhibition and, on the other hand, the inhibition by purine nucleosides of the response of these cells to a mitogenic stimulation under conditions of normal purine de novo synthesis. The use of 50 μM azaserine, a potent inhibitor of purine de novo synthesis, allowed us to define the physiologically active salvage pathways of purine bases, ribo- and deoxyribonucleosides in concanavalin-A-stimulated rat T-lymphocytes. Except for guanylic compounds, all purines completely restored cell proliferation at a concentration of 50 μM. Guanine, guanosine and 2′-deoxyguanosine at concentrations up to 500 μM did not allow us to restore more than 50% of the cell proliferation. In conditions of normal purine de novo synthesis, the addition of 1000 μM adenine, adenosine, 2′-deoxyadenosine or 100 μM 2′-deoxyguanosine inhibited rat T-lymphocyte proliferation. The differences between the degree of inhibition of cell proliferation could be explained only in part by the differences between the capacities of salvage of these compounds. Furthermore, the fact that 2′-deoxyguanosine toxicity was dependent and 2′-deoxyadenosine toxicity independent on the activation state of the cells provided more evidence that the biochemical mechanisms of inhibition of cell proliferation should be different for these two nucleosides.     

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.     

FUNCTIONALIZATION OF PYRIMIDINE AND PURINE NUCLEOSIDES AT C4 AND C6: C-NUCLEOPHILIC SUBSTITUTION OF THEIR C4- AND C6-(1,2,4-TRIAZOL-1-YL) DERIVATIVES

A study of C-nucleophilic substitution at the C4-position on pyrimidine and C6-position on 2′-deoxyguanosine to produce novel nucleosides is presented with the spectroscopic properties of their respective substitution products. C4-(1,2,4-triazol-1-yl) pyrimidine nucleosides 1 were treated with nitroalkanes, malononitrile, acetylacetone, ethyl nitroacetate, acetoacetate and cyanoacetate at 100°C in dioxane in the presence of DBU resulting in the production of novel nucleosides 2–11. To explore the application of this methodology to purine chemistry, this approach was used to produce novel analogs from 2′-deoxyguanosine. We found that the triazolo derivative 12 undergoes C-nucleophilic substitution with nitromethane, malononitrile, acetylacetone, ethyl nitroacetate and cyanoacetate in the presence of potassium carbonate (K₂CO₃) in DMF at 100°C to give novel nucleosides 13–7.     

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.     

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.     

Selective Deuteration Labelling of 2′-Deoxyguanosine at the Carbon C-4’ Position

Abstract

Selective incorporation of deuterium within the sugar moiety of nucleosides and oligonucleotides can be used for different purposes including isotopic effect determination in mechanistic studies, massspectrometry fragmentation investigations, nuclear magnetic resonance analyses. We wish to report a simple method which allows the selective deuteration labelling of 2'-deoxyguanosine at the C-4'position through the intermediary of 9-(2-deoxy-B-D-erythropento-1,5-dialdo-114-furanosyllquanine. Heating of aqueous pyridine solution [1:11 of 2′-deoxyguanosine-5′-aldehyde for 1 hr at 60°C leads to a partial epimerisation of carbon C-4' with subsequent formation of 9-(2-deoxy-α-L-threopento-1,5-dialdo-1,4-furanosyl)guanine in 40% yield. A likely intermediate of this reaction appears to be a 5'-enol derivative. Similar treatment of 2′-deoxyguanosine-5′-aldehyde in D₂0-pyridine [1-1] gives after NaBH₄ reduction 60% of 2′-deoxyguanosine which is selectively deuterated at the C-4′ position. The extend of the isotopic labelling was up to 95% as determined by high resolution electron impact mass spectrometry and ¹H NMR analyses. Heating of the aqueous pyridine solution of 2′-deoxyguanosine-5′-aldehyde for a longer period (3–4 hrs) gave rise to two other nucleosides which where assigned as 9-(2-deoxy-α-D-threo-pentofuranosy1)guanine and 9-(2-deoxy-n-L-erythro-pentofuranosyl)guanine. A retro-aldol mechanism appears to be involved in the epimerization reaction which takes place at carbon C-3′.     

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.     

Trimethylsilyl Trifluoromethanesulfonate-promoted Reductive 2′-O-arylmethylation of Ribonucleoside Derivatives

Arylmethyl groups such as benzyl, p-methoxybenzyl, and 1-pyrenylmethyl groups were introduced to the 2′-O-position of nucleosides by reductive etherification. Combining corresponding aromatic aldehydes with 2′-O-trimethylsilylnucleoside derivatives in the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) resulted in moderate to good yields of the 2′-O-arylmethyluridine derivatives, whereas the corresponding cytidine and adenosine derivatives were obtained in low yields. The reaction of ribonucleosides with aliphatic aldehydes did not proceed smoothly. Anomerization of the uridine derivatives by TMSOTf was observed in CH₂Cl₂, toluene, and CH₃CN, but was completely suppressed when the reactions were conducted in 1,4-dioxane.     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

2′-Modified Guanosine Analogs for the Treatment of HCV

Novel 2′-modified guanosine nucleosides were synthesized from inexpensive starting materials in 7–10 steps via hydroazidation or hydrocyanation reactions of the corresponding 2′-olefin. The antiviral effectiveness of the guanosine nucleosides was evaluated by converting them to the corresponding 5′-O-triphosphates (compounds 38–44) and testing their biochemical inhibitory activity against the wild-type NS5B polymerase.     

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.     

Some Examples of the Use of Trifluoromethane Sulfonic Anhydride in Nucleic Acid Chemistry

Abstract

Trifluoromethane sulfonic anhydride has been used for the inversion of configuration at the 3′-position of 2′-deoxypurine nucleosides, for the modification of the base moiety of purine and pyrimidine nucleosides, for nucleophilic substitution in the sugar moiety, for the synthesis of O²,3′-cyclothymidine and for sugar-base condensation reactions. Reaction can be carried out under very mild conditions. The conditions for these reactions are quite different so that a good selectivity can be obtained when different reactive groups are present.     

HPLC as a Tool to Study the Kinetics of Parallel and Consecutive Reactions: Competitive Interconversion,Dephosphorylation, Deamination and Depyrimidination of Cytidine 2′- and 3′-Monophosphates in Aqueous Acid

Abstract

The applicability of HPLC as a method to study the kinetics of complicated reaction systems of nucleosides and nucleotides has been demonstrated by using the hydrolytic reactions of cytidine 2′- and 3′-monophosphates as an example.     

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.     

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.     

Urinary Nucleosides as Biomarkers of Breast,Colon, Lung,and Gastric Cancer in Taiwanese

Urinary nucleosides are associated with many types of cancer. In this study, six targeted urinary nucleosides, namely adenosine, cytidine, 3-methylcytidine, 1-methyladenosine, inosine, and 2-deoxyguanosine, were chosen to evaluate their role as biomarkers of four different types of cancer: lung cancer, gastric cancer, colon cancer, and breast cancer. Urine samples were purified using solid-phase extraction (SPE) and then analyzed using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The Mann-Whitney U test and Principal Component Analysis (PCA) were used to compare differences in urinary nucleosides between patients with one of four types of cancer and healthy controls. The diagnostic sensitivity of single nucleosides for different types of cancer ranged from 14% to 69%. In contrast, the diagnostic sensitivity of a set of six nucleosides ranged from 37% to 69%. The false-positive identification rate associated with the set of six nucleosides in urine was less than 2% compared with that of less than 5% for a single nucleoside. Furthermore, combining the set of six urinary nucleosides with carcinoembryonic antigen improved the diagnostic sensitivity for colon cancer. In summary, the study show that a set of six targeted nucleosides is a good diagnostic marker for breast and colon cancers but not for lung and gastric cancers.     

15N-Labeled Oligodeoxynucleotides -Useful Probes for 1H-NMR Investigations

Abstract

The amino protons of ¹⁵N-labeled deoxyoligonucleotides were studied as possible structural probes for NMR investigations of the interaction between DNA and regulatory proteins. To apply this strategy, 6-¹⁵NH₂-2′ -deoxyadenoslne, 4-¹⁵NH -2′-deoxycytidine and 2-¹⁵NH -2′-deoxyguanosine were chemically synthesized. The labeled nucleosides were introduced into distinct positions of oligodeoxy-nucleotides by large-scale DNA synthesis. The behaviour of the ¹⁵N-coupled cytidine amino protons in a 18 base pair (bp) lac operator sequence were investigated using H-¹⁵N INDOR spectroscopy .