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14 results on '"Shafirovich V"'

1. DNA Lesions Derived from the Site Selective Oxidation of Guanine by Carbonate Radical Anions

Author

Joffe, A., Geacintov, N. E., and Shafirovich, V.

Abstract

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order:  5‘-d(CCATCCTACC) [(5.7 ± 0.6) × 106 M-1 s-1] < 5‘-d(TATAACGTTATA), self-complementary duplex [(1.4 ± 0.2) × 107 M-1 s-1] < 5‘-d(CCATCGCTACC [(2.4 ± 0.3) × 107 M-1 s-1] < 5‘-d(CCATC[8-oxo-G]CTACC) [(3.2 ± 0.4) × 108 M-1 s-1], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of ~2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40−60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

Published
2003

2. Oxidative Generation of Guanine Radicals by Carbonate Radicals and Their Reactions with Nitrogen Dioxide to Form Site Specific 5-Guanidino-4-nitroimidazole Lesions in Oligodeoxynucleotides

Author

Joffe, A., Mock, S., Yun, B. H., Kolbanovskiy, A., Geacintov, N. E., and Shafirovich, V.

Abstract

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5‘-d(ACC CG1C G*2TC CG*3C G4CC) and 5‘-d(ACC CG1C G*2TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G*2) and 158 (G*3) mutation hot spots in the former sequence with four Gs and the codon 157 (G*2) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20−40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2‘,3‘,5‘-tri-O-acetylguanosine, to 2.1−2.6 in the single-stranded oligodeoxynucleotides and to 0.8−1.1 in the duplexes. The adduct of the 5‘-d(ACC CG1C G*2TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G2) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

Published
2003

3. Photochemically Catalyzed Generation of Site-Specific 8-Nitroguanine Adducts in DNA by the Reaction of Long-Lived Neutral Guanine Radicals with Nitrogen Dioxide

Author

Shafirovich, V., Mock, S., Kolbanovskiy, A., and Geacintov, N. E.

Abstract

A novel photochemical approach is described for synthesizing site-specific 8-nitro-2‘-deoxyguanosine (8-nitro-dG) adducts DNA. The method is based on the bimolecular reaction of a neutral, deprotonated guanine radical [G(-H)•] in DNA and nitrogen dioxide (•NO2) radicals. This approach is illustrated using the single-stranded oligodeoxyribonucleotide 5‘-d(CCATCGCTACC) dissolved in an aqueous solution of nitrite and bicarbonate anions at pH 7.5. The photochemical synthesis was triggered by the selective photodissociation of persulfate anions to yield SO4•- radical anions by either 308 nm XeCl excimer laser pulses or by a continuous irradiation with 290−340 nm light from a 1000 W Xe lamp. The sulfate radicals formed generate the CO3•- and •NO2 radicals by one-electron oxidation of the bicarbonate and nitrite anions. In turn, the CO3•- radicals site-selectively generate G(-H)• radicals in DNA that combine with •NO2 to form 8-nitro-dG lesions in the oligonucleotide. The nitrated oligonucleotides were purified by reversed-phase HPLC techniques and are stable at 4 °C for at least 4 days, but depurinate at ambient temperatures of 23 °C at pH 7 with a half-life of ~20 h. The nature of the reaction and decomposition products were studied by a combination of ESI and MALDI-TOF mass spectrometric techniques.

Published
2002
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4. Nitrogen Dioxide as an Oxidizing Agent of 8-Oxo-7,8-dihydro-2‘-deoxyguanosine but Not of 2‘-Deoxyguanosine

Author

Shafirovich, V., Cadet, J., Gasparutto, D., Dourandin, A., and Geacintov, N. E.

Abstract

The redox reactions of guanine and its widely studied oxidation product, the 8-oxo-7,8-dihydro derivative, are of significant importance for understanding the mechanisms of oxidative damage in DNA. Employing 2‘-deoxyguanosine 5‘-monophospate (dGMP) and 8-oxo-7,8-dihydro-2‘-deoxyguanosine (8-oxo-dG) in neutral aqueous solutions as model systems, we have used nanosecond laser flash photolysis to demonstrate that neutral radicals, dGMP(−H)•, derived by the one-electron oxidation and deprotonation of dGMP, can oxidize nitrite anions (NO2-) to the nitrogen dioxide radical •NO2. In turn, we show that •NO2 can give rise to a one-electron oxidation of 8-oxo-G, but not of dGMP. The one-electron oxidation of dGMP was initiated by a radical cation generated by the laser pulse-induced photoionization of a pyrene derivative with enhanced water solubility, 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene (BPT). The dGMP(−H)• neutral radicals formed via deprotonation of the dGMP•+ radical cations and identified by their characteristic transient absorption spectrum (λmax ~ 310 nm) oxidize nitrite anions with a rate constant of (2.6 ± 0.3) × 106 M-1 s-1. The 8-oxo-dG is oxidized by •NO2 with a rate constant of (5.3 ± 0.5) × 106 M-1 s-1. The 8-oxo-dG(−H)• neutral radicals thus generated are clearly identified by their characteristic transient absorption spectra (λmax ~ 320 nm). The rate constant of 8-oxo-dG oxidation (k12) by the •NO2 one-electron oxidant (the •NO2/NO2- redox potential, E° ≈ 1.04 V vs NHE) is lower than k12 for a series of oxidizing aromatic radical cations with known redox potentials. The k12 values for 8-oxo-dG oxidation by different aromatic radical cations derived from the photoionization of their parent compounds depend on the redox potentials of the latter, which were in the range of 0.8−1.6 V versus NHE. The magnitude of k12 gradually decreases from a value of 2.2 × 109 M-1 s-1 (E° = 1.62 V) to 5.8 × 108 M-1 s-1 (E° = 1.13 V) and eventually to 5 × 107 M-1 s-1 (E° = 0.91 V). The implications of these results, including the possibility that the redox cycling of the •NO2/NO2- species can be involved in the further oxidative damage of 8-oxo-dG in DNA in cellular environments, are discussed.

Published
2001

5. Role of Hydrophobic Effects in the Reaction of a Polynuclear Aromatic Diol Epoxide with Oligodeoxynucleotides in Aqueous Solutions

Author

Pirogov, N., Shafirovich, V., Kolbanovskiy, A., Solntsev, K., Courtney, S. A., Amin, S., and Geacintov, N. E.

Abstract

The need for large-scale direct synthesis of stereochemically defined and site-specific benzo[a]pyrenediol epoxide−oligodeoxyribonucleotide adducts for detailed NMR and other biochemical and physicochemical studies has necessitated a better understanding of variables that lead to an enhancement of the reaction yields. It is shown that, in aqueous solution, the formation of noncovalent hydrophobic complexes between 7r,8t-dihydroxy-9t,10t-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and the single-stranded oligonucleotide 5‘-d(CCATCGCTACC) precedes the covalent binding reaction of BPDE with the single deoxyguanosine residue. The yield of covalent reaction products (involving reaction of BPDE at the C10 position with the exocyclic amino group of the dG residue) increases with increasing DNA concentration and tends toward saturation at oligonucleotide single-strand concentrations above ~3 mM. The addition of NaCl (0.3 M) also tends to enhance the adduct reaction yields. However, organic solvents such as tetrahydrofuran in the reaction mixtures (10−40%) decrease the stabilities of the noncovalent complexes, which in turn leads to reductions in the yields of covalent BPDE−dG oligonucleotide adducts. The efficiencies of formation of hydrophobic complexes were probed by fluorescence and UV absorption techniques using the BPDE tetrol hydrolysis product 7,8,9,10-tetrahydroxytetrahydrobenzo[a]pyrene as a model system.

Published
1998

6. Base and Nucleotide Excision Repair Pathways in DNA Plasmids Harboring Oxidatively Generated Guanine Lesions.

Author

Kolbanovskiy M, Aharonoff A, Sales AH, Geacintov NE, and Shafirovich V

Subjects
DNA chemistry, DNA Repair, Guanine chemistry, HeLa Cells, Humans, Nucleic Acid Conformation, Nucleotides chemistry, Oxidation-Reduction, Plasmids, DNA metabolism, Guanine metabolism, Nucleotides metabolism
Abstract

The base and nucleotide excision repair pathways (BER and NER, respectively) are two major mechanisms that remove DNA lesions formed by the reactions of genotoxic intermediates with cellular DNA. We have demonstrated earlier that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) are excised from double-stranded DNA by competing BER and NER in whole-cell extracts [Shafirovich, V., et al. (2016) J. Biol. Chem . 321 , 5309-5319]. In this work we compared the NER and BER yields with single Gh or Sp lesions embedded at the same sites in covalently closed circular pUC19NN plasmid DNA (cccDNA) and in the same but linearized form (linDNA) of this plasmid. The kinetics of the Sp and Gh BER and NER incisions were monitored in HeLa cell extracts. The yield of NER products is ∼5 times greater in covalently closed circular DNA than in the linearized form, while the BER yield is smaller by ∼20-30% depending on the guanine lesion. Control BER experiments with 8-oxo-7,8-dihydroguanine (8-oxoG) show that the BER yield is increased by a factor of only 1.4 ± 0.2 in cccDNA relative to linDNA. These surprising differences in BER and NER activities are discussed in terms of the lack of termini in covalently closed circular DNA and the DNA lesion search dynamics of the NER DNA damage sensor XPC-RAD23B and the BER enzyme OGG1 that recognizes and excises 8-oxoG.

Published
2021
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7. Excision of Oxidatively Generated Guanine Lesions by Competing Base and Nucleotide Excision Repair Mechanisms in Human Cells.

Author

Shafirovich V, Kropachev K, Kolbanovskiy M, and Geacintov NE

Subjects
Cells, Cultured, DNA Repair, Fibroblasts metabolism, Guanine chemistry, HeLa Cells, Humans, Kinetics, Molecular Structure, Oxidation-Reduction, Guanine metabolism
Abstract

The interchange between different repair mechanisms in human cells has long been a subject of interest. Here, we provide a direct demonstration that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) embedded in double-stranded DNA are substrates of both base excision repair (BER) and nucleotide excision repair (NER) mechanisms in intact human cells. Site-specifically modified, 32 P-internally labeled double-stranded DNA substrates were transfected into fibroblasts or HeLa cells, and the BER and/or NER mono- and dual incision products were quantitatively recovered after 2-8 h incubation periods and lysis of the cells. DNA duplexes bearing single benzo[ a]pyrene-derived guanine adduct were employed as positive controls of NER. The NER activities, but not the BER activities, were abolished in XPA -/- cells, while the BER yields were strongly reduced in NEIL1 -/- cells. Co-transfecting different concentrations of analogous DNA sequences bearing the BER substrates 5-hydroxyuracil diminish the BER yields of Sp lesions and enhance the yields of NER products. These results are consistent with a model based on the local availability of BER and NER factors in human cells and their competitive binding to the same Sp or Gh BER/NER substrates.

Published
2019
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8. Generation of guanine-thymine cross-links in human cells by one-electron oxidation mechanisms.

Author

Madugundu GS, Wagner JR, Cadet J, Kropachev K, Yun BH, Geacintov NE, and Shafirovich V

Subjects
Cross-Linking Reagents chemistry, DNA genetics, Free Radicals chemistry, HeLa Cells, Humans, Hydrogen-Ion Concentration, Lasers, Molecular Structure, Oxidation-Reduction, DNA chemistry, DNA Damage, Electrons, Guanine chemistry, Thymine chemistry
Abstract

The one-electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation produces cross-links between guanine and thymine bases (G*-T*), characterized by a covalent bond between C8 guanine (G*) and N3 thymine (T*) atoms. The DNA lesions were quantified by isotope dilution LC-MS/MS methods in the multiple reaction-monitoring mode using isotopically labeled [(15)N, (13)C]-nucleotides as internal standards. Among several known pyrimidine and 8-oxo-7,8-dihydroguanine lesions, the G*-T* cross-linked lesions were detected at levels of ~0.21 and 1.19 d(G*-T*) lesions per 10(6) DNA bases at laser intensities of 50 and 280 mJ/cm(2)/pulse, respectively.

Published
2013
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9. Sequence-dependent variation in the reactivity of 8-Oxo-7,8-dihydro-2'-deoxyguanosine toward oxidation.

Author

Lim KS, Taghizadeh K, Wishnok JS, Babu IR, Shafirovich V, Geacintov NE, and Dedon PC

Subjects
8-Hydroxy-2'-Deoxyguanosine, Base Sequence, Carbonates chemistry, Deoxyguanosine chemistry, Deoxyguanosine genetics, Deoxyguanosine radiation effects, Nitrates chemistry, Oxidation-Reduction, Photochemical Processes, Photosensitizing Agents chemistry, Photosensitizing Agents radiation effects, Riboflavin chemistry, Riboflavin radiation effects, Ultraviolet Rays, Deoxyguanosine analogs & derivatives, Oxidants chemistry
Abstract

The goal of this study was to define the effect of DNA sequence on the reactivity of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) toward oxidation. To this end, we developed a quadrupole/time-of-flight (QTOF) mass spectrometric method to quantify the reactivity of site specifically modified oligodeoxyribonucleotides with two model oxidants: nitrosoperoxycarbonate (ONOOCO(2)(-)), a chemical mediator of inflammation, and photoactivated riboflavin, a classical one-electron oxidant widely studied in mutagenesis and charge transport in DNA. In contrast to previous observations with guanine [ Margolin , Y. , ( 2006 ) Nat. Chem. Biol. 2 , 365 ], sequence context did not affect the reactivity of ONOOCO(2)(-) with 8-oxodG, but photosensitized riboflavin showed a strong sequence preference in its reactivity with the following order (8-oxodG = O): COA ≈ AOG > GOG ≥ COT > TOC > AOC. That the COA context was the most reactive was unexpected and suggests a new sequence context where mutation hotspots might occur. These results point to both sequence- and agent-specific effects on 8-oxodG oxidation.

Published
2012
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10. Generation of guanine-thymidine cross-links in DNA by peroxynitrite/carbon dioxide.

Author

Yun BH, Geacintov NE, and Shafirovich V

Subjects
Alkaline Phosphatase metabolism, Animals, Bicarbonates chemistry, Cattle, Chromatography, High Pressure Liquid, DNA metabolism, Guanidines chemistry, Guanine analogs & derivatives, Hydrogen-Ion Concentration, Nitroimidazoles chemistry, Oligonucleotides chemistry, Oxidation-Reduction, Tandem Mass Spectrometry, Water chemistry, Carbon Dioxide chemistry, DNA chemistry, Guanine chemistry, Peroxynitrous Acid chemistry, Thymidine chemistry
Abstract

Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO(3)(•-) and (•)NO(2) radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2'-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and the thymine N3 (T*) atoms (Crean Nucleic Acids Res. 2008, 36, 742-755). In this work, we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5-7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well-known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitro-G), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxo-G), and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled (15)N,(13)C-labeled 2'-deoxy oligoribonucleotides 5'-dGpT and 5'-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and trioligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction monitoring mode. The NIm and 8-nitro-G are the major products formed (∼0.05% each), and lesser amounts of 8-oxo-G (∼0.02%) and d(G*pT*) and d(G*-T*) enzymatic digestion products (∼0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both interstrand and intrastrand cross-linked products., (© 2011 American Chemical Society)

Published
2011
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11. Oxidation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by oxyl radicals produced by photolysis of azo compounds.

Author

Shao J, Geacintov NE, and Shafirovich V

Subjects
8-Hydroxy-2'-Deoxyguanosine, Deoxyguanosine chemistry, Kinetics, Lasers, Oxidation-Reduction, Peroxides toxicity, Photolysis, Amidines chemistry, Deoxyguanosine analogs & derivatives, Peroxides chemistry
Abstract

Oxidative damage to 8-oxo-7,8-dihydroguanine (8-oxoG) bases initiated by photolysis of the water-soluble radical generator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) has been investigated by laser kinetic spectroscopy. In neutral oxygenated aqueous solutions, 355 nm photolysis of AAPH initiates efficient one-electron oxidation of the 8-oxodG nucleosides directly monitored by the appearance of the 8-oxodG(*+)/8-oxodG(-H)* radicals at 325 nm. The reaction kinetics consist of a mechanism that includes the transformation of the 2-amidinoprop-2-peroxyl radicals (ROO*) derived from photolysis of AAPH to more reactive 2-amidinoprop-2-oxyl radicals (RO*), which directly react with the 8-oxoG bases. The major pathways for the formation of end products of 8-oxoG oxidation include the combination of the 8-oxodG(*+)/8-oxodG(-H)* radicals with superoxide (O(2)(*-)) and ROO* radicals in approximately 1:1 ratios, as demonstrated by experiments with Cu,Zn superoxide dismutase, to form dehydroguanidinohydantoin (Gh(ox)) derivatives. This mechanism was confirmed by analysis of the end products produced by the oxidation of two substrates: (1) the 8-oxoG derivative 2',3',5'-tri-O-acetyl-7,8-dihydroguanosine (tri-O-Ac-8-oxoG) and (2) the 5'-d(CCATC[8-oxoG]CTACC) sequence. The major products isolated by HPLC and identified by mass spectrometry methods were the tri-O-Ac-Gh(ox) and 5'-d(CCATC[Gh(ox)]CTACC products.

Published
2010
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12. Absolute configurations of spiroiminodihydantoin and allantoin stereoisomers: comparison of computed and measured electronic circular dichroism spectra.

Author

Ding S, Jia L, Durandin A, Crean C, Kolbanovskiy A, Shafirovich V, Broyde S, and Geacintov NE

Subjects
Guanosine chemistry, Molecular Conformation, Optical Rotatory Dispersion, Stereoisomerism, Allantoin chemistry, Circular Dichroism methods, Guanosine analogs & derivatives, Spiro Compounds chemistry
Abstract

The assignment of absolute configurations is of critical importance for understanding the biochemical processing of DNA lesions. The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG), and the absolute configurations of the two diastereomers, Sp1 and Sp2, have been evaluated by experimental and computational optical rotatory dispersion (ORD) methods. In order to support our previous assignments by the ORD method, we calculated the electronic circular dichroism spectra (ECD) of the Sp stereoisomers. Comparison of the experimentally measured and computed ECD spectra indicates that Sp1 has (-)-S absolute configuration, while Sp2 has (+)-R absolute configuration. Thus, the S and R assignments, based on the ECD spectra of Sp1 and Sp2, are consistent with our previous assignments of absolute configurations. To further test the validity of this approach, we performed a proof-of-principle computation of the ECD and ORD of the R and S enantiomers of allantoin (similar in chemical composition to Sp) of known absolute configurations. The calculations provide the correct assignment of the absolute configurations of the allantoin enantiomers, indicating that the computational TDDFT approach is robust for identifying the absolute configurations of allantoins and probably the Sp stereoisomers, as has been shown previously for other organic molecules.

Published
2009
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13. Pathways of arachidonic acid peroxyl radical reactions and product formation with guanine radicals.

Author

Crean C, Geacintov NE, and Shafirovich V

Subjects
Chromatography, High Pressure Liquid, Fatty Acids, Monounsaturated chemistry, Fatty Acids, Unsaturated chemistry, Linoleic Acid chemistry, Oxidation-Reduction, Photolysis, Spectrometry, Mass, Electrospray Ionization methods, Arachidonic Acid chemistry, Free Radicals chemistry, Guanine chemistry, Peroxides chemistry
Abstract

Peroxyl radicals were derived from the one-electron oxidation of polyunsaturated fatty acids by sulfate radicals that were generated by the photodissociation of peroxodisulfate anions in air-equilibrated aqueous solutions. Reactions of these peroxyl and neutral guanine radicals, also generated by oxidation with sulfate radicals, were investigated by laser kinetic spectroscopy, and the guanine oxidation products were identified by HPLC and mass spectrometry methods. Sulfate radicals rapidly oxidize arachidonic (ArAc), linoleic (LnAc), and palmitoleic (PmAc) acids with similar rate constants, (2-4) x 10 (9) M (-1) s (-1). The C-centered radicals derived from the oxidation of ArAc and LnAc include nonconjugated Rn(.) ( approximately 80%) and conjugated bis-allylic Rba(.) ( approximately 20%) radicals. The latter were detectable in the absence of oxygen by their prominent, narrow absorption band at 280 nm. The Rn(.) radicals of ArAc (containing three bis-allylic sites) transform to the Rba(.) radicals via an intramolecular H-atom abstraction [rate constant (7.5 +/- 0.7) x 10 (4) s (-1)]. In contrast, the Rn(.) radicals of LnAc that contain only one bis-allylic site do not transform intramolecularly to the Rba(.) radicals. In the case of PmAc, which contains only one double bond, the Rba(.) radicals are not observed. The Rn(.) radicals of PmAc rapidly combine with oxygen with a rate constant of (3.8 +/- 0.4) x 10(9) M(-1) s(-1). The Rba(.) radicals of ArAc are less reactive and react with oxygen with a rate constant of (2.2 +/- 0.2) x 10 (8) M (-1) s (-1). The ArAc peroxyl radicals formed spontaneously eliminate superoxide radical anions [rate constant = (3.4 +/- 0.3) x 10 (4) M (-1) s (-1)]. The stable oxidative lesions derived from the 2',3',5'-tri- O-acetylguanosine or 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine radicals and their subsequent reactions with ArAc peroxyl radicals were also investigated. The major products found were the 2,5-diamino-4 H-imidazolone (Iz), dehydroguanidinohydantoin (Gh ox), and diastereomeric spiroiminodihydantoin (Sp) nucleosides from 2',3',5'-tri- O-acetylguanosine and the Gh ox and Sp nucleosides from 2',3',5'-tri- O-acetyl-8-oxo-7,8-dihydroguanosine. In air-saturated aqueous solutions, covalent alkylated guanine adducts were not detected.

Published
2008
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14. Assignment of absolute configurations of the enantiomeric spiroiminodihydantoin nucleobases by experimental and computational optical rotatory dispersion methods.

Author

Durandin A, Jia L, Crean C, Kolbanovskiy A, Ding S, Shafirovich V, Broyde S, and Geacintov NE

Subjects
Guanosine chemistry, Molecular Conformation, Molecular Structure, Optical Rotatory Dispersion, Oxidation-Reduction, Stereoisomerism, Cell Nucleus chemistry, Computer Simulation, Guanosine analogs & derivatives, Nucleic Acids chemistry, Spiro Compounds chemistry
Abstract

The diastereomeric spiroiminodihydantoin (Sp) lesions are oxidation products of guanine and 8-oxo-7,8-dihydroguanine (8-oxoG) and have generated considerable interest because of their stereochemistry-dependent mutagenic properties in vivo (Henderson, P. T., et al. (2003) Biochemistry 42, 9257-9262). However, the absolute configurations of the two diastereomers have not yet been elucidated, and such information may prove valuable for understanding relationships between biological function and structure at the DNA level (Jia, L., Shafirovich, V., Shapiro, R., Geacintov, N. E., and Broyde, S. (2005) Biochemistry 44, 13342-13353). We have synthesized the two chiral Sp nucleobases by hydrolysis of the nucleosides denoted by dSp1 and dSp2 according to their elution order in HPLC experiments using a Hypercarb column, and determined their absolute configurations using a combination of experimentally measured optical rotatory dispersion (ORD) spectra in aqueous solutions and computed ORD specific rotations using density functional theory (DFT). Recent developments have shown that DFT methods are now sufficiently robust for predicting ORD values of chiral molecules (Polavarapu, P. L. (2002) Chirality 14, 768-781). The nucleobases Sp1 and Sp2 exhibit experimentally measured CD and ORD spectra that are very close to those of the respective precursor nucleosides dSp1 and dSp2 in shape and sign. The first nucleoside stereoisomer (dSp1) to elute from a typical Hypercarb HPLC column has (-)-S, while the second (dSp2) has (+)-R absolute configuration. The R and S assignments are applicable to the amino tautomeric forms in each case.

Published
2006
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