Chemistry of neocarzinostatin-mediated cleavage of oligonucleotides. Competitive ribose C5' and C4' hydroxylation

Sugiyama, Hiroshi; Fujiwara, Tsuyoshi; Kawabata, Hiroshi; Yoda, Naoya; Hirayama, Noriaki; Saito, Isao

doi:10.1021/ja00040a014

Cited by 36 publications

(24 citation statements)

References 3 publications

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“…This disulfide bridge is conserved in all chromoprotein antibiotics and may be a common stabilizing feature. A second, distinct, cycloaromatization pathway has been recently observed when the NCS chromophore is incubated with 2-mercaptoethanol in the presence of the apoprotein in which the zwitterionic intermediate 9 (Figure , path B) is indicated, , although this mechanism probably does not operate for the free chromophore. Since it is thought likely that dissociation of the NCS chromophore from the apoprotein and subsequent DNA binding precedes activation of the chromophore, the biological relevance of this second mechanism seems dubious, and Goldberg et al .…”

Section: Neocarzinostatinmentioning

confidence: 88%

The Enediyne Antibiotics

1996

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Section: Neocarzinostatinmentioning

confidence: 88%

The Enediyne Antibiotics

1996

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“…An oligodeoxynucleotide standard containing a nucleoside 5’-aldehyde terminus, 5’-TGC-3’ ( 4 ), was prepared as described by Sugiyama et al 27 Briefly, a 90 µM solution of a self-complementary hexadeoxynucleotide, 5’-GCATGC-3’, in potassium phosphate buffer (50 mM, pH 7.4) was treated with 46 µM neocarzinostatin and 10 mM glutathione, the latter to activate the drug for DNA cleavage. After 12 h at 0 °C, the cleavage mixture was resolved by reversed phase HPLC using the conditions described above, with collection of the nucleoside 5’-aldehyde-containing TGC fragment eluting at 15.2 min.…”

Section: Experimental Methodsmentioning

confidence: 99%

Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe²⁺−EDTA

et al. 2010

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The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1’-oxidation and the nucleoside 5’-aldehyde of 5’-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5’-aldehyde lesions. Further, the well defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1I, was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5’-aldehyde per 106 nt per µM in accord with its established minor 1’- and major 5’-oxidation chemistry. Calicheamicin unexpectedly caused 1’-oxidation at a low level of 10 2-deoxyribonolactone per 106 nt per µM in addition to the expected predominance of 5’-oxidation at 560 nucleoside 5’-aldehyde per 106 nt per µM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe2+-EDTA, produced nucleoside 5’-aldehyde at a frequency of 57 per 106 nt per Gy (G-value 74 nmol/J) and 3.5 per 106 nt per µM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe2+-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per µM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5’-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a ~250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.

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“…1-7 However, the factors that control the efficiency of hydrogen atom abstraction are still not well understood, in spite of the importance of this knowledge on many research areas, such as radical-induced cleavage of nucleic acids initiated by hydrogen atom abstraction from the sugar-phosphate backbone. 8,9 For example, the enediyne anti-tumor antibiotics, such as neocarzinostatin 10,11 and calicheamicin, 12 undergo rearrangement and aromatization to form carbon-centered σ,σ-biradical intermediates, which abstract hydrogen atoms from sugar-moieties in DNA and lead to double-strand scission. Although these enediyne compounds are potent drugs, they are also highly toxic to normal cells.…”

Section: Introductionmentioning

confidence: 99%

Direct Comparison of Solution and Gas-Phase Reactions of the Three Distonic Isomers of the Pyridine Radical Cation with Methanol

et al. 2012

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In order to directly compare the reactivity of positively charged carbon-centered aromatic σ-radicals toward methanol in solution and in the gas phase, the 2-, 3-, and 4-dehydropyridinium cations (distonic isomers of the pyridine radical cation) were generated by ultraviolet photolysis of the corresponding iodo-precursors in a mixture of water and methanol at varying pH. The reaction mixtures were analyzed by using liquid chromatography/mass spectrometry. Hydrogen atom abstraction was the only reaction observed for the 3- and 4-dehydropyridinium cations (and –pyridines) in solution. This also was the major reaction observed earlier in the gas phase. Depending on the pH, the hydrogen atom can be abstracted from different molecules (i.e., methanol or water) and from different sites (in methanol) by the 3- and 4-dehydropyridinium cations/-pyridines in solution. In the pH range of 1 – 4, the methyl group of methanol is the main hydrogen atom donor site for both 3- and 4-dehydropyridinium cations (just like in the gas phase). At higher pH, the hydroxyl groups of water and methanol also act as hydrogen atom donors. This finding is rationalized by a greater abundance of the unprotonated radicals that preferentially abstract hydrogen atoms from the polar hydroxyl groups. The percentage yield of hydrogen atom abstraction by these radicals was found to increase with lowering the pH in the pH range of 1.0-3.2. This pH effect is rationalized by polar effects: the lower the pH, the greater the fraction of protonated (more polar) radicals in the solution. This finding is consistent with previous results obtained in the gas phase and suggests that gas-phase studies can be used to predict solution reactivity, but only as long as the same reactive species is studied in both experiments. This was found not to be the case for the 2-iodopyridinium cation. Photolysis of this precursor in solution resulted in the formation of two major addition products, 2-hydroxy- and 2-methoxypyridinium cations, in addition to the hydrogen atom abstraction product. These addition products were not observed in the earlier gas-phase studies on 2-dehydropyridinium cation. Their observation in solution is explained by the formation of another reactive intermediate, the 2-pyridyl cation, upon photolysis of 2-iodopyridinium cation (and 2-iodopyridine). The same intermediate was observed in the gas phase but it was removed before examining the reactions of the desired radical, 2-dehydropyridinium cation (which cannot be done in solution).

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Chemistry of neocarzinostatin-mediated cleavage of oligonucleotides. Competitive ribose C5' and C4' hydroxylation

Cited by 36 publications

References 3 publications

The Enediyne Antibiotics

The Enediyne Antibiotics

Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe²⁺−EDTA

Direct Comparison of Solution and Gas-Phase Reactions of the Three Distonic Isomers of the Pyridine Radical Cation with Methanol

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Chemistry of neocarzinostatin-mediated cleavage of oligonucleotides. Competitive ribose C5' and C4' hydroxylation

Cited by 36 publications

References 3 publications

The Enediyne Antibiotics

The Enediyne Antibiotics

Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe2+−EDTA

Direct Comparison of Solution and Gas-Phase Reactions of the Three Distonic Isomers of the Pyridine Radical Cation with Methanol

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Quantification of the 2-Deoxyribonolactone and Nucleoside 5′-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of γ-Radiation and Fe²⁺−EDTA