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 gamma-Radiation and Fe2+-EDTA

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.National Institute of Environmental Health Sciences (ES002109)National Center for Research Resources (U.S.) (RR023783-01)National Center for Research Resources (U.S.) (RR017905-01)National Cancer Institute (U.S.) (CA103146

Cleavage of double-stranded DNA by 'metalloporphyrin-linker-oligonucleotide' molecules: influence of the linker.

Manganese porphyrin-linker-triple-helix-forming oligonucleotide molecules were prepared and their ability to cleave in vitro a double-stranded DNA target present in the HIV-1 genome was studied. The nature of the linker is a determining factor of the cleavage efficiency. Cleavage yields as high as 80% were observed when the linker was a spermine residue and in the absence of a large excess of free spermine known to stabilize triplex structures. The hydrophobic nature of aliphatic diamine linker modified the cleaver-DNA interactions and reduced the efficiency of DNA cleavage

Pyridine-3,4-dicarboximide as starting material for the total synthesis of the natural product eupolauramine and its isomer iso-eupolauramine endowed with anti-tubercular activities

A direct and convenient synthetic pathway for preparation of the key intermediate aza-isoindolinone 4a/4b, which could be ready obtained from pyridine-3,4-dicarboximide was described. This approach was valorized in the total synthesis of the natural product eupolauramine 7b and in the first synthesis of the position analog iso-eupolauramine 7a, which was obtained after 6 steps in an overall yield of 13%. The developed strategy represents the first synthetic approach employing a starting material already embedding the pyridine and the lactam cycles (cycles D and C, respectively) of the azaphenanthrene lactam framework. These compounds, mainly the new non-natural product, showed a good inhibitory activity against Mycobacterium tuberculosis growth

Ethionamide biomimetic activation and an unprecedented mechanism for its conversion into active and non-active metabolites

International audienceEthionamide (ETH), a second-line anti-tubercular drug that is regaining a lot of interest due to the increasing cases of drug-resistant tuberculosis, is a pro-drug that requires an enzymatic activation step to become active and to exert its therapeutic effect. The enzyme responsible for ETH bioactivation in Mycobacterium tuberculosis is a monooxygenase (EthA) that uses flavin adenine dinucleotide (FAD) as a cofactor and is NADPH- and O2-dependant to exert its catalytic activity. In this work, we investigated the activation of ETH by various oxygen-donor oxidants and the first biomimetic ETH activation methods were developed (KHSO5, H2O2, and m-CPBA). These simple oxidative systems, in the presence of ETH and NAD+, allowed the production of short-lived radical species and the first non-enzymatic formation of active and non-active ETH metabolites. The intermediates and the final compounds of the activation pathway were well characterized. Based on these results, we postulated a consistent mechanism for ETH activation, not involving sulfinic acid as a precursor of the iminoyl radical, as proposed so far, but putting forward a novel reactivity for the S-oxide ethionamide intermediate. We proposed that ETH is first oxidized into S-oxide ethionamide, which then behaves as a "ketene-like" compound via a formal [2 + 2] cycloaddition reaction with peroxide to give a dioxetane intermediate. This unstable 4-membered intermediate in equilibrium with its open tautomeric form decomposes through different pathways, which would explain the formation of the iminoyl radical and also that of different metabolites observed for ETH oxidation, including the ETH-NAD active adduct. The elucidation of this unprecedented ETH activation mechanism was supported by the application of isotopic labelling experiments