5‘-(2-Phosphoryl-1,4-dioxobutane) as a Product of 5‘-Oxidation of Deoxyribose in DNA:  Elimination as trans-1,4-Dioxo-2-butene and Approaches to Analysis

Abstract

Oxidation of deoxyribose in DNA leads to the formation of a spectrum of electrophilic products unique to each position in the sugar. For example, chemical reactions following abstraction of the C5‘-hydrogen atom partition to form either a nucleoside 5‘-aldehyde residue attached to the 5‘-end of the DNA strand or a 5‘-formyl phosphate residue attached to the 3‘-end of the DNA strand that is accompanied by a four-carbon fragment on the 5‘-end. We now present two approaches that both identify the latter fragment as 5‘-(2-phosphoryl-1,4-dioxobutane) and provide a means to quantify the formation of this residue by different oxidizing agents. The first approach involves oxidation of DNA followed by reaction with O-benzylhydroxylamine to form stable dioxime derivatives of the putative 5‘-(2-phosphoryl-1,4-dioxobutane) residues. The β-elimination product of this dioxime proved to be the expected trans-1,4-dioxo-2-butene, as judged by gas chromatographic and mass spectrometric (GC/MS) comparison to authentic dioximes of cis- and trans-1,4-dioxo-2-butene, which revealed a unique pattern of three signals for each isomer, and by X-ray crystallography. Using a benzylhydroxylamine dioxime derivative of [2H4]-labeled cis-1,4-dioxo-2-butene as an internal standard, the dose−response for the formation of 5‘-(2-phosphoryl-1,4-dioxobutane) was determined to be linear for γ-radiation, with ∼6 lesions per 106 nt per Gy, and nonlinear for Fe2+-EDTA. A comparison of 5‘-(2-phosphoryl-1,4-dioxobutane) formation to total deoxyribose oxidation suggests that γ-radiation produces ∼0.04 lesions per deoxyribose oxidation event. As a positive control for 5‘-oxidation of deoxyribose, the enediyne calicheamicin was observed to produce 5‘-(2-phosphoryl-1,4-dioxobutane) at the rate of ∼9 lesions per 106 nt per μM. A second approach to identifying and quantifying the sugar residue involved derivatization with hydrazine and β-elimination to form pyridazine followed by quantification of the pyridazine by GC/MS. Using this approach, it was observed that the enediyne, neocarzinostatin, produced a linear dose−response for pyridazine formation, as expected given the ability of this oxidant to cause 1‘-, 4‘-, and 5‘-oxidation of deoxyribose in DNA. The antitumor antibiotic, bleomycin, on the other hand, produced pyridazine at a 10-fold lower rate, which is consistent with 4‘-chemistry as the predominant mode of deoxyribose oxidation by this agent. These results provide novel insights into the chemistry of deoxyribose oxidation in DNA and two approaches to quantifying the 5‘-(2-phosphoryl-1,4-dioxobutane) precursor of trans-1,4-dioxo-2-butene, an electrophile known to react with nucleobases to form novel DNA adducts.

GC/MS Methods To Quantify the 2-Deoxypentos-4-ulose and 3′-Phosphoglycolate Pathways of 4′ Oxidation of 2-Deoxyribose in DNA: Application to DNA Damage Produced by γ Radiation and Bleomycin

“…The instability of the oxAB (t 1/2 ~20 hr at 37 °C; refs. 39,44) necessitated chemical stabilization in quantitative fashion, so we chose to convert it to a stable 3′-pyridazine moiety by derivatization with hydrazine as originally described by Hecht and coworkers (45) Each of the steps in the method was optimized for parameters such as concentration, reaction time, temperature and pH, as previously described (34). The ethyl acetate extraction of HMP (3X, pH 7) was 99.5% efficient and silylation was most efficient at 25 °C and 30 min reaction time with ratios of ethyl acetate to BSTFA between 50:50 to 70:30; there was a significant reduction in silylation when ethyl acetate was reduced below 30% (data not shown).…”

Formation of 1,4-Dioxo-2-butene-Derived Adducts of 2‘-Deoxyadenosine and 2‘-Deoxycytidine in Oxidized DNA

“…As shown in Scheme 1, the chemistry of 5′-oxidation of deoxyribose in DNA by a variety of DNA-cleaving agents, including enediynes (8) and manganese porphyrins (9), partitions to form strand breaks containing 3′-phosphate and 5′-nucleoside-5′-aldehyde residues, or a 3′-formylphosphate species accompanied by a four-carbon fragment that we and others have identified as a 5′-(2-phosphoryl-1,4-dioxobutane) residue (7,10). We recently showed that this dialdehydic residue undergoes β-elimination to form trans-1,4-dioxo-2-butene (7), a highly reactiveα,β-unsaturated dicarbonyl species.…”

An Oxidized Abasic Lesion as an Intramolecular Source of DNA Adducts

“…[7] The trans isomer ( trans - 1 ), which also forms adducts with nucleotides in DNA, is released from 5′-(2-phosphoryl-1,4-dioxobutane) (DOB), an oxidized abasic lesion resulting from C5′-hydrogen-atom abstraction. [8,11] The DOB lesion is produced by several antitumour agents and by γ radiation. [11–14] This lesion is a potent irreversible inhibitor of DNA polymerase β, an enzyme that is a primary component of base excision repair that is overexpressed in many cancers.…”

“…[8,11] The DOB lesion is produced by several antitumour agents and by γ radiation. [11–14] This lesion is a potent irreversible inhibitor of DNA polymerase β, an enzyme that is a primary component of base excision repair that is overexpressed in many cancers. [15–19] Hence, DOB formation may provide a chemical basis for the potent cytotoxicity of the antitumor agents that produce it.…”