Background:. Deoxyribonucleotide radicals resulting from formal C1'-hydrogen atom abstraction are important reactive intermediates in a variety of DNA-damage processes. The reactivity of these radicals can be affected by the agents that generate them and the environment in which they are produced. As an initial step in determining the factors that control the reactivity of these important radical species, we developed a mild method for their generation at a defined site within a biopolymer. Results:. Irradiation of oligonucleotides containing a photolabile nucleotide produced C1'-DNA radicals. In the absence of potential reactants other than O2, approximately 90% of the damage events involve formation of alkaline-labile lesions, with the remainder resulting in direct strand breaks. The ratio of alkaline-labile lesions to direct strand breaks ( approximately 9:1) is independent of whether the radical is generated in single-stranded DNA or double-stranded DNA. Strand damage is almost completely quenched under anaerobic conditions in the presence of low thiol concentrations. Competition studies with O2 indicate that the trapping rate of C1'-DNA radicals by beta-mercaptoethanol is approximately 1.1 x 10(7) M-1 s-1. Conclusions:. The mild generation of the C1'-DNA radical in the absence of exogenous oxidants makes it possible to examine their intrinsic reactivity. In the absence of other reactants, the formation of direct strand breaks from C1'-radicals is, at most, a minor pathway. Competition studies between beta-mercaptoethanol and O2 indicate that significantly higher thiol concentrations than those in vivo or some means of increasing the effective thiol concentration near DNA are needed for these reagents to prevent the formation of DNA lesions arising from the C1'-radical under aerobic conditions.

The ability of tirapazamine (1, 3-amino-1,2,4-benzotriazine 1, 4-dioxide, SR4233) to fix DNA radical lesions is demonstrated by studying the reaction between the antitumor drug and an oligonucleotide radical that is independently produced at a defined site within a biopolymer. Using beta-mercaptoethanol as a competitor, it was determined that tirapazamine traps a C1'-nucleotide radical with a rate constant of approximately 2 x 10(8) M-1 s-1. Product and isotopic labeling studies suggest that tirapazamine reacts with the radical via covalent adduct formation, resulting primarily from reaction at the N-oxide oxygen. Intermediate covalent adducts could not be observed, but are postulated to decompose to the alkaline labile 2'-deoxyribonolactone lesion. These experiments affirm recent proposals suggesting that tirapazamine can serve as a surrogate for O2 in converting DNA radicals into toxic strand damage events.

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5,6-Dihydrothymidin-5-yl (1) and 2′-deoxyuridin-1′-yl (3) were independently generated in solution under aerobic conditions. The release of superoxide (O 2 •-) from the respective peroxyl radicals derived from 1 and 3 was determined spectrophotometrically. Competition studies enable one to estimate that the rate constant for elimination of O 2 •from the peroxyl radical (4) derived from 3 is ∼1 s -1 . This process is competitive with the anticipated rate of trapping of 4 in DNA by glutathione. Relative rate studies indicate that O 2 •generation resulting from the formation of 1 under aerobic conditions competes effectively with trapping of the peroxyl radical by Bu 3 SnH. Superoxide elimination from the peroxyl radical of 1 (2) restores the damaged nucleoside to its unaltered form, implying that this reactive intermediate has a naturally occurring detoxification pathway available to it. However, the freely diffusible superoxide can react further to generate other reactive species capable of damaging nucleic acids, suggesting that the elimination of O 2 •from 2 is a potential double-edged sword.

Laser-flash photolysis methods were used to determine Arrhenius functions for cyclizations of the 4,4-diphenyl-3-butenyl (2) and trans-4-phenyl-3-butenyl (5) radicals to the 1,1-diphenylcyclopropylcarbinyl (1) and 1-phenylcyclopropylcarbinyl (4) radicals, respectively. At 20 °C, the cyclization rate constants are 1.7 × 107 and 5.4 × 106 s-1. Equilibrium constants for the two processes were estimated and evaluated with thermochemical data and via computational methods, and Arrhenius functions for the ring-opening reactions of the cyclopropylcarbinyl radicals were calculated. The cyclization reactions of 2 and 5 are strongly enthalpy controlled. Production of radicals 1 and 2 from the corresponding tert-butylperoxy esters in the presence of Et3SnH gave diphenylcyclopropylmethane and 1,1-diphenyl-1-butene from H-atom trapping of radicals 1 and 2 and 4-phenyl-1,2-dihydronaphthalene which derives from the product radical formed by addition of the radical moiety in 2 to the cis-phenyl group. Rate constants for the latter cyclization of 2 and for reactions of radicals 1 and 2 with Et3SnH were obtained from the indirect kinetic studies.