The carbonate radical anion (CO 3 . ) is believed to be an . anions and the formation of G(؊H) ⅐ radicals are correlated with one another on the millisecond time scale, whereas the neutral guanine radicals decay on time scales of seconds. Alkali-labile guanine lesions are produced and are revealed by treatment of the irradiated oligonucleotides in hot piperidine solution. The DNA fragments thus formed are identified by a standard polyacrylamide gel electrophoresis assay, showing that strand cleavage occurs at the guanine sites only. The biological implications of these oxidative processes are discussed.There is growing evidence that bicarbonate and carbon dioxide, both present in biological systems in significant amounts, can alter the mechanisms and reaction pathways of reactive oxygen (1-4) and nitrogen (5-13) species formed during normal metabolic activity and under conditions of oxidative stress. It has been proposed that the mechanism of generation of carbonate radical anions (CO 3 . ) 1 from bicarbonate (HCO 3 Ϫ ) or CO 2 can involve the one-electron oxidation of HCO 3 Ϫ at the active site of copper-zinc superoxide dismutase (3, 4) and homolysis of the nitrosoperoxycarbonate anion (ONOOCO 2 Ϫ ) formed by the reaction of peroxynitrite with carbon dioxide (14 -18).The carbonate radical anion is a strong one-electron oxidant that oxidizes appropriate electron donors via electron transfer mechanisms (19). Detailed pulse radiolysis studies have shown that carbonate radicals can rapidly abstract electrons from aromatic amino acids (tyrosine and tryptophan). However, reactions of CO 3 . with sulfur-containing methionine and cysteine are less efficient (20 -22). Hydrogen atom abstraction by carbonate radicals is generally very slow (19), and their reactivities with other amino acids are negligible (20 -22). It is well established that carbonate radicals can play an important role in the modification of selective amino acids in proteins in cellular environments under conditions of oxidative stress, aging, and inflammatory processes (1,11,12). The role of HCO 3 Ϫ /CO 2 in potentiating oxidative DNA damage has received relatively little attention. It has been shown that the presence of HCO 3 Ϫ /CO 2 inhibits direct strand cleavage of DNA induced by ONOO Ϫ but enhances the formation of 8-nitroguanine, alkali-labile and formamidopyrimidine glycosylase-labile DNA lesions (23-25). Peroxynitrite causes direct DNA strand cleavage by oxidizing deoxyribose. However, in the presence of HCO 3 Ϫ /CO 2 there is a shift in product distribution from direct strand cleavage to the formation of oxidative modifications of guanines (26), suggesting that the carbonate radical anion could play an important role in this phenomenon (24). Although guanine is indeed the most easily oxidized base in DNA, the reactions of the carbonate radical anions with the different aromatic DNA residues have not yet been characterized.In this work, we explore the electron transfer reactions from guanine electron donor residues embedded in the self-comp...

Photoionization of free 2-aminopurine (2AP) in aqueous solutions, or of 2AP residues in oligonucleotides, is observed upon excitation with 308 nm XeCl excimer laser pulses (fwhm = 12 ns, ≤100 mJ/cm2/pulse) and involves the consecutive absorption of two photons. The absorption of light by the normal DNA bases at this wavelength is negligible under the same conditions. The kinetics and transient absorption spectra of the hydrated electrons and of 2AP radicals resulting from the deprotonation of the 2AP radical cations formed initially, have been investigated using standard spectroscopic methods. The 2AP radicals in aqueous solutions reversibly oxidize 2‘-deoxyguanosine and guanosine 5‘-monophosphate but do not react with the other three DNA nucleosides or nucleotides. The efficiency of photoionization of 2AP decreases in the following order:  free 2AP > 2AP incorporated into single-stranded oligonucleotide > 2AP in double-stranded oligonucleotides. Photoionization of single 2AP residues incorporated into 18-mer single- and double-stranded oligonucleotides results in the oxidation of guanine residues in GG doublets. Employing gel electrophoresis methods, strand cleavage at these GG sites and at the site of the 2AP residue is observed after treatment of the irradiated oligonucleotides with hot piperidine. Using nanosecond transient absorption techniques, it is shown that the unimolecular oxidation of guanine by 2AP radicals at a distance can also be monitored directly in single-stranded oligonucleotides containing GG-doublets. It is found that increasing the number of nucleic acid bases between the 2AP radicals and the GG-doublet from 0 to 5 results in a decrease in the rate constant of guanine radical formation by a factor greater than 103.

Electron transfer from guanine donor to 2-aminopurine radical acceptor at various distances from one another in oligonucleotides was investigated using transient absorption techniques that allow for the direct monitoring of both the acceptor and oxidized donor short-lived species. The oligonucleotides (15-mers) containing one GG doublet and a 2-aminopurine (2AP) base analog, separated by 0È4 thymine bases, were studied either in the single-stranded form (ss), or in the double-stranded form (ds) with T opposite the 2AP residue in the complementary strand. The 2AP residues were selectively photoionized by a two-photon excitation with intense 308 nm XeCl excimer laser pulses (FWHM \ 12 ns, D60 mJ pulse~1 cm~2). The oxidation of guanine by the 2AP radicals was monitored by the evolution of the transient absorption spectra of 2AP radicals (absorption band at 360 nm, bleaching at 310 nm) and guanine radicals (narrow absorption band at 312 nm). The fast (\100 ns) and slow ([100 ns) kinetic components of guanine radical formation were observed. The time dependence of the fast component, attributed to the oxidation of guanines by the radical cation 2AP~`, was not resolved. At neutral pH, rapidly (D30 ns) deprotonates to the neutral radical 2AP(-H) which 2AP~`~, oxidizes the guanines on the 0.1È500 ls timescale and gives rise to the neutral G(-H) radical (the slow component). Both the prompt (\100 ns) relative yield of the guanine radicals, and the rate constant of the U G , slow electron transfer decrease with the number of bridging thymine bases on the strand bearing both the 2AP and GG units (the attenuation parameter b in dsDNA is 0.75 ^0.20When four thymine bridging bases A ~1). between the 2AP and GG units are replaced by four adenine bases (with normal complementary strands in both cases), the rate of the electron transfer is increased from \500 s~1 to 1.7 ] 106 s~1 (U G D 0) (U G \ 0.36), indicating that the electron transfer rates can strongly dependent on the base sequence. The slow kinetic component is discussed in terms of a proton-coupled electron transfer mechanism in which electron transfer from G to 2AP(-H) is coupled with deprotonation of and protonation of 2AP(-H)~. ~G 1.