The thermodynamic properties of aqueous nitroxyl (HNO) and its anion (NO ؊ ) have been revised to show that the ground state of NO ؊ is triplet and that HNO in its singlet ground state has much lower acidity, pKa( 1 HNO͞ 3 NO ؊ ) Ϸ 11.4, than previously believed. These conclusions are in accord with the observed large differences between 1 HNO and 3 NO ؊ in their reactivities toward O2 and NO. Laser flash photolysis was used to generate 1 HNO and 3 NO ؊ by photochemical cleavage of trioxodinitrate (Angeli's anion). The spin-allowed addition of 3 O2 to 3 NO ؊ produced peroxynitrite with nearly diffusion-controlled rate (k ‫؍‬ 2.7 ؋ 10 9 M ؊1 ⅐s ؊1 ). In contrast, the spin-forbidden addition of 3 O2 to 1 HNO was not detected (k Ͻ Ͻ 3 ؋ 10 5 M ؊1 ⅐s ؊1 N itroxyl (HNO, also known as nitrosyl hydride) and its anion, NO Ϫ , are the simplest molecules with nitrogen in the ϩ1 oxidation state and yet their aqueous chemistry is not well understood. Recent suggestions that these redox neighbors of the biologically important NO radical may play a role in cellular metabolism (1-4) and in aerobic environments may be precursors to cytotoxic peroxynitrite, ONOO Ϫ , (5, 6) have engendered considerable interest in the chemistry of HNO͞NO Ϫ . The characterization of these species is complicated by their instability with respect to formation of nitrous oxide (7,8). In most cases where nitroxyl has been invoked as an intermediate, the rate-determining step was its generation, a situation that allows little insight into the properties and reactivities of HNO͞NO Ϫ themselves. The NO Ϫ anion is isoelectronic with O 2 and, like O 2 , should have a triplet ground state, whereas the ground state of HNO should be a singlet. Indeed, these ground state assignments have been well established for HNO͞NO Ϫ in the gas phase (9, 10).A frequently used source for aqueous HNO͞NO Ϫ is trioxodinitrate (N 2 O 3 2Ϫ , also known as Angeli's anion), whose conjugate acid (H 2 N 2 O 3 ) has consecutive pKa values of 2.5 and 9.7 (11). It is widely accepted (7, 8) that slow decomposition of the monoprotonated anion occurs through heterolytic NON bond cleavageSubsequent addition of O 2 could yield peroxynitriteHowever, nitrate, which is the peroxynitrite decomposition product, was not detected among the end products of HN 2 O 3 Ϫ decay (12). This result was interpreted as evidence against the occurrence of reaction 2. On the other hand, the same researchers reported peroxynitrite formation during N 2 O 3 2Ϫ photolysis in alkaline solution (13). To reconcile the data, it was suggested that thermal reaction 1 followed by deprotonation of HNO produces singlet NO Ϫ , which is the ground state in water, and that 1 NO Ϫ is unreactive toward O 2 . In contrast, photochemical cleavage of N 2 O 3 2Ϫ was thought to generate the long-lived triplet excited state of NO Ϫ , which reacted with O 2 . However, it seems unlikely that hydration can reverse a gas-phase energy gap of about 70 kJ͞mol between the ground state 3 NO Ϫ and the excited state 1 NO Ϫ (10). Moreover, by ana...

The mechanism and dynamics of photoinduced charge separation and charge recombination have been investigated in synthetic DNA hairpins possessing donor and acceptor stilbenes separated by one to seven A:T base pairs. The application of femtosecond broadband pump-probe spectroscopy, nanosecond transient absorption spectroscopy, and picosecond fluorescence decay measurements permits detailed analysis of the formation and decay of the stilbene acceptor singlet state and of the charge-separated intermediates. When the donor and acceptor are separated by a single A:T base pair, charge separation occurs via a single-step superexchange mechanism. However, when the donor and acceptor are separated by two or more A:T base pairs, charge separation occurs via a multistep process consisting of hole injection, hole transport, and hole trapping. In such cases, hole arrival at the electron donor is slower than hole injection into the bridging A-tract. Rate constants for charge separation (hole arrival) and charge recombination are dependent upon the donor-acceptor distance; however, the rate constant for hole injection is independent of the donor-acceptor distance. The observation of crossover from a superexchange to a hopping mechanism provides a "missing link" in the analysis of DNA electron transfer and requires reevaluation of the existing literature for photoinduced electron transfer in DNA.

No abstract

Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The latter were monitored by their characteristic transient absorption band at 600 nm. The rate constants of reactions of carbonate radicals with oligonucleotides increase in the ascending order: 5'-d(CCATCCTACC) [(5.7 +/- 0.6) x 10(6) M(-)(1) s(-)(1)] < 5'-d(TATAACGTTATA), self-complementary duplex [(1.4 +/- 0.2) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATCGCTACC [(2.4 +/- 0.3) x 10(7) M(-)(1) s(-)(1)] < 5'-d(CCATC[8-oxo-G]CTACC) [(3.2 +/- 0.4) x 10(8) M(-)(1) s(-)(1)], where 8-oxo-G is 8-oxo-7,8-dihydroguanine, the product of a two electron oxidation of guanine. This remarkable enhancement of the rate constants is correlated with the presence of either G or 8-oxo-G bases in the oligonucleotides. The rate constant for the oxidation of G in a single-stranded oligonuclotide is faster by a factor of approximately 2 than in the double-stranded form. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects and biological implications of these site selective reactions in DNA initiated by carbonate radicals are discussed.

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...

The exposure of guanine in the oligonucleotide 5’-d(TCGCT) to one-electron oxidants leads initially to the formation of the guanine radical cation G•+, its deptotonation product G(−H)• and, ultimately, to various two- and four-electron oxidation products via pathways that depend on the oxidants and reaction conditions. We utilized single or successive multiple laser pulses (308 nm, 1 Hz rate) to generate the oxidants CO3•− and SO4•− (via the photolysis of S2O82− in aqueous solutions in the presence and absence of bicarbonate, respectively) at concentrations/pulse that were ~20-fold lower than the concentration of 5’-d(TCGCT). Time-resolved absorption spectroscopy measurements following single-pulse excitation show that the G•+ radical (pKa = 3.9) can be observed only at low pH and is hydrated within ≥ 3 ms at pH 2.5, thus forming the two-electron oxidation product 8-oxo-7,8-dihydroguanosine (8-oxoG). At neutral pH, and single pulse excitation, the principal reactive intermediate is G(−H)• that at best, reacts only slowly with H2O, and lives for ≥ 70 ms in the absence of oxidants/other radicals to form base sequence-dependent intrastrand cross-links via the nucleophilic addition of N3-thymidine to C8-guanine (5'-G*CT* and 5'-T*CG*). Alternatively, G(−H)• can be oxidized further by reaction with CO3•− generating the two electron products 8-oxoG (C8 addition), and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih, by C5 addition). The four-electron oxidation products, guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp), appear only after a second (or more) laser pulses. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses.

The mechanistic aspects of hydration of guanine radical cations, G•+ in double- and single-stranded oligonucleotides were investigated by direct time-resolved spectroscopic monitoring methods. The G•+ radical one-electron oxidation products were generated by SO4•– radical anions derived from the photolysis of S2O82– anions by 308 nm laser pulses. In neutral aqueous solutions (pH 7.0), after the complete decay of SO4•– radicals (∼5 μs after the actinic laser flash) the transient absorbance of neutral guanine radicals, G(-H)• with maximum at 312 nm, is dominant. The kinetics of decay of G(-H)• radicals depend strongly on the DNA secondary structure. In double-stranded DNA, the G(-H)• decay is biphasic with one component decaying with a lifetime of ∼2.2 ms and the other with a lifetime of ∼0.18 s. By contrast, in single-stranded DNA the G(-H)• radicals decay monophasically with a ∼ 0.28 s lifetime. The ms decay component in double-stranded DNA is correlated with the enhancement of 8-oxo-7,8-dihydroguanine (8-oxoG) yields which are ∼7 greater than in single-stranded DNA. In double-stranded DNA, it is proposed that the G(-H)• radicals retain radical cation character by sharing the N1-proton with the N3-site of C in the [G•+:C] base pair. This [G(-H)•:H+C ⇆ G•+:C] equilibrium allows for the hydration of G•+ followed by formation of 8-oxoG. By contrast, in single-stranded DNA, deprotonation of G•+ and the irreversible escape of the proton into the aqueous phase competes more effectively with the hydration mechanism, thus diminishing the yield of 8-oxoG, as observed experimentally.

The carbonate radical anion is a biologically important one-electron oxidant that can directly abstract an electron from guanine, the most easily oxidizable DNA base. Oxidation of the 5′-d(CCTACGCTACC) sequence by photochemically generated CO3·− radicals in low steady-state concentrations relevant to biological processes results in the formation of spiroiminodihydantoin diastereomers and a previously unknown lesion. The latter was excised from the oxidized oligonucleotides by enzymatic digestion with nuclease P1 and alkaline phosphatase and identified by LC-MS/MS as an unusual intrastrand cross-link between guanine and thymine. In order to further characterize the structure of this lesion, 5′-d(GpCpT) was exposed to CO3·− radicals, and the cyclic nature of the 5′-d(G*pCpT*) cross-link in which the guanine C8-atom is bound to the thymine N3-atom was confirmed by LC-MS/MS, 1D and 2D NMR studies. The effect of bridging C bases on the cross-link formation was studied in the series of 5′-d(GpCnpT) and 5′-d(TpCnpG) sequences with n = 0, 1, 2 and 3. Formation of the G*-T* cross-links is most efficient in the case of 5′-d(GpCpT). Cross-link formation (n = 0) was also observed in double-stranded DNA molecules derived from the self-complementary 5′-d(TTACGTACGTAA) sequence following exposure to CO3·− radicals and enzymatic excision of the 5′-d(G*pT*) product.