Peroxynitrite generation from a NO-releasing nitrobenzene derivative in response to photoirradiation

Photocontrollable ONOO(-) generation from a nitrobenzene derivative was demonstrated. The designed compound released NO in response to photoirradiation, and the resulting semiquinone reduced molecular oxygen to generate O(2)˙(-); reaction of the two generated ONOO(-), as confirmed with an ONOO(-) fluorescent probe, HKGreen-3.     

GC-MS and HPLC methods for peroxynitrite (ONOO- and O15NOO-) analysis: a study on stability, decomposition to nitrite and nitrate, laboratory synthesis, and formation of peroxynitrite from S-nitrosoglutathione (GSNO) and KO2

Nitric oxide (˙NO) and superoxide (O(2)(-)˙) are ubiquitous in nature. Their reaction product peroxynitrite (ONOO(-)) and notably its conjugated peroxynitrous acid (ONOOH) are highly unstable in aqueous phase. ONOO(-)/ONOOH (referred to as peroxynitrite) isomerize and decompose to NO(3)(-), NO(2)(-) and O(2). Here, we report for the first time GC-MS and HPLC methods for the analysis of peroxynitrite in aqueous solution. For GC-MS analysis peroxynitrite in alkaline solution was derivatized to a pentafluorobenzyl derivative using pentafluorobenzyl bromide. O(15)NOO(-) was synthesized from H(2)O(2) and (15)NO(2)(-) and used as internal standard. HPLC analysis was performed on stationary phases consisting of Nucleosil? 100-5C(18)AB or Nucleodur? C(18) Gravity. The mobile phase consisted of a 10 mM aqueous solution of tetrabutylammonium hydrogen sulfate and had a pH value of 11.5. UV absorbance detection at 300 nm was used. HPLC allows simultaneous analysis of ONOO(-), NO(2)(-) and NO(3)(-). The GC-MS and HPLC methods were used to study stability, synthesis, formation from S-[(15)N]nitrosoglutathione (GS(15)NO) and KO(2), and isomerization/decomposition of peroxynitrite to NO(2)(-) and NO(3)(-) in aqueous buffer.     

One-electron oxidation of acetohydroxamic acid: the intermediacy of nitroxyl and peroxynitrite

The pharmacological effects of hydroxamate derivatives have been attributed not only to metal chelation or enzyme inhibition but also to their ability to serve as nitroxyl (HNO/NO(-)) and nitric oxide (NO) donors. However, the mechanism underlying the formation of these reactive nitrogen species is not clear and requires further elucidation. In the present study, one-electron oxidation of acetohydroxamic acid (aceto-HX) by (?)OH, (?)N(3), (?)NO(2), CO(3)(?-), and O(2)(?-) radicals was investigated using pulse radiolysis. It is demonstrated that only (?)OH, (?)N(3), and CO(3)(?-) radicals attack effectively and selectively the deprotonated form of the hydroxamate moiety, yielding the respective transient nitroxide radical. This nitroxide radical is a weak acid (CH(3)C(O)NHO(?), pK(a) = 9.1), which decays via a pH-dependent second-order reaction, 2k(2CH(3)C(O)NO(?-)) = (5.6 ± 0.4) × 10(7) M(-1) s(-1) (I = 0.002 M), 2k(CH(3)C(O)NO(?-) + CH(3)C(O)NHO(?)) = (8.3 ± 0.5) × 10(8) M(-1) s(-1)), and 2k(2CH(3)C(O)NHO(?)) = (8.7 ± 1.3) × 10(7) M(-1) s(-1). The second-order decomposition of the nitroxide yields transient species, one of which decomposes via a first-order reaction whose rate increases linearly upon increasing [CH(3)C(O)NHO(-)] or [OH(-)]. One-electron oxidation of aceto-HX under anoxia does not give rise to nitrite even after exposure to O(2), indicating that NO is not formed during the decomposition of the nitroxide radical. The presence of oxidants such as Tempol or O(2) during CH(3)C(O)NO(?-) decomposition had no effect on the reaction kinetics. Nevertheless, in the presence of Temopl, which does not react with NO but does with HNO, the formation of the hydroxylamine Tempol-H was observed. In the presence of O(2), about 60% of CH(3)C(O)NO(?-) yields ONOO(-), indicating that 30% NO(-) is formed in this system. It is concluded that under pulse radiolysis conditions, the transient nitroxide radicals derived from one-electron oxidation of aceto-HX decompose bimoleculary via a complex mechanism forming nitroxyl rather than NO.     

Mechanism of nitrite formation by nitrate photolysis in aqueous solutions: the role of peroxynitrite, nitrogen dioxide, and hydroxyl radical

Photolysis of aqueous NO3(-) with lambda > or = 195 nm is known to induce the formation of NO2(-) and O2 as the only stable products. The mechanism of NO3- photolysis, however, is complex, and there is still uncertainty about the primary photoprocesses and subsequent reactions. This is, in part, due to photoisomerization of NO3(-) to ONOO(-) at lambda < 280 nm, followed by the formation of *OH and *NO2 through the decomposition of ONOOH (pKa = 6.5-6.8). Because of incomplete information concerning the mechanism of peroxynitrite (ONOOH/ONOO(-)) decomposition, previous studies were unable to account for all observations. In the present study aqueous nitrate solutions were photolyzed by monochromatic light in the range of 205-300 nm. It is shown that the main primary processes at this wavelength range are NO3(-) hv-->*NO2 + O*(-) (reaction 1) and NO3(-) hv--> ONOO(-) (reaction 2). Based on recent knowledge on the mechanisms of peroxynitrite decomposition and its reactions with reactive nitrogen and oxygen species, we determined Phi(1) and Phi(2) using different experimental approaches. Both quantum yields increase with decreasing the excitation wavelength, approaching Phi(1) = 0.13 and Phi(2) = 0.28 at 205 nm. It is also shown that the yield of nitrite increases with decreasing the excitation wavelength. The implications of these results on UV disinfection of drinking water are discussed.     

Reactivities of superoxide and hydroperoxyl radicals with disubstituted cyclic nitrones: a DFT study

The unique ability of nitrone spin traps to detect and characterize transient free radicals by electron paramagnetic resonance (EPR) spectroscopy has fueled the development of new spin traps with improved properties. Among a variety of free radicals in chemical and biological systems, superoxide radical anion (O(2)(?-)) plays a critical role as a precursor to other more oxidizing species such as hydroxyl radical (HO(?)), peroxynitrite (ONOO(-)), and hypochlorous acid (HOCl), and therefore the direct detection of O(2)(?-) is important. To overcome the limitations of conventional cyclic nitrones, that is, poor reactivity with O(2)(?-), instability of the O(2)(?-) adduct, and poor cellular target specificity, synthesis of disubstituted nitrones has become attractive. Disubstituted nitrones offer advantages over the monosubstituted ones because they allow bifunctionalization of spin traps, therefore accommodating all the desired spin trap properties in one molecular design. However, because of the high number of possible disubstituted analogues as candidate, a systematic computational study is needed to find leads for the optimal spin trap design for biconjugation. In this paper, calculation of the energetics of O(2)(?-) and HO(2)(?) adduct formation from various disubstituted nitrones at PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory was performed to determine the most favorable disubstituted nitrones for this reaction. In addition, our results provided general trends of radical reactivity that is dependent upon but not exclusive to the charge densities of nitronyl-C, the position of substituents including stereoselectivities, and the presence of intramolecular H-bonding interaction. Unusually high exoergic ΔG(298K,aq)'s for O(2)(?-) and HO(2)(?) adduct formation were predicted for (3S,5S)-5-methyl-3,5-bis(methylcarbamoyl)-1-pyrroline N-oxide (11-cis) and (4S,5S)-5-dimethoxyphosphoryl-5-methyl-4-ethoxycarbonyl-1-pyrroline N-oxide (29-trans) with ΔG(298K,aq) = -3.3 and -9.4 kcal/mol, respectively, which are the most exoergic ΔG(298K,aq) observed thus far for any nitrone at the level of theory employed in this study.     

Control of the evolution of iron peroxide intermediate in superoxide reductase from Desulfoarculus baarsii. Involvement of lysine 48 in protonation

Superoxide reductase is a nonheme iron metalloenzyme that detoxifies superoxide anion radicals O(2)(?-) in some microorganisms. Its catalytic mechanism was previously proposed to involve a single ferric iron (hydro)peroxo intermediate, which is protonated to form the reaction product H(2)O(2). Here, we show by pulse radiolysis that the mutation of the well-conserved lysine 48 into isoleucine in the SOR from Desulfoarculus baarsii dramatically affects its reaction with O(2)(?-). Although the first reaction intermediate and its decay are not affected by the mutation, H(2)O(2) is no longer the reaction product. In addition, in contrast to the wild-type SOR, the lysine mutant catalyzes a two-electron oxidation of an olefin into epoxide in the presence of H(2)O(2), suggesting the formation of iron-oxo intermediate species in this mutant. In agreement with the recent X-ray structures of the peroxide intermediates trapped in a SOR crystal, these data support the involvement of lysine 48 in the specific protonation of the proximal oxygen of the peroxide intermediate to generate H(2)O(2), thus avoiding formation of iron-oxo species, as is observed in cytochrome P450. In addition, we proposed that the first reaction intermediate observed by pulse radiolysis is a ferrous-iron superoxo species, in agreement with TD-DFT calculations of the absorption spectrum of this intermediate. A new reaction scheme for the catalytical mechanism of SOR with O(2)(?-) is presented in which ferrous iron-superoxo and ferric hydroperoxide species are reaction intermediates, and the lysine 48 plays a key role in the control of the evolution of iron peroxide intermediate to form H(2)O(2).     

Lacidipine,a potential peroxynitrite scavenger: investigation of activity by liquid chromatography and mass spectrometry

Inflamed tissues are often characterised by the production of *NO and O(2)(-) radicals, which are known to react at an extremely fast rate to produce peroxynitrite (ONOO(-)). This highly oxidising entity reacts with protein-bound tyrosine to give 3-nitrotyrosine, which is considered a biochemical marker of peroxynitrite-induced damage. Lacidipine is a calcium antagonist indicated for the treatment of mild to moderate hypertension. In the present work, electrospray mass spectrometry with and without liquid chromatography was used to evaluate the capability of lacidipine and two other related molecules as ONOO(-) scavengers. This capability is compared with that associated with a number of commercial polyphenols described in the literature as efficient scavengers of this cytotoxic agent. The use of mass spectrometry provided rapid quantitative assessment of both the nitration and its reduction, and showed that lacidipine possesses a reasonable capability for reducing in vitro nitration of superoxide dismutase.     

Photoinduced NO release by visible light irradiation from pyrazine-bridged nitrosyl ruthenium complexes

The binuclear complex [RuII(NH3)5(pz)RuII(bpy)2(NO)](PF6)5 was prepared and characterized by elemental analysis, UV-vis, and IR spectroscopy. The complex UV-vis spectrum has presented bands at 242, 286, and 530 nm in acetate buffer solution at pH 4.5. The photochemical study by laser flash-photolysis at 532 nm showed the NO release account from the NO measured by a NO sensor. The quantum yield for NO release (0.025 +/- 0.004 mol einsten-1) was determined with a laser flash-photolysis apparatus (Continuum Q-switched Nd:YAG laser). The major irradiation product of the [RuII(NH3)5(pz)RuII(bpy)2(NO)]5+ complex besides nitric oxide is [RuIII(NH3)5(pz)RuII(bpy)2(H2O)]5+.     

Reaction of superoxide radical with quinone molecules

When the superoxide radical O(2)(?-) is generated on reaction of KO(2) with water in dimethyl sulfoxide, the decay of the radical is dramatically accelerated by inclusion of quinones in the reaction mix. For quinones with no or short hydrophobic tails, the radical product is a semiquinone at much lower yield, likely indicating reduction of quinone by superoxide and loss of most of the semiquinone product by disproportionation. In the presence of ubiquinone-10, a different species (I) is generated, which has the EPR spectrum of superoxide radical. However, pulsed EPR shows spin interaction with protons in fully deuterated solvent, indicating close proximity to the ubinquinone-10. We discuss the nature of species I, and possible roles in the physiological reactions through which ubisemiquinone generates superoxide by reduction of O(2) through bypass reactions in electron transfer chains.     

Delineation of the chemical pathways underlying nitric oxide-induced homologous recombination in mammalian cells

Inflammation is an important risk factor for cancer. During inflammation, macrophages secrete nitric oxide (NO*), which reacts with superoxide or oxygen to create ONOO- or N2O3, respectively. Although homologous recombination causes DNA sequence rearrangements that promote cancer, little was known about the ability of ONOO- and N2O3 to induce recombination in mammalian cells. Here, we show that ONOO- is a potent inducer of homologous recombination at an integrated direct repeat substrate, whereas N2O3 is relatively weakly recombinogenic. Furthermore, on a per lesion basis, ONOO(-)-induced oxidative base lesions and single-strand breaks are significantly more recombinogenic than N2O3-induced base deamination products, which did not induce detectable recombination between plasmids. Similar results were observed in mammalian cells from two different species. These results suggest that ONOO(-)-induced recombination may be an important mechanism underlying inflammation-induced cancer.     

Lifetimes and reaction pathways of guanine radical cations and neutral guanine radicals in an oligonucleotide in aqueous solutions

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, 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 CO(3)(?-) and SO(4)(?-) (via the photolysis of S(2)O(8)(2-) 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 (pK(a) = 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)(?), which, at best, reacts only slowly with H(2)O 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 CO(3)(?-), generating the two-electron oxidation 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 pulse. The levels of all products, except 8-oxoG, which remains at a low constant value, increase with the number of laser pulses.     

Predicting solvent stability in aprotic electrolyte Li-air batteries: nucleophilic substitution by the superoxide anion radical (O2(•-))

There is increasing evidence that cyclic and linear carbonates, commonly used solvents in Li ion battery electrolytes, are unstable in the presence of superoxide and thus are not suitable for use in rechargeable Li-air batteries employing aprotic electrolytes. A detailed understanding of related decomposition mechanisms provides an important basis for the selection and design of stable electrolyte materials. In this article, we use density functional theory calculations with a Poisson-Boltzmann continuum solvent model to investigate the reactivity of several classes of aprotic solvents in nucleophilic substitution reactions with superoxide. We find that nucleophilic attack by O(2)(?-) at the O-alkyl carbon is a common mechanism of decomposition of organic carbonates, sulfonates, aliphatic carboxylic esters, lactones, phosphinates, phosphonates, phosphates, and sulfones. In contrast, nucleophilic reactions of O(2)(?-) with phenol esters of carboxylic acids and O-alkyl fluorinated aliphatic lactones proceed via attack at the carbonyl carbon. Chemical functionalities stable against nucleophilic substitution by superoxide include N-alkyl substituted amides, lactams, nitriles, and ethers. The results establish that solvent reactivity is strongly related to the basicity of the organic anion displaced in the reaction with superoxide. Theoretical calculations are complemented by cyclic voltammetry to study the electrochemical reversibility of the O(2)/O(2)(?-) couple containing tetrabutylammonium salt and GCMS measurements to monitor solvent stability in the presence of KO(2)(?) and a Li salt. These experimental methods provide efficient means for qualitatively screening solvent stability in Li-air batteries. A clear correlation between the computational and experimental results is established. The combination of theoretical and experimental techniques provides a powerful means for identifying and designing stable solvents for rechargeable Li-air batteries.     

Scanning electrochemical microscopy studies of glutathione-modified surfaces. An erasable and sensitive-to-reactive oxygen species surface

A surface sensitive to reactive oxygen species (ROS) was prepared by reduction of a diazonium salt on glassy carbon electrode followed by the chemical coupling of glutathione (GSH) playing the role of an antioxidant species. The presence of active GSH was characterized through spectroscopic studies and electrochemical analysis after labeling of the -SH group with ferrocene moieties. The specific reactivity of GSH vs ROS was evaluated with scanning electrochemical microscopy (SECM) using the reduction of O(2) to superoxide, O(2)(?-), near the GSH-modified surface. Approach curves show a considerable decrease of the blocking properties of the layer due to reaction of the immobilized GSH with O(2)(?-) and the passage of GSH to the glutathione disulfide (GSSG). The initial surface could be regenerated several times with no significant variations of its antioxidant capacity by simply using the biological system glutathione reductase (GR)/NADPH that reduces GSSG back to GSH. SECM imaging shows also the possibility of writing local and erasable micropatterns on the GSH surface by production of O(2)(?-) at the tip probe electrode.     

Release of oxygen atoms and nitric oxide molecules from the ultraviolet photodissociation of nitrate adsorbed on water ice films at 100 K

Production of O((3)P(J), J = 2, 1, 0) atoms from the 295-320 nm photodissociation of NO(3)- adsorbed on water polycrystalline ice films at 100 K was directly confirmed using the resonance-enhanced multiphoton ionization technique. Detection of the O atom signals required an induction period after deposition of HNO3 onto the ice film held at 130 K due to the slow ionization rate of HNO(3) to H+ and NO(3)- with a rate constant of k = (5.3 +/- 0.2) x 10(-3)s(-1). Translational energy distributions of the O atoms were represented by a combination of two Maxwell-Boltzmann energy distributions with translational temperatures of 2000 and 100 K. Direct detection of NO from the secondary photodissociation process was also successful. On the atmospheric implications, the influence of the direct release of the oxygen atoms into the air from NO(3)- adsorbed on the natural snowpack was included in an atmospheric model calculation on the mixing ratios of ozone and nitric oxide at the South Pole, and the results compared favorably with the field data.     

CH3与NO2的反应

The elementary reaction of the CH3 radical with NO2 was investigated by time-resolved FTIR spectroscopy and quantum chemical calculations. The CH3 radical was produced by laser photolysis of CH3Br or CH3I at 248 nm. Vibrationally excited products OH, HNO and CO2 were observed by the time-resolved spectroscopy for the first time. The formation of another product NO was also verified. According to these observations, the product channels leading to CH3O+NO, CH2NO+OH and HNO+H2CO were identified. The channel of CH3O+NO was the major one. The reaction mechanisms of the above channels were studied by quantum chemical calculations at CCSD(T)/6-311++G(df,p)//MP2/6-311G(d,p) level. The calculated results fit with the experimental observations well.     

Seminaphthofluorescein-based fluorescent probes for imaging nitric oxide in live cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.     

Hydroxyl radical formation by O-O bond homolysis in peroxynitrous acid

Peroxynitrite decay in weakly alkaline media occurs by two concurrent sets of pathways which are distinguished by their reaction products. One set leads to net isomerization to NO(3)(-) and the other set to net decomposition to O(2) plus NO(2)(-). At sufficiently high peroxynitrite concentrations, the decay half-time becomes concentration-independent and approaches a limiting value predicted by a mechanism in which reaction is initiated by unimolecular homolysis of the peroxo O-O bond, i.e., the following reaction: ONOOH --> (*)OH + (*)NO(2). This dynamical behavior excludes alternative postulated mechanisms that ascribe decomposition to bond rearrangement within bimolecular adducts. Nitrate and nitrite product distributions measured at very low peroxynitrite concentrations also correspond to predictions of the homolysis model, contrary to a recent report from another laboratory. Additionally, (1) the rate constant for the reaction ONOO(-) --> (*)NO + (*)O(2)(-), which is critical to the kinetic model, has been confirmed, (2) the apparent volume of activation for ONOOH decay (DeltaV() = 9.7 +/- 1.4 cm(3)/mol) has been shown to be independent of the concentration of added nitrite and identical to most other reported values, and (3) complex patterns of inhibition of O(2) formation by radical scavengers, which are impossible to rationalize by alternative proposed reaction schemes, are shown to be quantitatively in accord with the homolysis model. These observations resolve major disputes over experimental data existing in the literature; despite extensive investigation of these reactions, no verifiable experimental evidence has been advanced that contradicts the homolysis model.     

Evaluation of the anti-oxidant properties of a SOD-mimic Mn-complex in activated macrophages

SOD-mimics are small complexes that reproduce the activity of superoxide dismutases, natural proteins that catalytically dismutate the superoxide anion. Activated macrophages, which produce ROS and RNS fluxes, constitute a relevant model to challenge antioxidant activity in a cellular context and were used to test a Mn-complex which was shown to efficiently alter the flow of O(2)(-), ONOO(-) and H(2)O(2).     

Reactions of CH3SH and CH3SSCH3 with gas-phase hydrated radical anions (H2O)n(•-), CO2(•-)(H2O)n, and O2(•-)(H2O)n

The chemistry of (H(2)O)(n)(?-), CO(2)(?-)(H(2)O)(n), and O(2)(?-)(H(2)O)(n) with small sulfur-containing molecules was studied in the gas phase by Fourier transform ion cyclotron resonance mass spectrometry. With hydrated electrons and hydrated carbon dioxide radical anions, two reactions with relevance for biological radiation damage were observed, cleavage of the disulfide bond of CH(3)SSCH(3) and activation of the thiol group of CH(3)SH. No reactions were observed with CH(3)SCH(3). The hydrated superoxide radical anion, usually viewed as major source of oxidative stress, did not react with any of the compounds. Nanocalorimetry and quantum chemical calculations give a consistent picture of the reaction mechanism. The results indicate that the conversion of e(-) and CO(2)(?-) to O(2)(?-) deactivates highly reactive species and may actually reduce oxidative stress. For reactions of (H(2)O)(n)(?-) with CH(3)SH as well as CO(2)(?-)(H(2)O)(n) with CH(3)SSCH(3), the reaction products in the gas phase are different from those reported in the literature from pulse radiolysis studies. This observation is rationalized with the reduced cage effect in reactions of gas-phase clusters.