Production of reactive oxygen species on photolysis of dilute aqueous quinone solutions

We have examined the generation of the reactive oxygen species (ROS) superoxide and hydrogen peroxide (H2O2) by irradiation of dilute aqueous solutions of disodium anthraquinone-2-6-disulfonate (AQDS) with simulated sunlight. Irradiating a solution of AQDS in 2 mM NaHCO3 and 0.01 M NaCl produced superoxide and H2O2 at nanomolar concentrations. Experiments in which initial concentrations of dioxygen, H2O2, the superoxide radical trap nitroblue tetrazolium and the electron donor dimethyl sulfoxide were varied suggested that the interaction of solvent water with photo-excited quinone moieties produces dioxygen-reducing radicals, and that these are the primary source of ROS in the system. A kinetic model for ROS production is proposed based on our experimental data.     

Reactivity of superoxide anion radical with a perchlorotriphenylmethyl (trityl) radical

The reaction of superoxide radical with a tricarboxylate derivative of perchlorotriphenylmethyl radical (PTM-TC) is studied. PTM-TC is a stable ("inert") free radical, which gives a single sharp electron paramagnetic resonance (EPR) peak in aqueous solutions. PTM-TC also gives a characteristic optical absorption at 380 nm. Superoxide, on reaction with PTM-TC, induced a decrease in the intensity of the EPR signal and optical absorption of PTM-TC at 380 nm. The signal loss was specific to superoxide and linearly dependent on the superoxide flux in the system. Competitive kinetics experiments revealed that PTM-TC reacts with superoxide with an apparent second-order rate constant of 8.3x10(8) M(-1) s(-1). Electrochemical and mass spectrometric analyses of the reaction suggested the formation of perchlorotriphenylmethane and molecular oxygen as products. The high sensitivity of detection of PTM-TC combined with the high rate constant of the reaction of superoxide with PTM-TC may offer a potential opportunity for measurement of superoxide in biological systems. In conclusion, the PTM-TC molecule has high sensitivity and specificity for superoxide radicals and thus may enable quantitative detection of superoxide generation in biological systems using EPR and/or spectrophotometric methods.     

PRIMARY PROCESSES IN THE PHOTOCHEMISTRY OF AQUEOUS SULPHACETAMIDE: A LASER FLASH PHOTOLYSIS and PULSE RADIOLYSIS STUDY

Abstract— The spectra have been measured of the transient species formed in the nanosecond flash photolysis of aqueous solutions of sulphacetamide under a variety of conditions. In addition to the excited triplet state, the cation radical and the solvated electron were observed. The ionisation of aqueous sulphacetamide was found to occur by a biphotonic process. The extinction coefficient of the cation radical of sulphacetamide was determined by both laser flash photolysis and pulse radiolysis techniques, a value of 1.9 times 10₃ dm₃mol_-1cm_-1 being obtained. The rate of electron reaction with sulphacetamide and the anion radical spectrum were also determined by the two techniques, good agreement being obtained. The spectrum of the product of the reaction of the superoxide anion radical and the corresponding rate constant have also been determined. A possible mechanism of photosensitized skin reaction due to sulphacetamide is discussed.     

Chemical and photochemical electron transfer of new helianthrone derivatives: aspects of their photodynamic activity

Helianthrones 2-4 are a new class of synthetic photosensitizers, which have a molecular skeleton related to that of hypericin. We established that irradiation of heliantrones with visible light leads to the formation of semiquinone radicals and reactive oxygen species. The structures of the paramagnetic anion species produced by electron transfer were calculated on the density functional level and investigated by cyclovoltammetry, UV/vis, and EPR/ENDOR spectroscopy. As with hypericin, the pi system of the helianthrones was found to be considerably deviated from planarity, and, upon electron transfer, deprotonation in the bay region occurs. The structure of the semiquinone radicals was found to be identical in THF, DMF, and aqueous buffered solutions regardless of the means by which reduction was achieved. Semiquinone radicals can be formed via self-electron transfer between the excited state and the ground state or via electron transfer from an electron donor to the excited state of helianthrone. Therefore, the presence of an electron donor significantly enhanced the photogeneration of semiquinone and superoxide radical. The kinetic studies showed that no significant photochemical destruction of helianthrones occurred upon irradiation. Generation of superoxide and singlet oxygen upon irradiation of helianthrones was established by spin trapping techniques. This shows that both type I and type II mechanisms are of importance for the photodynamic action of these compounds.     

Thermodynamics for nonequilibrium solvation and numerical evaluation of solvent reorganization energy

This work presents a thermodynamic method for treating nonequilibrium solvation. By imposing an extra electric field onto the nonequilibrium solvation system, a virtual constrained equilibrium state is prepared. In this way, the free energy difference between the real nonequilibrium state and the con-strained equilibrium one is simply the potential energy of the nonequilibrium polarization in the extra electronic field, according to thermodynamics. Further, new expressions of nonequilibrium solvation energy and solvent reorganization energy have been formulated. Analysis shows that the present formulations will give a value of reorganization energy about one half of the traditional Marcus theory in polar solvents, thus the explanation on why the traditional theory tends to overestimate this quantity has been found out. For the purpose of numerical determination of solvent reorganization energy, we have modified Gamess program on the basis of dielectric polarizable continuum model. Applying the procedure to the well-investigated intramolecular electron transfer in biphenyl-androstane-naphthyl and biphenyl-androstane-phenanthryl systems, the numerical results of solvent reorganization energy have been found to be in good agreement with the experimental fittings.     

Design of photoactivated DNA oxidizing agents: synthesis and study of photophysical properties and DNA interactions of novel viologen-linked acridines

A new series of photoactivated DNA oxidizing agents in which an acridine moiety is covalently linked to viologen by an alkylidene spacer was synthesized, and their photophysical properties and interactions with DNA, including DNA cleaving properties, were investigated. The fluorescence quantum yields of the viologen-linked acridines were found to be lower than that of the model compound 9-methylacridine (MA). The changes in free energy for the electron transfer reactions were found to be favorable, and the fluorescence quenching observed in these systems is explained by an electron transfer mechanism. Intramolecular electron transfer rate constants were calculated from the observed fluorescence quantum yields and singlet lifetime of MA and are in the range from 1.06x10(10) s(-1) for 1 a (n=1) to 6x10(8) s(-1) for 1 c (n=11), that is, the rate decreases with increasing spacer length. Nanosecond laser flash photolysis of these systems in aqueous solutions showed no transient absorption, but in the presence of guanosine or calf thymus DNA, transient absorption due to the reduced viologen radical cation was observed. Studies on DNA binding demonstrated that the viologen-linked acridines bind effectively to DNA in both intercalative and electrostatic modes. Results of PM2 DNA cleavage studies indicate that, on photoexcitation, these molecules induce DNA damage that is sensitive to formamidopyrimidine DNA glycosylase. These viologen-linked acridines are quite stable in aqueous solutions and oxidize DNA efficiently and hence can be useful as photoactivated DNA-cleaving agents which function purely by the co-sensitization mechanism.     

THE ROLE OF O2- IN THE CHEMILUMINESCENCE OF LUMINOL*

Abstract— The chemiluminescence of luminol in buffered aqueous solutions is inhibited by superoxide dismutase. This occurs whether the luminescence is induced by ferricyanide, persulfate, hypochlorite, or by the action of xanthine oxidase on xanthine. Since superoxide dismutase inhibits reactions which involve O₂^-, we conclude that this radical is a constant factor in the chemiluminescence of luminol in aqueous solutions. The kinetics of light production are discussed in terms of hypothetical mechanisms that fit the available data. The strong luminescence of luminol in aprotic solvents or in aqueous systems containing relatively high concentrations of H₂O₂ could not be explored in this way, because superoxide dismutase is inactive under such conditions.     

Radiation Chemistry of Neptunium Ions in Aqueous Carbonate and Bicarbonate Solutions*

The radiolytic reactions of neptunium ions in aqueous carbonate, alkaline carbonate, and bicarbonate solutions were examined. It was found that Np(VI) is not oxidized to Np(VII) in carbonate and bicarbonate solutions saturated with nitrous oxide, whereas this oxidation process takes place in alkaline carbonate solutions, in which Np(VI) occurs as a hydroxo complex. It was also found that Np(V) is a radiation-stable neptunium species in carbonate solutions, whereas Np(VI) is stable in bicarbonate solutions.     

The mechanism of the electrogenerated chemiluminescence of luminol in aqueous alkaline solution

The mechanism of the electrogenerated chemiluminescence of luminol in aqueous alkaline solution based on the rotating ring—disc electrode system is discussed. The disc electrode is maintained at a negative potential and the ring electrode at a symmetrically changing double-step potential. Hydrogen peroxide generated at the disc electrode by the reduction of oxygen is immediately transported to the ring electrode because of electrode rotation. Hydrogen peroxide and luminol are oxidized at the ring electrode during the positive pulse of the double-step potential. These oxidation processes generate a superoxide radical and a luminol radical as intermediates. The luminol radical reacts with the superoxide radical (or oxygen) emitting light.     

Singlet oxygen generated from the decomposition of peroxymonocarbonate and its observation with chemiluminescence method

The decomposition of peroxymonocarbonate (HCO(4)(-)) has been investigated by flow-injection chemiluminescence (CL) method. An ultraweak CL was observed during mixing the bicarbonate and hydrogen peroxide solution in organic cosolvent. An appropriate amount of fluorescent organic compounds, such as dichlorofluorescein (DCF), was added to the HCO(4)(-) solution, a strong CL was recorded. Based on studies of the spectrum of fluorescence, CL and UV-vis spectra, electron spin trapping (ESR) technique, mass spectra (MS) and comparison with H(2)O(2)/hypochlorite (ClO(-)) and H(2)O(2)/molybdate (MoO(4)(-)) systems, the CL mechanism was proposed. The reaction is initiated by unimolecular homolysis of the peroxo O-O bond in HO-OCOO(-) molecule. It was suggested that the bond rearrangement within radicals yield superoxide ion (O(2)(*-)). The interaction of superoxide ion with perhydroxyl radical produces singlet oxygen ((1)O(2)). The energy transfers from singlet oxygen to DCF forming an excited energy acceptor (DCF*). Luminescence (lambda(max)=509 nm) was emitted during the relaxation of the energy acceptor to the ground state.     

Metal bacteriochlorins which act as dual singlet oxygen and superoxide generators

A series of stable free-base, Zn(II) and Pd(II) bacteriochlorins containing a fused six- or five-member diketo- or imide ring have been synthesized as good candidates for photodynamic therapy sensitizers, and their electrochemical, photophysical, and photochemical properties were examined. Photoexcitation of the palladium bacteriochlorin affords the triplet excited state without fluorescence emission, resulting in formation of singlet oxygen with a high quantum yield due to the heavy atom effect of palladium. Electrochemical studies revealed that the zinc bacteriochlorin has the smallest HOMO-LUMO gap of the investigated compounds, and this value is significantly lower than the triplet excited-state energy of the compound in benzonitrile. Such a small HOMO-LUMO gap of the zinc bacteriochlorin enables intermolecular photoinduced electron transfer from the triplet excited state to the ground state to produce both the radical cation and the radical anion. The radical anion thus produced can transfer an electron to molecular oxygen to produce superoxide anion which was detected by electron spin resonance. The same photosensitizer can also act as an efficient singlet oxygen generator. Thus, the same zinc bacteriochlorin can function as a sensitizer with a dual role in that it produces both singlet oxygen and superoxide anion in an aprotic solvent (benzonitrile).     

Primary photophysical properties of ofloxacin

Steady-state fluorescence has been used to study the excited singlet state of ofloxacin (OFLX) in aqueous solutions. Fluorescence emission was found to be pH dependent, with a maximum quantum yield of 0.17 at pH 7. Two pKa*s of around 2 and 8.5 were obtained for the excited singlet state. Laser flash photolysis and pulse radiolysis have been used to study the excited states and free radicals of OFLX in aqueous solutions. OFLX undergoes monophotonic photoionization from the excited singlet state with a quantum yield of 0.2. The cation radical so produced absorbs maximally at 770 nm with an extinction coefficient of 5000 +/- 500 dm3 mol-1 cm-1. This is confirmed by one-electron oxidation in the pulse radiolysis experiments. The hydrated electron produced in the photoionization process reacts with ground state OFLX with a rate constant of 2.0 +/- 0.2 x 10(10) dm3 mol-1 s-1, and the anion thus produced has two absorption bands at 410 nm (extinction coefficient = 3000 +/- 300 dm3 mol-1 cm-1) and at 530 nm. Triplet-triplet absorption has a maximum at 610 nm with an extinction coefficient of 11,000 +/- 1500 dm3 mol-1 cm-1. The quantum yield of triplet formation has been determined to be 0.33 +/- 0.05. In the presence of oxygen, the triplet reacts to form both excited singlet oxygen and superoxide anion with quantum yields of 0.13 and < or = 0.2, respectively. Moreover, superoxide anion is also formed by the reaction of oxygen with the hydrated electron from photoionization. Hence the photosensitivity due to OFLX could be initiated by the oxygen radicals and/or by OFLX radicals acting as haptens.     

Near 0 eV electrons attach to nucleotides

To elucidate the mechanism of the nascent stage of DNA strand breakage by low-energy electrons, theoretical investigations of electron attachment to nucleotides have been performed by the reliably calibrated B3LYP/DZP++ approach (Chem. Rev. 2002, 102, 231). The 2'-deoxycytidine-3'-monophosphate (3'-dCMPH) and its phosphate-deprotonated anion (3'-dCMP(-)) have been selected herein as models. This investigation reveals that 3'-dCMPH is able to capture near 0 eV electrons to form a radical anion which has a lower energy than the corresponding neutral species in both the gas phase and aqueous solution. The excess electron density is primarily located on the base of the nucleotide radical anion. The electron detachment energy of this pyrimidine-based radical anion is high enough that subsequent phosphate-sugar C-O sigma bond breaking or glycosidic bond cleavage is feasible. Although the phosphate-centered radical anion of 3'-dCMPH is not stable in the gas phase, it may be stable in aqueous solution. However, an incident electron with kinetic energy less than 4 eV might not be able to effectively produce the phosphate-centered radical anion either in solution or in the gas phase. This research also suggests that the electron affinity of the nucleotides is independent of the counterion in aqueous solution.     

Excited-state behavior and photoionization of 1,8-acridinedione dyes in micelles--comparison with NADH oxidation

The photophysics and photochemistry of 1,8-acridinedione dyes, which are analogues of reduced nicotinamide adenine dinucleotide (NADH), are studied in anionic and cationic micelles. Acridinedione dyes (ADDs) are solubilized in micelles at the micelle-water interface and are in equilibrium between the aqueous and micellar phase. The binding of the ADDs with micelles is attributed to hydrophobic interactions and the binding constants are determined with steady-state and time-resolved techniques. Nanosecond laser flash photolysis studies are carried out in aqueous, anionic, and cationic micellar solutions. The ADD undergoes photoionization in the excited state to give a solvated electron. The solvated electron reacts with the ADD to give an anion radical. In anionic micelles, the yield of the solvated electron increases because of the efficient separation of the cation radical and the electron. Cation radicals derived from the photooxidation of ADDs are involved in keto-enol tautomerization. Under acidic conditions, an enol radical cation of the acridinedione dye is formed from the keto form of the cation radical by intramolecular hydrogen atom transfer. In cationic micelles, due to electrostatic attraction, the electron cannot escape from the micelle and recombination of the cation radical and the electron results in the formation of a triplet state. For the first time, a solvated electron is observed in the laser flash photolysis of ADDs in anionic micelles. The photoionization of ADDs depends on the excitation wavelength and is biphotonic at 355 nm and monophotonic at 248 nm. From the results with this NADH model compound, the sequential electron-proton-electron transfer oxidation of NADH is confirmed and the nature of the intermediates involved in the oxidation is unraveled; these intermediates are found to depend on the pH value of the medium.     

Coumarin 314 free radical cation: formation, properties, and reactivity toward phenolic antioxidants

We have explored the photogeneration of the coumarin 314 radical cation by using nanosecond laser excitation at wavelengths longer than 400 nm in benzene, acetonitrile, dichloromethane, and aqueous media. In addition, time-resolved absorption spectroscopy measurements allowed detection of the triplet excited state of coumarin 314 (C(314)) with a maximum absorption at 550 nm in benzene. The triplet excited state has a lifetime of 90 μs in benzene. It is readily quenched by oxygen (k(q) = 5.0 × 10(9) M(-1) s(-1)). From triplet-triplet energy transfer quenching experiments, it is shown that the energy of this triplet excited state is higher than 35 kcal/mol, in accord with the relatively large singlet oxygen quantum yield (Φ(Δ) = 0.25). However, in aqueous media, the coumarin triplet was no longer observed, and instead of that, a long-lived (160 μs in air-equilibrated solutions) free radical cation with a maximum absorbance at 370 nm was detected. The free radical cation generation, which has a quantum yield of 0.2, occurs by electron photoejection. Moreover, density functional theory (DFT) calculations indicate that at least 40% of the electronic density is placed on the nitrogen atom in aqueous media, which explains its lack of reactivity toward oxygen. On the other hand, rate constant values close to the diffusion rate limit in water (>10(9) M(-1) s(-1)) were found for the quenching of the C(314) free radical cation by phenolic antioxidants. The results have been interpreted by an electron-transfer reaction between the phenolic antioxidant and the radical cation where ion pair formation could be involved.     

An electron paramagnetic resonance study of the influence of spermine on the photophysical reactions of tryptophan in aqueous solution at 77–200 K

The influence of the antioxidant spermine of the UV-induced formation of free radicals from tryptophan in frozen aqueous solutions was studied by electron paramagnetic resonance (EPR) instrumentation, and the stability of the radicals was investigated in the range 95–200 K. Without spermine, the tryptophan cation and neutral tryptophan radical were stabilized at 77 K; cations were formed by electron ejection from an excited singlet state, and neutral radicals by hydrogen donation from tryptophan in the triplet state. When present, spermine trapped the ejected photoelectrons; the rates of the two photoreactions of tryptophan were also influenced by spermine. Firstly, at low tryptophan concentrations, the yield of cations was reduced, due to diminished charge transfer from the excited singlet state to the solvation shell. Secondly, at high concentrations, minute additions of spermine enhanced intersystem crossing (which is quenched, in the absence of spermine, by dimerization) and, consequently, the yield of neutral radicals was increased. At 180 K, the electrons trapped by spermine were released and reacted with molecular oxygen to form the superoxide radical; at 190 K, the tryptophan radicals were thermally annealed.     

Near-ultraviolet photolysis of L-mandelate, formation of reactive oxygen species, inactivation of phage T7 and implications on human health

Compared with ultraviolet B and C, UVA is considered to have little direct effects on biological systems. However, damaging effects of UVA on biological systems are often synergistically enhanced in the presence of sensitizers. Production of reactive oxygen species (ROS) has been implicated in the process. Several ROS have been identified but their involvement in inducing cellular damage is yet to be fully evaluated. Although membranes and proteins are affected, DNA is an important target and a variety of types of damage have been reported. Here, we present evidence that L-mandelate can act as a near UV (NUV) sensitizer, when activated by a lamp emitting 99% UVA and 1% UVB. Although evidence is available that H(2)O(2) and a small amount of *OH are produced, an alternative effect of the sensitization reaction may involve direct electron transfer. Studies have shown that NUV photolysis of mandelate can inactivate phage T7. Employment of tetrazolium blue test to detect superoxide anion may not be sufficient evidence as this agent may be reduced by alternative routes.     

Generation of free radicals by photoexcitation of pheophorbide alpha, haematoporphyrin and protoporphyrin

An investigation of the production of radical species by photoexcitation pheophorbide alpha, haematoporphyrin and protoporphyrin was performed. In an aqueous solution containing different amounts of ethanol, the superoxide radical was detected by the spin trapping technique. In addition, secondary radicals were observed. The generation of oxygen radicals was found to dominate in solutions with a low ethanol content.     

Pulse radiolysis of tetrazolium violet in aqueous and aqueous-alcoholic solutions under oxidative and reductive conditions

The radiolytic reduction of colourless tetrazolium salts to coloured formazans in liquid and solid state is suggested for dosimetry purposes. In order to clarify the reaction mechanism, a pulse radiolysis study was conducted in aqueous and aqueous-alcoholic solutions under oxidative and reductive conditions. Under reducing conditions, fast formation of the electron adduct tetrazolinyl radical was observed: coloured formazan final product formed during the decay of electron adduct. Both the decay of the tetrazolinyl radical and the formation of the formazan were found to be second order. The spectra of the formazan were similar in neutral and alkaline solutions, but with higher absorbance in the latter solutions due to the higher molar absorption coefficient. Under oxidative conditions formazan did not form; hydroxylated products through OH-adducts were observed in the pH range studied.     

Reductive halogen elimination from phenols by organic radicals in aqueous solutions; chain reaction induced by proton-coupled electron transfer

Gamma-radiolysis and measurements of halide ions by means of ion chromatography have been employed to investigate reductive dehalogenation of chloro-, bromo-, and iodophenols by carbon-centered radicals, *CH(CH(3))OH, *CH(2)OH, and *CO(2)-, in oxygen-free aqueous solutions in the presence of ethanol, methanol, or sodium formate. While the reactions of 4-IC(6)H(4)OH with *CH(CH(3))OH and *CH(2)OH radicals are endothermic in water/alcohol solutions, the addition of bicarbonate leads to iodide production in high yields, indicative of a chain reaction. The maximum effect has been observed with about 10 mM sodium bicarbonate present. The complex formed from an alpha-hydroxyalkyl radical and a bicarbonate anion is considered to cause the enhancement of the reduction power of the former to the extent at which the reduction of the iodophenol molecule becomes exothermic. No such effect has been observed with phosphate, which is a buffer with higher proton affinity, when added in the concentration of up to 20 mM at pH 7. This indicates that one-electron reduction reactions by alpha-hydroxyalkyl radicals occur by the concerted proton-coupled electron transfer, PCET, and not by a two-step ET/PT or PT/ET mechanisms. The reason for the negative results with phosphate buffer could be thus ascribed to a less stable complex or to the formation of a complex with a less suitable structure for an adequate support to reduce iodophenol. The reduction power of the carbonate radical anion is shown to be high enough to reduce iodophenols by a one-electron-transfer mechanism. In the presence of formate ions as H-atom donors, the dehalogenation also occurs by a chain reaction. In all systems, the chain lengths depend on the rate of reducing radical reproduction in the propagation step, that is, on the rate of H-atom abstraction from methanol, ethanol, or formate by 4-*C(6)H(4)OH radicals liberated after iodophenol dehalogenation. The rate constants of those reactions were determined from the iodide yield measurements at a constant irradiation dose rate. They were estimated to be 6 M(-1)(s-1) for methanol, 140 M(-1)(s-1) for ethanol, and 2100 M(-1)(s-1) for formate. Neither of the tested reducing C-centered radicals was able to dehalogenate the bromo or chloro derivative of phenol.