Electron spin resonance studies of azasemiquinone free radical intermediates in the oxidation of hydroxypyridones

The oxidation of hydroxy-2-pyridones by a number of one-electron oxidants has been studied, and intermediate free radicals observed by ESR spectroscopy. In alkaline media, azasemiquinones arising from electron transfer, solvation and oxidative coupling processes have been detected. The ESR hyperfine splittings are assigned with the aid of Me substitution, and the spin densities can be understood by considering the perturbation of the N atom within the semiquinone nucleus. In acid solution, the protonation of nitrogen produces structural changes in the semiquinone nucleus, which greatly affect the spin density distributions. The apparent lifetimes of the various radical species can be correlated with their expected tendencies to direct free radical dimerisation.     

A REACTION BETWEEN PHEOPHYTIN and CHLOROPHYLL ADSORBED TO POLYETHYLENE-TETRADECANE PARTICLES*

Abstract— When chlorophyll is adsorbed to polyethylene-tetradecane particles along with ligating amphiphiles, and pheophytin is present also, a slow but irreversible photobleaching of the chlorophyll is observed in suspensions of the particles in aqueous media. It is suggested that the reaction starts by transfer of an electron from singlet excited chlorophyll to pheophytin and concludes by hydration and reduction of the chlorophyll radical cation.     

ESR studies of chlorophyll one-electron photochemistry in solution: kinetics of reversible quinone reduction and general redox mechanisms

Abstract— ESR studies have been made of the kinetics of semiquinone radical formation and disappearance resulting from the reversible photosensitization of reduction or oxidation, by chlorophyll, pheophytin or hematoporphyrin, of several quinone-hydroquinone pairs in various solvents. The rate of radical decay was found to be second order with respect to the radical concentration in all systems. Radical formation rates were determined by the initial production rate minus the decay rate. The kinetic constants for single electron transfer between triplet porphyrins and quinones or hydroquinones were determined usingβ-carotene as a quencher in aqueous pyridine, and by measuring the initial rate of radical formation at various concentrations of quinones and hydroquinones in methanol and ethanol. These constants were found to be approximately the same in a given solvent for benzoquinone and hydroquinone with all porphyrins, though the rates differed in different solvents: pyridine-water ～ 10⁶I./mole sec, and methanol and ethanol ～ 5X 10⁴l./mole sec. Trimethylquinone and its hydroquinone also give similar rate constants for radical formation in pyridine-water, ～ 10⁶ l./mole sec. The second order radical decay constants for both benzoquinone and hydroquinone in pyridine-water were the same, ～ 10⁵ I./mole sec, with either chlorophyll, pheophytin or hematoporphyrin as sensitizer. The same activation energy, 6900 cal/mole, was found for chlorophyll-benzoquinone and hydroquinone in aqueous pyridine; 5500 cal/mole was obtained for these systems in ethanol. In methanol and ethanol solutions of chlorophyll, the same radical decay rate constants, ～10⁶ I./mole sec, were observed for both benzoquinone and hydroquinone. Also, the same decay constants, ～ 10⁶ I./mole sec, were found for trimethylquinone and its hydroquinine in pyridine-water. These latter two compounds gave extremely small steady-state ESR signals in ethanol compared with aqueous pyridine. We have also observed that the steady-state signal obtained with chlorophyll-menadione in ethanol-water was much enhanced by the presence of NADH. In contrast, NAD⁺ was found to decrease radical production, by increasing the decay rate, in the chlorophyll-hydroquinone system in aqueous pyridine. These results are discussed in terms of possible mechanisms for radical formation and disappearance. The most likely possibility is considered to be a one-electron oxidation or reduction of the porphyrin triplet, followed by radical disproportionation and redox reactions between the disproportionation products.     

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.     

Electron-transfer oxidation properties of unsaturated fatty acids and mechanistic insight into lipoxygenases

Rate constants of photoinduced electron-transfer oxidation of unsaturated fatty acids with a series of singlet excited states of oxidants in acetonitrile at 298 K were examined and the resulting electron-transfer rate constants (k(et)) were evaluated in light of the free energy relationship of electron transfer to determine the one-electron oxidation potentials (E(ox)) of unsaturated fatty acids and the intrinsic barrier of electron transfer. The k(et) values of linoleic acid with a series of oxidants are the same as the corresponding k(et) values of methyl linoleate, linolenic acid, and arachidonic acid, leading to the same E(ox) value of linoleic acid, methyl linoleate, linolenic acid, and arachidonic acid (1.76 V vs SCE), which is significantly lower than that of oleic acid (2.03 V vs SCE) as indicated by the smaller k(et) values of oleic acid than those of other unsaturated fatty acids. The radical cation of linoleic acid produced in photoinduced electron transfer from linoleic acid to the singlet excited state of 10-methylacridinium ion as well as that of 9,10-dicyanoanthracene was detected by laser flash photolysis experiments. The apparent rate constant of deprotonation of the radical cation of linoleic acid was determined as 8.1 x 10(3) s(-1). In the presence of oxygen, the addition of oxygen to the deprotonated radical produces the peroxyl radical, which has successfully been detected by ESR. No thermal electron transfer or proton-coupled electron transfer has occurred from linoleic acid to a strong one-electron oxidant, Ru(bpy)3(3+) (bpy = 2,2'-bipyridine) or Fe(bpy)3(3+). The present results on the electron-transfer and proton-transfer properties of unsaturated fatty acids provide valuable mechanistic insight into lipoxygenases to clarify the proton-coupled electron-transfer process in the catalytic function.     

Comparison of photo- and radiation-induced radical formation in polyethylene by ESR spectroscopy

The behaviors of free radicals produced in polyethylene irradiated with ultraviolet light and electron beams were compared in connection with primary processes of radical formation and trapping regions of free radicals. In the case of irradiation with ultraviolet light, an ESR spectrum observed at ?196°C immediately after irradiation is an eight-line spectrum due to alkyl radicals of the type ? CH₂? ?H? CH₃, while in the case of ionizing radiation, a six-line spectrum due to ? CH₂? ?H? CH₂? was observed. The former radicals are produced by the Norrish type I reaction of the carbonyl groups contained in the polymer, followed by radical rearrangement; and the latter are formed by dissociation of hydrogen atom from the excited state of the polymer or ion-molecular reactions. From the sensitivity to oxygen molecules, it was deduced that free radicals are trapped in amorphous regions after ultraviolet irradiation, but mainly in crystalline regions after irradiation with electron beams. Saturation studies of ESR spectra seem to support this conclusion.     

ESR EVIDENCE FOR CHLOROPHYLL-PHOTOSENSITIZED ONE-ELECTRON OXIDATION OF WATER BY BENZOQUINONE*

Abstract —Illumination with red light of degassed solutions of chlorophyll and benzoquinone in dry acetone does not result in quinone radical formation, even though the presence of quinone greatly enhances the photoproduction of chlorophyll cation radical at low temperatures. However, when small amounts of water (or alcohols) are added to this system, large benzoquinone anion radical electron spin resonance (ESR) signals are generated. The effects on the low-temperature ESR signals of microwave power saturation, replacement of chlorophyll and quinone by their deuterated analogs, and replacement of H₂O by D₂O, lead to the suggestion that a one-electron oxidation of water is occurring. Pheophytin and bacteriochlorophyll are shown to sensitize a similar photoreaction. These results could lead to new insights into the mechanism of oxygen formation in photosynthesis.     

The effect of axial Mg ligation on the geometry and spin density distribution of chlorophyll and bacteriochlorophyll cation free radical models: a density functional study.

Density functional calculations are performed on models of chlorophyll and bacteriochlorophyll to examine the effect of Mg ligation on the geometry and spin density distribution of the cation free radicals formed. It is shown that, whereas the properties of the bacteriochlorophyll model can be explained on the basis of the electron density distribution of the highest occupied molecular orbital (HOMO), for the chlorophyll model the geometry and spin density properties of the ligated species do not follow this trend. For the ligated chlorophyll models it is shown that, due to the closeness in energy of the HOMO and HOMO-1 orbitals, a Jahn-Teller distortion occurs on one-electron oxidation, leading to an admixed hybrid orbital for the cation radical form. Orbital mixing is shown to lead to significant changes in the geometry and spin density distribution of the cation free radical formed. It is also shown that orbital mixing does not lead to an increase in the magnitude of the (14)N hyperfine couplings thereby invalidating reports in the literature which have dismissed mixed orbital states for the primary donor cation radicals of photosynthetic reaction centers based on this criterion.     

LASER PHOTOLYSIS STUDIES OF QUINONE REDUCTION BY PHEOPHYTIN a IN ALCOHOL SOLUTION

Abstract- Upon laser photolysis of pheophytin-benzoquinone solutions in ethanol, transients due to the pheophytin triplet state (P_t), an exciplex (Pδ⁺ Qδ^-), the pheophytin cation radical (P⁺) and the semiquinone radical (Q^-) can be observed. Kinetic analysis indicates that the evolution of these transients at times longer than one microsecond is due to the decay of the exciplex with the concomitant formation of P⁺ and Q^-, reverse electron transfer to form P and Q, solvent oxidation by P⁺, and Q^- disproportionation. In support of the suggested solvent oxidation reaction, a large deuterium isotope effect is observed upon changing the solvent from methanol to its fully-deuterated counterpart. Comparisons are made between these results and those obtained with chlorophyll as described in the preceding paper.     

LONG-LIVED AFTERGLOW OF PHOTOSYNTHETIC PIGMENTS IN SOLUTION: PHOTOCHEMILUMINESCENCE

Abstract —Our recent research on photochemiluminescence (PCL) of pigments in solutions is reviewed. PCL was observed in the course of photooxidation by oxygen of chlorophyll a , bacteriochlorophyll, protochlorophyll, their analogs, synthetic dyes and aromatic hydrocarbons. The PCL of chlorophyll was studied in detail. It depends on oxygen concentration, intensity of exciting light, pH, nature of pigments, solvents etc. The thermochemiluminescence was observed after illumination of liquid and solid pigment solutions at low temperature (down to - 170C). The excitation spectra of PCL coincide with the pigment absorption spectra. The PCL emission spectra in most cases differ from those of pigment fluorescence. Electron acceptors, electron donors, radical inhibitors and β-carotene quench PCL. The quenching efficiency of electron acceptors is similar to their action on the chlorophyll triplet state. The quenching effect of radical inhibitors and β-carotene correlates with their activity in reaction with singlet oxygen. The effect of quenchers on the chlorophyll fluorescence, photobleaching and pigment sensitized oxygenation was studied. Analysis of experimental data allowed the assumption that chemiluminescence accompanies the decomposition of labile pigment peroxides. The accumulation of peroxides is probably due to the reaction in the complex of pigment and singlet oxygen, formed as a result of energy transfer from photoexcited (triplet) pigment molecules to oxygen. The terminal chemiluminescence emission proceeds from the singlet excited states of molecules of pigments and products of their oxidation.     

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.     

Anomalous excimer formation of a pyrenyl derivative having a mesogen group of alkoxycyanobiphenyl in its smectic mesophase

The direct photolysis of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) in aqueous solution was investigated under monochromatic ultraviolet (UV) irradiation at 254 nm. ABTS was found to be directly photolyzed by UV irradiation to yield the one-electron oxidized radical, ABTS⁺, which is a blue-green colored persistent radical species that has strong visible absorption bands. The photochemical production of ABTS⁺ was strongly dependent on the solution pH and the presence of dissolved oxygen. The presence of dissolved oxygen increased the quantum yields at pH 3, whereas it inhibited the production of ABTS⁺ at pH 6.5. Spectrophotometric and spectrofluorometric data indicated that ABTS photolysis may occur as a result of the transfer of one-electron between the singlet excited state and the ground state of ABTS. Observations made during UV/H₂O₂ experiments with ABTS suggested that the dependence of the photoloysis of ABTS on the solution pH and the presence of dissolved oxygen is related to the role of the hydroperoxyl/superoxide radical (HO₂/O₂⁻), which appears to be formed via a secondary reaction of the reduced intermediate of ABTS with dissolved oxygen. The proposed photolytic reactions were supported by the observed stoichiometry between the amount of ABTS⁺ radicals produced and the amount of ABTS molecules decomposed.     

氧化锌分散体光反应产生的自由基中间体

溶液中光诱导的电子转移反应已进行了大量的研究。而半导体粉末在水相或非水溶剂中的光化学研究也与自俱增^[1-3]。这种光化学与成像体系、太阳能转换以及光催化或污物的光降解有关。因此,越来越引起人们的重视。     

Autocatalytic processes during oxidation of lignin in alkaline media

Results are presented that characterize features of the oxidation of lignin in alkaline media. The prerequisites are considered and proofs are given of the appearance of autocatalytic reactions on the oxidation of lignin at the level of one-electron transfer between the phenoxyl radicals being formed and the initial fragments and between chromophores in triplet electronically excited states and other fragments and of the development of concerted oxidation reactions through the appearance of active forms of oxygen, and vibrationally excited states. The results obtained indicate that on the oxidation of lignin in alkaline media a network of chemical reactions bearing the autocatalytic nature of chain processes with degenerate energy branching is formed.Siberian Scientific Research Institute of Cellulose and Board, Bratsk. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 426–433, May–June, 1988.     

Photosensitive decomposition of organic solvents at 77 degrees K by an aromatic amine, diphenylparaphenylenediamine

Abstract— –By e.s.r. we have studied the photoexcitation of an aromatic amine to its triplet state at 77°K, its photoionization to a radical cation and the simultaneous formation of solvent radicals proceeding from the photosensitization of the organic glassy matrix. In the case of methanol and ethanol matrix we observe approximately one solvent radical per solute radical cation. In the case of isopropanol and methyltetrahydrofuran we find respectively three and two solvent radicals per solute radical cation. The results suggest two possible processes of photosensitization. By successive absorption of two photons, the amine reaches an excited triplet state which is able either to dissociate giving one electron and one cation radical or to transfer its energy to the solvent, this last being decomposed. It is assumed that in the case of methanol and ethanol, the radicals from the solvent are only formed by reaction on the matrix by the released electron, whereas in the case of isopropanol and methyltetrahydrofuran, the second process is prevalent or exclusive.     

CHLOROPHYLL ONE-ELECTRON PHOTOCHEMISTRY-II. PHOTOSENSITIZATION BY CHLOROPHYLL,PHEOPHYTIN AND BACTERIOCHLOROPHYLL OF A ONE-ELECTRON TRANSFER REACTION BETWEEN ETHANOL AND QUINONE*

Abstract— Chlorophyll, pheophytin and bacteriochlorophyll sensitize a one-electron transfer in the presence of quinones in ethanol, to produce a ternary complex of ground state porphyrin analog, alcohol cation radical and semiquinone anion radical as the primary photo-product at low temperature. A similar photo-oxidation of alcohol to produce a binary complex is caused by direct excitation of quin-one in the absence of chlorophyll. The mechanisms of these reactions and their implications for photosynthesis are discussed.     

Direct photooxidation and xanthene-sensitized oxidation of naphthols: quantum yields and mechanism

The photoinduced oxidation of 1-naphthol to 1,4-naphthoquinone and of 5-hydroxy-1-naphthol to 5-hydroxy-1,4-naphthoquinone was studied by steady-state and time-resolved techniques. The direct photooxidation of naphthols in methanol or water takes place by reaction of the naphoxyl radical ((?)ONaph) with the superoxide ion radical (O(2)(?-)), the latter of which results from the reaction of the solvated electron with oxygen after photoionization. The sensitized oxidation takes place by energy transfer from the xanthene triplet state to oxygen. From the two oxygen atoms, which are consumed, one is incorporated into the naphthol molecule giving naphthoquinone and the second gives rise to water. The effects of eosin, erythrosin, and rose bengal in aqueous solution, pH, and the oxygen and naphthol concentrations were studied. The quantum yield of the photosensitized transformation was determined, which increases with the naphthol concentration and is largest at pH > 10. The quantum yield of oxygen uptake is similar. The pathway involving singlet molecular oxygen is suggested to operate for the three sensitizers. The alternative pathway via electron transfer from the naphthol to the xanthene triplet state and subsequent reaction of (?)ONaph with O(2)(?-), the latter of which is formed by scavenging of the xanthene radical anion by oxygen, does also contribute.     

PHOTOOXIDATION OF CHLOROPHYLL AND PHEOPHYTIN. QUENCHING OF SINGLET OXYGEN AND INFLUENCE OF THE MICELLAR STRUCTURE

Abstract— When chlorophyll(Chl) and pheophytin(Phn) are irradiated in Triton X-100 water binary solvents, singlet oxygen is formed in the medium in a higher yield for Phn than for Chi. Chlorophyll shows an irreversible photooxidation reaction and a chemical oxidation reaction when 1,3-diphenyliso-benzofuran (DPBF) is added to the solution. During the chemical oxidation, Chi is destroyed by an oxidizing agent that is a reaction product of the endoperoxide formed in the medium by the addition of singlet oxygen to DPBF. This reaction depends on the structure of the medium and has some characteristics of an oxidation by hydroxyl radicals. The highest yield is obtained with the micellar structure. Chlorophyll and Phn are readily oxidized by hydroxyl radicals generated using the Fenton reagent. This suggests that in the presence of Triton X-100, the Mg²⁺ ion of a Chi molecule plays a key role in the irreversible oxidation of the pigment.     

REVERSIBLE LIGHT-TNDUCED SINGLE ELECTRON TRANSFER REACTIONS BETWEEN CHLOROPHYLL AND HYDROQUINONE IN SOLUTION*

Abstract— EPR and optical studies demonstrate the occurrence of reversible temperature-independent light-induced single electron transfer reactions between chlorophyll (and pheophytin) and hydroquinone in degassed ethanol solutions. Oxygen is shown to quench this process, presumably by interacting with chlorophyll excited states. Pyridine and isoquino-line increase the effectiveness of the hydroquinone as an electron donor to chlorophyll, probably by acting as bases to form hydroquinone anions (or perhaps ion-pairs) which are more easily oxidized. Hydroquinone is found to be more effective in electron transfer than is benzoquinone, suggesting that the chlorophyll excited state is a better oxidizing agent than it is a reducing agent.     

Activation of 3-amino-1,2,4-benzotriazine 1,4-dioxide antitumor agents to oxidizing species following their one-electron reduction

The mechanism by which a benzotriazine 1,4-dioxide class of anticancer drugs produce oxidizing radicals following their one-electron reduction has been investigated using tirapazamine (3-amino-1,2,4-benzotriazine 1,4-dioxide, 1) and its 6-methoxy (6), 7-dimethylamino (7), and 8-methyl (8) analogues. By measuring the changes in absorption with pH, we found that the radical anions undergo protonation with radical pK(r) values of 6.19 +/- 0.05, 6.10 +/- 0.03, 6.45 +/- 0.04, and 6.60 +/- 0.04, respectively. The one-electron reduced species underwent a first-order reaction, with increased rate constants from 112 +/- 23 s(-)(1) for 1 to 777 +/- 12 s(-)(1)(6), 1120 +/- 29 s(-)(1) (7), and 825 +/- 89 s(-)(1) (8) at pH 7. No overall change in conductance was observed following the one-electron reduction of 6, and 8 at pH 4.5, consistent with the protonation of the radical anions, but a loss in conductance was seen for one-electron reduced 7 because of further protonation of the initially formed radical. This is assigned to the protonation of the dimethylamino group of the radical species, which has a pK(a) of 8.8 +/- 0.3. All conductance changes take place on a time-scale shorter than those of the above first-order reactions, which are not associated with the formation or loss of charged species. The absorption spectra present at the end of the unimolecular reactions were found to be similar to those formed immediately upon the one-electron oxidation of the respective substituted 3-amino-1,2,4-benzotriazine 1-oxides, and it is suggested that common benzotriazinyl radicals are formed by both routes. All these intermediate radicals underwent dismutation to produce final spectra matched by equal contributions of the parent compound and their respective substituted 3-amino-1,2,4-benzotriazine 1-oxides. By establishing redox equilibria between the intermediate radicals formed on the one-electron oxidation of the respective 3-amino-1,2,4-benzotriazine 1-oxides of the compounds and reference compounds, we found the one-electron reduction potential of the oxidizing radicals to range from 0.94 to 1.31 V. The benzotriazinyl radical of tirapazamine was found to oxidize dGMP and 2-deoxyribose with rate constants of (1.4 +/- 0.2) x 10(8) M(-)(1) s(-)(1) and (3.7 +/- 0.5) x 10(6) M(-)(1) s(-)(1), respectively.