REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

CHEMILUMINESCENCE IN THE PROCESS OF OXIDATIVE RING-OPENING OF PURPUROGALLINQUINONES

Abstract— The oxidation of purpurogalline (PPG) by alkaline solution of H₂O₂ pH 9–11 at 298°K is accompanied by chemiluminescence (CL) in the spectral range 400–600 nm with the maximum at 500 nm and quantum yield about 10^-6. The optimal concentrations of reactants with respect to maximal intensity are: 2 × 10^-4 M PPG, 10^-2 M NaOH, 1 M H₂O₂. Activation energy calculated from the maximum intensity of CL is 8.1×0.4 kcal/mole. Light emission occurs only when OH-groups of the phenolic ring of PPG undergo oxidation and the blue anion of o -PPG-quinone is formed. The rate that determines step in the reaction associated with luminescence is the nucleophilic attack of OOH^- ion on the blue anion of o -PPG-quinone. In this exergonic step (-ΔH = 63 to 230kcal/mole) the o - and/or p -quinone ring is opened and carbonyl derivatives of α-tropolone are produced. They display fluorescence in the region 400–600 nm. The fluorescence spectrum of the reaction mixture after oxidation of PPG is very close to that of CL. It is likely that carbonyl derivatives of α-tropolone are emitters of CL.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

AN ULTRASENSITIVE BIOASSAY FOR THE DETECTION OF FUROCOUMARINS AND OTHER PHOTOSENSITIZING MOLECULES

Abstract— A photobiological assay based upon inhibition of growth in the DNA repair-deficient bacterium E. coli B _s-1, is described for the analysis of a number of photosensitizing agents. The lower limits of detection were as follows: psoralen 5 × 10^-11g; 5-methoxypsoralen 1 × 10^-9 g; 8-methoxypsoralen 1 × 10^-9 g; 4,5',8-trimethylpsoralen 1 × 10-¹¹ g; angelicin 5 × 10^-9 g; 5,7-di-methoxycoumarin 1 × 10^-7 g; isoimperatorin 5 × 10^-9 g; dictamnine 1 × 10^-8 g; oxypeucedanin 5 × 10^-7 g; 5-nitroxanthotoxin 5 × 10^-7 g; and α-terthienyl 1 × 10^-6 g. All active compounds with the exception of α-terthienyl were more easily detected by several orders of magnitude by E. coli B _s-1 than with the normal wild type E. coli. 5—Geranoxypsoralen and isopimpinellin were not active. The application of this technique, after TLC, to the analysis of complex mixtures from lemon oil, oil of bergamot, Heracleum lanatum, Angelica dawsonii , and celery and parsnip is illustrated. The bioassay described is more rapid and sensitive than previously published methods, permits replica plates to be made, and allows tentative identification of the photosensitized molecular target.     

KINETICS OF BACTERIAL BIOLUMINESCENCE AND THE FLUORESCENT TRANSIENT

Abstract— The addition of FMNH₂ to Vibrio harveyi luciferase at 2°C in the presence of tetradecanal results in the formation of a highly fluorescent transient species with a spectral distribution indistinguishable from that of the bioluminescence. The bioluminescence reaches maximum intensity in 1.5 s and decays in a complex manner with exponential components of 10^-1s^-1, 7 × 10^-3s^-1, and 7 × 10 ⁴s^-1. The fluorescent transient rises exponentially at 7 × 10^-2s^-3 and decays at 3 × 10^-4s^-1. The slowest bioluminescence component, comprising the bulk of the bioluminescence, decays at twice the rate of the fluorescent transient under all variations of reaction conditions: concentration of reactants, temperature 2–20°C, and aldehyde chain length—decanal, dodecanal and tetradecanal. The activation energy for both the slowest bioluminescence decay and the transient fluorescence decay is 80 kJ-mol^-1. An energy transfer scheme is proposed to explain the results where two distinct chemically energized species utilize the fluorescent transient as emitter for the slower bioluminescences, and for the faster process a fluorophore present in the protein preparation. Kinetic observations suggest that typical preparations of V. harveyi luciferase comprise 15% active protein.     

THE AQUEOUS PHOTOLYSIS OF ETHYLENE GLYCOL ADSORBED ON GOETHITE

Abstract— Suspensions of goethite (α-FeOOH) were photolyzed in aerated ethylene glycol-water solutions at pH 6.5, with ultraviolet light in the wavelength range300–400 nm. Under these conditions, formaldehyde and glycolaldehyde were detected as photoproducts. Quantum yields of formaldehyde production ranged from 1.9 7times; 10^-5 to 2.9 × 10^-4 over the ethylene glycol concentration range of 0.002-2.0 mol/ℓ, and gave evidence that the reaction occurred at the goethite surface. Quantum yields of glycolaldehyde were 20% less than those of formaldehyde, and displayed a concentration-dependent relationship with ethylene glycol similar to that of formaldehyde. Immediately after photolysis, Fe²⁺ was measured to be 4.6 × 10^-7 mol/ℓ in an aerated suspension containing 1.3 mol/ℓ ethylene glycol, and 8.5 × 10^-6 mol/ℓ in the corresponding deoxygenated suspension. Glycolaldehyde was not generated in the deoxygenated suspensions. These results are consistent with a mechanism involving the transfer of an electron from an adsorbed ethylene glycol molecule to an excited state of Fe³⁺ (Iron[III]) in the goethite lattice, to produce Fe²⁺ and an organic cation. In a series of reactions involving O₂, FeOOH, and Fe²⁺, the organic cation decomposes to form formaldehyde and the intermediate radicals "OH and" CH₂OH. OH reacts further with ethylene glycol in the presence of O₂ to yield glycolaldehyde. Aqueous photolysis of ethylene glycol sorbed onto goethite is typical of reactions that can occur in the aquatic environment.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

PHOTOFRAGMENTATION OF α-OXOCARBOXYLIC ACIDS IN AQUEOUS SOLUTION. AN EPR STUDY. FORMATION OF SEMIDIONE RADICALS BY DECARBOXYLATIVE SUBSTITUTION OF α-OXOCARBOXYLIC ACIDS BY ACYL RADICALS-I: GLYOXYLIC, PYRUVIC, α-OXOBUTYRIC, α-OXOGLUTARIC AND α-OXOISOCAPROlC ACIDS

Abstract— By means of in situ photolysis EPR of aqueous solutions of α-oxocarboxylic acids (RCO-CO₂H) at pH values above 5, semidione radical anions [RC(O^-)=C(O')R] and α-hydroxy-α-carboxy alkyl radicals [RC(OH)CO₂-] were detected. C0₂ was identified as a reaction product. On photolysis of mixtures of α-oxocarboxylic acids (RCOCO₂H and R'COCC₂H), "mixed" semidione radical anions [RC(O^->=C(O)R'] were observed in addition to RC(O^-)=C(O')R, R'C(O^-)=C(O')R', RC(OH)CO₂^- and R'C(OH)CO₂^-. The experimental results are explained in terms of photodecarboxylation (α-clea-vage) of electronically excited RCOCOJ to yield RCO and CO₂. The radicals RC(OH)CO₂^- are formed by reduction of RCOCO₂^- by CO₂^-. The semidione radicals are produced by addition of RCO to RCOCO₂^- followed by decarboxylation of the intermediate adduct. This mechanism was confirmed by generating acyl radicals independently and reacting them with α-oxocarboxylic acids. Selected product studies support the mechanism suggested.     

ON THE REACTION OF SINGLET MOLECULAR OXYGEN WITH N-ALLYLTHIOUREA IN AQUEOUS SOLUTION

Abstract— Experiments on the photooxidation of N -allylthiourea, thiourea, and N-allylurea sensitized by the dye phenosafranine show that in N -allylthiourea the thiourea group is the site of singlet oxygen attack, while the allyl moiety neither reacts with nor quenches this metastable form of O₂ (in neutral aqueous solutions). Low concentrations of N^-₃ (a known quencher of singlet oxygen) strongly reduce the photooxidation of allylthiourea by a mechanism which apparently obeys simple competition kinetics. From these results the rate constant of the reaction between allylthiourea and singlet oxygen is obtained ( k = 4 × 10⁶ M ^-1 s^-1; pH = 7.1).     

DETECTION OF THE EXCITED SINGLET STATE OF A DEPROTONATED, REDUCED FLAVIN

Abstract— The excited singlet state of a deprotonated, reduced flavin [1, 5-dihydro- N (3)-carboxymethyllumiflavin] in aqueous solution at pH 8 has been detected by laser flash photolysis. The broad absorption band maximized at ∼ 490 nm (ε= 9.9 × 10³ M ^-1 cm^-1). The lifetime of the transient was found to be 100 ± 15 ps. The lifetime was not affected by the presence of pyrimidine dimers, which would be monomerized under these conditions. A longer-lived transient, tentatively identified as the solvated electron, was also detected. The neutral reduced flavin did not give a detectable transient.     

MECHANISMS OF THE PHOTOSENSITIZED OXIDATION OF TYRAMINE

Abstract— …According to the criteria of enhancement in D₂O and inhibition by sodium azide, the oxidation of tyramine photosensitized by methylene blue is largely a singlet oxygen or Type II process. Its quantum yield approximates 0.3 in D₂O at pH 10. There is a less efficient reaction not quenched by azide, which is assigned to a dye-substrate or Type I process. It gives rise to products with distinct bands at 320 and 285nm. Products of the Type I reaction are further oxidized by singlet oxygen and thereby compete with tyramine for this reagent. Kinetic parameters were estimated by computer simulation of the dependence of quantum yield on extent of reaction. The rate constant for reaction of O₂ (¹Δ_g) with tyramine was estimated to be 2.8 × 10⁸ M ^-1 s ^-1± 20% at pH 10. The reaction was also sensitized by hypericin in what appears to be a Type II process.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

FLUORESCENCE OF 5-METHYLCYTOSINE

Abstract— 5-Methylcytosine and 5-methyldeoxycytidylic acid are fluorescent in aqueous solution at room temperature and neutral pH. 5-Methylcytosine, 10^-3M, pH 8.5, 25°C, has a quantum yield of 5 ×10^-4, 5-Methyldeoxycitydylic acid, 10^-4M, pH 7.5, 20°C, has a quantum yield of 8 × 10^-4. Emission maxima are 2.91 and 2.80μ^-1. At pH 14, the quantum yield of 5-methylcytosine is 1.6 × 10^-2; the emission maximum is 2.82μ^-1. At pH I, the quantum yield of both compounds is less than or equal to 10^-4. Both compounds were chromatographically homogeneous, had absorption spectra which agreed with published data, and excitation spectra which agreed closely with absorption spectra.     

TRANSIENT PHOTOCHEMISTRY OF NEUTRAL RED

Abstract— Flash photolysis of neutral red between pH 1.3 and pH 11 yields the triplet species ³DH₂⁺²³DH⁺ and ³D. Both ³DH₂⁺² and ³D exhibit first order decay with rate constants of 1.6 ± 0.3 × 10⁴ s^-1 but ³DH⁺ decays within the lifetime of the flash. Over the entire pH range, ascorbic acid quenches the triplet, forming the semireduced radicals DH₃⁺² DH₂⁺ and DH, all of which exhibit second order decay with k = 1.8 ± 0.4 ± 10⁸ M ^-1s^-1 most probably by recombination with semioxidized ascorbic acid. The dependence of the rate of decay of radical neutral red on the identity of reversible reductants supports the back-electron transfer mechanism, as does digital simulation of complex radical disproportionation schemes. In contrast to the efficient reduction of triplet neutral red by ascorbic acid, its reduction by EDTA is quite inefficient.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

BIOLOGICAL CHEMILUMINESCENCE

Abstract
The nucleobase 5-methylcytosine ( I ) is a minor component of eukaryotic DNA thought to be important in regulation of gene expression. The photochemical reactions of this nucleobase and its 2'-deoxyribonucleoside, 5-methyl-2'-deoxycytidine ( II ), in water have been studied. These reactions lead, respectively, to 3-amino-2-methylacrylamidine ( Ib ) and 3-(2- erythro - d -pentopyranos-1-yl) amino-2-methylacrylamidine ( IIb ) as the main photoproducts. The structure of the photoproducts was established by spectroscopic methods (¹H and ¹³C NMR, UV spectroscopy, electron impact and liquid secondary ion mass spectrometry); in the case of Ib , confirmatory evidence was obtained by chemical methods (photolysis of 5-methyl[2–¹³C]cytosine, hydrolysis of N -carbomethoxy-3-amino-2-methylacryl-amidine and reaction of Ib with 1,1'-carbonyldiimidazole to give I ). The quantum yield for formation of Ib was determined to be 1.8 × 10^-3at pH 7.5 while the quantum yield for formation of IIb has a lower value of 0.2 × 10^-3 at pH 7.5. These quantum yields depend strongly on pH and reach maximum values of 2.0 × 10^-3 at pH 7.0 ( Ib ) and 0.6 × 10^-3 at pH 5.0 ( IIb ). The mechanism of formation of Ib (or IIb ) is proposed to involve nucleophilic attack of water on the C-2 position of photoexcited I (or II ), followed by ring opening and decarboxylation of an intermediate carbamic acid.     

THE PHOTOCHEMICAL INTERACTION BETWEEN THE TRIPLET STATE OF 8-METHOXYPSORALEN AND THE MELANIN PRECURSORL–3, 4 DIHYDROXYPHENYLALANINE

Abstract— The photochemical interaction between 8-methoxypsoralen (8-MOP) and the melanin precursorL–3,4-dihydroxyphenylalanine(dopaH₂) has been studied using laser flash photolysis. Triplet excited 8-MOP was thus found to abstract electrons from dopaH₂ ( k ∼ 2 × 10⁹ dm³ mol^-1 s^-1) to form semireduced 8-MOP and semioxidised dopaH₂.The technique of pulse radiolysis was used to establish separately the spectra of (a) the semi-reduced form of 8-MOP at pH 6.5 and (b) the semioxidised forms of dopaH₂ at pH 6.5, 5.8, 4.6 and 3.3. The corresponding λ_max and extinction coefficients found were: for 8-MOP at pH 6.5, λ_max= 350 nm (= 9050 dm³ mol^-1 cm^-1); for dopa at pH 6.5, λ_max= 305 nm (ε= 12000 dm³ mol^-1 cm^-1) and for dopaH at pH 3.3, λ= 305 nm (ε= 5900 dm³ mol^-1 cm^-1).     

ON THE PRODUCTION OF NITROXIDE RADICALS BY SINGLET OXYGEN REACTION: AN EPR STUDY

Abstract— The production of free radicals by reaction of 2,2,6,6-tetramethyl-4-piperidinol with singlet oxygen was studied by EPR spectroscopy. The rate constant of the amine was found to be equal to 8 ×10⁵ M ^-1s^-1 in ethanol and to 4 × 10⁷M^-1s^-1 in phosphate buffer (pH 8). Competition experiments were performed with singlet oxygen quenchers such as NaN₃, DABCO and the quenching rate constants were found to be consistent with the literature values. The EPR method proved to be a valuable technique to study the reaction of singlet O₂ with the sterically hindered amine without any interfering effect.     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.