UVA self-photosensitized oxygenation of beta-ionone

The steady-state UVA (350 nm) photolysis of ( E )-β-ionone ( 1 ) in aerated toluene solutions was studied by ¹H NMR spectroscopy. The formation of the 1,2,4-trioxane ( 2 ) and 5,8-endoperoxide ( 5 ) derivatives in the ratio of 4:1 was observed. Time-resolved laser induced experiments at 355 nm, such as laser-flash photolysis, photoacoustic and singlet oxygen ¹O₂ phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield Φ _T = 0.50 as the precursor for the generation of singlet oxygen ¹O₂ ( Φ _Δ = 0.16) and the isomeric α-pyran derivative ( 3 ), which was a reaction intermediate detected by NMR. In turn, the reaction of ¹O₂ with 1 and 3 occurred with rate constants of 1.0 × 10⁶ and 2.5 × 10⁸  m ⁻¹s⁻¹ to yield the oxygenated products 5 and 2 , respectively, indicating the relevance of the fixed s-cis configuration in the α-pyran ring in the concerted [2+4] cycloaddition of ¹O₂.     

STEROL METABOLISM. XLII. ON THE INTERCEPTION OF SINGLET MOLECULAR OXYGEN BY STEROLS

Abstract— The use of cyclic 1,3-dienes and polycyclic aromatic hydrocarbons as xenobiotic substrates for the interception of electronically excited (singlet) molecular oxygen (¹O₂) in biological systems is reviewed and criticized, and the possibility of utilization of reactive endogenous substrates for ¹O₂ interception is considered. The common sterols, cholesterol, 5α-cholest-7-en-3β-ol, and 5α-lanost-8-en-3β-ol each give oxidation products with ¹O₂ different from those with ground-state molecular oxygen that can be distinguished from one another by simple chromatographic means.     

MESODIPHENYLHELIANTHRENE—THE MOST REACTIVE SINGLET OXYGEN ACCEPTOR

Abstract— The self-sensitized photooxidation of mesodiphenylhelianthrene in various solvents has been investigated. The involvement of ¹O₂ as the reactive intermediate in the formation of the endoperoxide has been demonstrated by the quenching of the photooxidation by the efficient ¹O₂-quencher β-carotene. The rate constant for the addition of ¹O₂ to mesodiphenylhelianthrene has been determined to be k _R≅ 10¹⁰ M ^-1 s^-1, which is the highest value hitherto known in the literature. The probability factor p , which describes the concentration independent part of the overall quantum yield, has been measured to be p =0.17.     

FURTHER IN VIVO STUDIES ON THE PARTICIPATION OF SINGLET OXYGEN IN THE PHOTODYNAMIC INACTIVATION AND INDUCTION OF GENETIC CHANGES IN SACCHAROMYCES CEREVISIAE

Abstract —In vivo participation of singlet excited oxygen (¹O₂, ¹Δ9) in the photodynamic inactivation and induction of genetic changes (gene conversion) in acridine orange-sensitized yeast cells was investigated by using N₃^-, an efficient ¹O₂ quencher, and D²O, a known agent for the enhancement of the lifetime of ¹O₂. The addition of N₃^- protected the cells from both photodynamic actions. From an analysis of the concentration-dependent protection, about 80% of the induction of the genetic change is explainable on the basis of ¹O₂ mechanism. The quantitative estimation of the N₃^- protection in the inactivation was not possible because of the sigmoidal nature of the inactivation curve. The replacement of H₂O with D₂O during illumination was effective in enhancing the photodynamic inactivation but almost completely ineffective for the gene conversion induction. The deuterium effect with the cell system was clearly not as large as would be expected from in vitro experiments. This, however, could be explained from the kinetic consideration that natural quenchers of ^lO₂ in the cell would mask the deuterium effect. By experiments with different cell stages it was demonstrated that these two modifying effects were dependent on the intracellular reaction environment. The conclusion is that ¹O₂ must be the major intermediate responsible for the photodynamic actions in acridine orangesensitized yeast cells.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

Near-infrared Light Emission from Nanomolar-level Singlet Molecular Oxygen Generated with an Ultra low Concentration Mixture of Hypochlorite and Hydrogen Peroxide

Abstract— We report the detection of a weak near-infrared light emission originating from 8 nM singlet molecular oxygen (¹O₂) produced in a mixture of 1 m M hypochlorite (OC1^-) and 8 n M hydrogen peroxide (H₂O₂). The measurements were made with a highly sensitive detection system for ultraweak light emission in the 1.0-1.5 μm wavelength region. The emission intensity exhibited linear dependence for H₂O₂ concentrations in the range of 8-670 n M . The mixture containing a lower concentration (33 μ M ) of OCl^- pseudocontinuously emitted near-infrared light for 5 s. The rate constant for ¹O₂ production obtained from the kinetic analysis agrees with that previously reported. Our results demonstrate the possibility of measuring very low concentrations of ¹O₂ in a OCi-/H₂O² mixture as well as ¹O₂ production in intact living systems.     

RATE CONSTANTS FOR INTERACTION OF 1O21Δg) WITH AZIDE ION IN WATER

Abstract— The rate constant for quenching of ¹O₂ by azide ion in water was determined to be (5.0 ± 0.4) × 10⁸ M ⁻¹ s⁻¹ using a variety of sensitizers (including humic acids) and ¹O₂ acceptors. The apparent second-order rate constant decreases with pH below pH 5.5 in accordance with the protonation of azide ion to form hydrazoic acid (p K _a= 4.6). Quenching by hydrazoic acid is at least 2 orders of magnitude slower than by azide ion. Greater than 99% of all interactions between ¹O₂ and azide ion involve physical quenching rather than chemical reaction. Humic acid triplets are not significantly quenched by azide ion at concentrations less than 2 m M , allowing azide ion quenching to be used as a diagnostic test for the intermediacy of ¹O₂ in photosensitized oxidations in natural surface waters.     

EFFECT OF SODIUM AZIDE ON PHOTODYNAMIC INDUCTION OF GENETIC CHANGES IN YEAST

Abstract— In view of the increasing attention to ¹O₂ (¹Δ_g) participation in the photodynamic action, different types of genetic changes in Saccharomyces cerevisiae by acridine orange sensitization were compared with respect to the response to N₃^-, a well known quencher of ¹O₂. The induction of mitotic crossing over with respect to ade 2 locus and mitotic gene conversion at trp 5 locus were suppressed by the addition of N₃^- suggesting the involvement of ¹O₂ as a major intermediate. However, the induction of reverse mutation at ilv 1 was only slightly suppressed. These results may indicate that there are two types of photodynamic DNA damage; one is produced via ¹O₂ and the other via non-¹O₂ reaction pathway which lead to mitotic gene conversion and mitotic crossing over, and to mutation, respectively.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

STEADY-STATE NEAR-INFRARED DETECTION OF SINGLET MOLECULAR OXYGEN: A STERN-VOLMER QUENCHING EXPERIMENT WITH SODIUM AZIDE

Abstract— A sensitive near-infrared detection system incorporating improvements to existing methodologies has been used to characterize the sodium azide quenching of the steady-state luminescence of singlet molecular oxygen at 1270 nm. Stern-Volmer plots which were linear up to 80% quenching of the ¹O₂ generated by rose bengal and eosin Y yielded a rate constant of 5.8 ± 0.1 times 10⁸ M ⁻¹ s⁻¹ for the quenching of ¹O₂ in water, while the rate constants obtained in deuterium oxide with the same sensitizers were 6.28 times 10⁸ M ⁻¹ s⁻¹ and 6.91 times 10⁸ M ⁻¹ s⁻¹ respectively. A flow system minimized the effects of photobleaching of the rose bengal. With a mercury arc light source, the instrument can be used in photosensitization experiments to detect low levels of ¹O₂ production in aqueous media.     

Photochemical Reactions Involved in the Phototoxicity of the Anticonvulsant and Antidepressant Drug Lamotrigine (Lamictal®)

Lamotrigine (LTG) [3,5-diamino-6-(2,3-dichlorophenyl)-1,2,4-triazine], an anticonvulsant and antidepressant drug Lamictal^®, produces a (photo)toxic response in some patients. LTG absorbs UV light, generating singlet oxygen (¹O₂) with a quantum yield of 0.22 in CH₂Cl₂, 0.11 in MeCN and 0.01 in D₂O. A small production of superoxide radical anion was also detected in acetonitrile. Thus, LTG is a moderate photosensitizer producing phototoxicity and oxidizing linoleic acid. LTG is a weak ¹O₂ quencher ( k _q = 3.2 × 10⁵  m ⁻¹ s⁻¹ in MeCN), but its photodecomposition products in dimethyl sulfoxide (DMSO) quenched ¹O₂ very efficiently. Upon intense UV irradiation from a xenon lamp, LTG was photobleached rapidly in DMSO and slowly in acetonitrile, alcohol and water. The rate increased significantly when laser pulses at 266 nm were employed. The photobleaching products generated ¹O₂ twice as strongly as LTG. Photobleaching was usually accompanied by the release of chloride anions, which increased in the presence of ascorbic acid. This suggests the formation of aryl radicals via dechlorination, a process which may be responsible for the photoallergic response observed in some patients. Our results demonstrate that LTG is a moderate generator of ¹O₂ prone to photodechlorination, especially in a reducing environment, which can contribute to the reported phototoxicity of LTG.     

DETERMINATION OF SINGLET OXYGEN RATE CONSTANTS IN AQUEOUS SOLUTIONS

Abstract— A very efficient quenching of singlet oxygen (¹O₂) by N₃^- ions has been applied to the determination of rate constants of reactions of ¹O₂ with various substrates (A). This determination has been made possible by choosing experimental conditions which give simple competition between N₃^- and A for ¹O₂ formed in the steady state irradiation of convenient sensitizing dye (S). The consumption of oxygen by the substrate, as followed with an oxygen analyzer, decreases in the presence of low concentrations of N₃^-. Using neutral air saturated aqueous solutions containing the dye phenosafranine + A and varying concentrations of N₃^-, the ¹O₂ rate constants for reactions with biological substrates and some radiation protective agents have been determined.     

KINETIC STUDIES OF SELF-SENSITIZED PHOTOOXYGENATION IN H2O AND D2O OF A WATER-SOLUBLE RUBRENE DERIVATIVE

Abstract A continuous argon laser has been used to study the self-sensitized photooxidation of potassium rubrene-2,3,8,9-tetracarboxylate in oxygen-saturated H₂O and D₂O. An analysis of the data obtained in concentrated solutions leads to an unexpected high value of the ratio of ¹O₂ lifetimes in D₂O and H₂O, ^T d ₂o/^T h ₂o =17 ± 1. Results obtained in diluted aqueous solutions are interpreted in terms of a re-encounter of ¹O₂ and ground state substrate molecules generated in the same triplet—triplet annihilation act.     

HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (¹O₂) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D₂O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that ¹O₂ was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D₂O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Ų for the inactivation of subtilisin BPN' by ¹O₂ and 20 Ų for the oxidation of tryptophan in BSA.     

PHOTOOXIDATION OF A REFINED PETROLEUM OIL: INHIBITION BY β-CAROTENE AND ROLE OF SINGLET OXYGEN

Abstract— β-Carotene, at concentrations 0.1 m M , inhibits the formation of hydroperoxides and other oxidation products in a refined petroleum oil exposed to Pyrex-filtered UV. The effect appears to be due to ¹O₂ quenching. A mechanism incorporating ¹O₂ and radical processes is proposed as a model for environmental photooxidation of petroleum.     

PHOTO-OXIDATION EFFECTS ON β-HYDROXYBUTYRATE DEHYDROGENASE: STUDIES OF MEMBRANE FRAGMENTS AND INTACT MITOCHONDRIA

The influence of singlet oxygen (¹O₂), generated by red light irradiation of oxygenated suspensions containing aluminium phthalocyanine sulphonate, on the membrane bound enzyme β-hydroxybutyrate dehydrogenase was investigated. The inactivation rates were measured using a spectrophotometry assay which involves disruption of the mitochondria. A novel NMR assay was also used to measure the activity of the enzyme in intact mitochondria. Relatively high inactivation rates of around 10⁹ M ⁻¹ s⁻¹ were observed in H₂O buffer, and rates in D₂O were a factor of 1.7 faster. Significant differences in enzyme inactivation rates by ¹O₂ were observed, not only between disrupted and intact mitochondria but also between the NMR assay results and the spectrophotometric assay results. The results indicate the value of a specific assay which does not require the disruption of the biological system.     

RELATION BETWEEN SOME PHOTOBIOLOGICAL PROPERTIES OF FUROCOUMARINS AND THEIR EXTENT OF SINGLET OXYGEN PRODUCTION

Abstract— The production of singlet oxygen (¹O₂) by a series of furocoumarins with different skin sensitizing abilities has been investigated with methods already proven to be suitable to establish the ability of 8-methoxypsoralen (8-MOP) to generate ¹O₂.
The following compounds: 5-methoxypsoralen (5-MOP), psoralen, 4,5',8-trimethylpsoralen (TMP) and 5,8–dimethoxypsoralen (5,8–DMOP), are able to generate ¹O₂ when irradiated with long–wave ultraviolet light. With the photobiologically inactive angelicin no ¹O₂ production has been found. The relative extent of ¹O₂ formation has been determined for the various furocoumarins and has been compared with literature data for the skin photosensitizing effect. The observed relation between experimental data on the one side and the literature data on the other side is discussed.     

PRODUCTS AND RELATIVE REACTION RATES OF THE OXIDATION OF TOCOPHEROLS WITH SINGLET MOLECULAR OXYGEN

Abstract— The relative reactivity of singlet molecular oxygen, 0₂(¹Δ_g), α-,β-,Γ-and δ with -tocopherol (vitamin E) was investigated using microwave discharge generation as a uniquely clean source of singlet oxygen and using a hydrocarbon solvent to approximate the membrane environment. The relative efficiencies of the tocopherols for O₂(¹Δg) were found to decrease in the order: D-α-tocopherol > D-β-tocopheroI > D–Γ-tocopherol > D-δ-tocopherol. The reaction products in all cases were found to be mixtures of quinone and quinone epoxides apparently resulting from decomposition of the primary product, the hydroperoxydienone.     

CHEMILUMINESCENCE FROM AUTOXIDIZING 6-HYDROXYDOPAMINE: THE INVOLVEMENT OF ACTIVATED FORMS OF OXYGEN

Abstract— The autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (20 μ M ) gave rise to a chemiluminescence which was greatly stimulated by FeSO₄ (20 μ M ) or by hydrogen peroxide addition (20 μ M to 2 m M ). The luminescence of both 6-hydroxydopamine alone or 6-hydroxydopamine plus hydrogen peroxide was strongly inhibited by catalase and by superoxide dismutase (both at 10 μg/m/); bovine serum albumin at 10 μg/m/ had no inhibitory effect. The luminescence was also strongly inhibited by several potent hydroxyl radical trapping agents and also by low concentrations of the ¹O₂ quencher DABCO (l,4-diazabicyclo-2.2.2.-octane). Chemiluminescence was greatly enhanced in D₂O, a solvent in which ¹O₂ has a prolonged lifetime. These data demonstrate the involvement of hydrogen peroxide, the superoxide radical and the hydroxyl radical in the chemiluminescence. The data are also consistent with some role for ¹O₂.     

A NEW METHOD FOR THE DETECTION OF SINGLET OXYGEN IN AQUEOUS SOLUTIONS

Abstract— In experiments on the interception of reactive intermediates of strongly oxidizing character in dye (S) sensitized photooxidations using p -nitrosodimethylaniline (RNO) as a selective scavenger, it has been observed that some substrates (A) or ¹O₂ acceptors (like imidazole derivatives) induce the bleaching of RNO as followed spectrophotometrically at 440 nm. Since singlet oxygen (¹O₂) does not react chemically with RNO, this bleaching is a consequence of ¹O₂ capture by the imidazole ring which results in the formation of a trans-annular peroxide intermediate [¹O₂] capable of inducing the bleaching of RNO (-RNO). In the absence of RNO, [¹O₂] decomposes or rearranges into the final oxygenation product ¹O₂: ¹Δ_g Thus, the system imidazole plus RNO can be used as a sensitive and selective test for the presence of ¹O₂ in aqueous solutions. The method can also be applied in the presence of sensitizing dyes which, under visible irradiation, can partially bleach RNO even in the absence of imidazole derivatives. In such a case, the bleaching of RNO is strongly increased by the presence of imidazoles with a characteristic dependence on their concentration. The separation of the product of RNO bleaching by thin layer chromatography can serve as additional proof of the presence of ¹O₂ in the system. The imidazole plus RNO method has been applied to a number of sensitizing and non-sensitizing dyes.