A PRELIMINARY INVESTIGATION OF THE USE OF LIMONENE FOR DETECTING SINGLET OXYGEN AS A COMPONENT OF POLLUTED AIR

Abstract— Exploratory experiments using simulated conditions indicate that the terpene, (+)-limonene can serve to detect O₂(¹Δ_g) in polluted atmospheres. ¹O₂ attack on limonene results in the formation of specific oxidation products in a specific distribution; quantitative identification of these products is a highly specific test for ¹O₂.     

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.     

CHEMILUMINESCENCE IN THE PEROXIDATION OF TANNIC ACID

Abstract— Peroxidation of tannins with alkaline H₂O₂ is accompanied by weak chemiluminescence in the spectral region 480–800 nm. o-Di and tri-hydroxy groups of polyphenols undergo oxidation by a free-radical mechanism and a green intermediate anion-radical with absorption Δ_max= 600 nm is formed. The radical mechanism is supported by the low activation energy 14–20 kJ/mol and the quenching effect of radical scavengers. The reaction of the green intermediate with peroxy anions is the chemiluminescence rate limiting step. In the presence of a-hydroxy-methylperoxide formed from H₂O₂ and formaldehyde, the alkaline peroxidation of tannins is accompanied by strong red luminescence (420–800 nm). The base catalyzed decomposition of peroxides gives only a weak red emission (460–800 nm). Light intensity is enhanced in D₂O by a factor 6.5. Quenchers of O₂(¹Δ_g) and 1,3-di-phenylisobenzofurane diminish light intensity in non-aqueous solutions. The data suggest ¹O₂ participation in the observed chemiluminescence. Thermo-chemical calculations give —ΔH values from 250–1000 kJ/mol for one elementary reaction step which limits the mechanism of chemi-enereization. Chemiexcitation of tannins is relevant to biochemical mechanisms of aerobic degradation of aromatic compounds, energy utilization as well as to defense and resistance processes in plants.     

A MORE EXACT THEORETICAL INTERPRETATION OF RECENT EXPERIMENTAL DATA OBTAINED USING THE SEPARATED SENSITIZER AND SUBSTRATE METHOD TO INVESTIGATE O2(lδg) INTERACTIONS. POSSIBLE IMPORTANCE OF O2(1σg+)

Abstract— Recent experimental data obtained using the separated sensitizer and substrate method to investigate the interaction of O₂(¹δ_g) with various substances has been re-interpreted by means of a more complete theory. Comparison of experimental and recalculated values of the dependence of relative reaction rates on the sensitizer-substrate separation indicate general accord for experiments in which singlet oxygen acceptors in aqueous solution were used. The presumption is therefore that singlet molecular oxygen O₂(¹δ_g) is indeed the active oxidizing agent and that the theory presented and experiment are entirely in agreement.
For experiments in which bacterial targets were used a very distinct disagreement between theory and experiment is evident, the conclusion being that the kill rate does not depend linearly on the O₂(¹δ_g) concentration in the immediate proximity of the bacteria. However, the data is consistent with a quadratic dependence on the ¹δ_g concentration. A possible conclusion therefore is that the cytotoxic species is actually O₂(¹σ⁺_g), formed by an energy pooling reaction involving two O₂(¹δ_g) molecules.     

THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS

Abstract— The physical quenching of singlet molecular oxygen (¹Δ_g) by amino acids and proteins in D₂O solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1–06 μm output of a Nd-YAG laser, which absorbs directly in the electronic transition ¹Δ_g+ 1 v →³Σ_g^-. The rate of quenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-blue-photosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in D₂O, pD 8, are (10⁷ℒ mol^-1 s^-1): alanine 0–2, methionine 3, tryptophan 9, histidine 17, carbonic anhydrase 85, lysozyme 150, superoxide dismutase 260, aposuperoxide dismutase 250.     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

PHOTOKINETIC AND PHOTOPHYSICAL MEASUREMENTS OF THE SENSITIZED PHOTOOXIDATION OF THE TRYPTOPHYL RESIDUE IN N-ACETYL TRYPTOPHANAMIDE AND IN HUMAN SERUM ALBUMIN

Abstract— The photosensitized oxidation of 10–100 μ M N -acetyl-L-tryptophanamide (NATA) in neutral aqueous solution and in the presence of various dyes proceeds by a pure O₂(¹Δ_g)-involving mechanism. Incorporation of the tryptophyl (Trp) residue into the polypeptide chain of human serum albumin (HSA) has no influence on the mechanism and efficiency of Trp photooxidation when sensitized either by methylene blue, a non-binding dye, or by rose bengal, a dye that gives non-covalent 1: 1 complexes with HSA. This is due to the location of the Trp residue in close proximity of the protein surface and, in the case of rose bengal, to the coincidence of the photophysical properties (including the quantum yield of O₂(¹Δ_g) generation) for the free and HSA-bound dye. Hematoporphyrin also binds to HSA with 1: 1 stoichiometry, although at a different site from rose bengal. Bound Hp again displays photophysical properties very similar with those of free Hp; however, the efficiency of Trp photo-oxidation in HSA is about 5-fold higher than in NATA owing to a limited rearrangement of the protein structure, induced by Hp binding, which enhances the probability of chemical quenching of O₂(¹Δ_g) by the indole ring.     

EFFECT OF AGGREGATION ON THE HEMATOPORPHYRIN-SENSITIZED PRODUCTION OF SINGLET MOLECULAR OXYGEN

Abstract— The hematoporphyrin-sensitized production of singlet molecular oxygen, O₂(¹Δ_g), has been investigated in methanol and in aqueous solution. The quantum yield for formation of O₂(¹Δ_g) (Φ_Δ) has been measured by both steady-state (oxygen consumption) and time-resolved (near-infrared luminescence) methods. In methanol, both techniques indicate that Φ_Δ= 0.76 and the value remains independent of sensitizer concentration over a wide range. This finding is consistent with the dye persisting in a monomelic form in methanol solution. In contrast, Φ_Δ decreases markedly with increasing sensitizer concentration in water due to dimerization of the dye. Analysis of the steady-state data indicates Φ_Δ values of 0.74 and 0.12, respectively, for monomer and dimer. It is further shown that the efficiency whereby quenching of the triplet state by O₂ results in generation of O₂(¹Δ_g) is substantially lower for the dimer than for the corresponding monomer. Because monomer and dimer possess quite different absorption spectral profiles, the efficacy for photodynamic action with hematoporphyrin exhibits a pronounced wavelength dependence.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

ACTIVATED OXYGEN: SINGLET MOLECULAR OXYGEN AND SUPEROXIDE ANION

Abstract— Elusive processes associated with molecular oxygen in chemical and biological systems are interpreted in terms of two activated oxygen species, singlet molecular oxygen (¹Σ⁺_g/¹Δ_g) and superoxide anion (X²π_g). The generation and deactivation of singlet oxygen by interaction with organic triplet states are discussed within a comprehensive theoretical framework. Experimental results indicate the anomalous molecular oxygen enhanced luminescence from organic chromophores in polymer matrices results from the deactivation of singlet (¹Δ_g) oxygen by energy transfer to electronically excited states of the chromophore, and three types of oxygen enhanced luminescence have been identified in these systems. Properties of the superoxide anion relevant to its solution chemistry are briefly discussed. Electron transfer theory is used to theoretically examine the generation of singlet oxygen in disproportionation reactions of the superoxide anion, predicting that, depending on the number of water molecules present, the disproportionation reaction is a proficient source of singlet oxygen. A competing quenching process imposes a limit to the steady state concentration of singlet oxygen in most chemical systems. Available experimental results on the quenching of singlet oxygen by superoxide anion are in good agreement with theoretical results obtained via application of electron transfer theory.     

UPCONVERSION OF THE SINGLET OXYGEN 1269 nm EMISSION TO THE VISIBLE

Abstract— Experiments are described that enable the kinetic behavior of singlet oxygen, O₂(^IΔ_g), to be monitored in the time-resolved mode using a photomultiplier to detect deep orange light (γ_max 660 nm). This orange light is a consequence of the upconversion of the natural emission of O₂(^IΔ_g) at 1269 nm.     

FORMATION AND CHEMILUMINESCENT DECOMPOSITION OF DIOXETANES IN THE GAS PHASE

Abstract— High resolution chemiluminescence spectra have been obtained of the singlet electronically excited products of O₂(¹Δ) plus alkene, dioxetane forming, reactions. The experiments were conducted in a flow apparatus at pressures of 1–5 torr. The spectra are a measure of the unrelaxed initial distribution of energy in the excited product. Results are reported for ethylene, 1, 1-difluoroethylene. methyl vinyl ether, ethyl vinyl ether, n -butyl vinyl ether, ketene, ketene-d₂, allene, unsymdimethyl allene, dimethyl ketene, 2-methoxy propene, 1-ethoxy propene, 2-bromo propene, and N, N- dimethyl isobutenyl amine. Chemiluminescence activation energies, representing the cycloaddition process, and absolute quantum yields for singlet excited product, ranging from 10^--⁴ to 2.5 × 10^--². are reported for 10 alkenes. Several of the reactions, 1,1-difluoroethylene, ketene, ethylene and allene give formaldehyde ¹ nπ* product with excess vibrational-rotational energy and a higher quantum yield than reactions not displaying this phenomenon. This is an indication of at least partially statistical partitioning of the energy in excess of that needed to electronically excite the formaldehyde. The experiments with ketene and ketene-d₂ provide the first evidence for the existence of unsubstituted 1,2-dioxetanone. The results from several of the experiments, particularly those with 2-methoxy propene and I-ethoxy propene are consistent with the mechanism of Goddard, which predicts regioselective and stereoselective attack of O₂(¹Δ) upon alkoxy substituted alkenes having allylic hydrogen.     

SPECTRAL CHARACTERISTICS AND MECHANISM OF CHEMILUMINESCENCE FROM TRYPTOPHAN SOLUTIONS INDUCED BY UV-IRRADIATION

The spectral distribution of the chemiluminescence, fluorescence and phosphorescence of tryptophan aqueous solutions irradiated with high and low pressure mercury lamps has been measured. The blue emission bands in the region of 380–520 nm observed both in the chemi- and photoluminescence, as well as an absorbance increase at 230 and 330 nm, indicate oxidative degradation of tryptophan leading to the formation of derivatives of N-formylkynurenine, xanthurenic and anthranilic acids. Red emission bands at 630 and 705 nm in the spectrum of the chemiluminescence, an enhancement of light intensity by D₂O and its decrease by NaN₃ and DABCO suggest a partial contribution of O₂(¹Δ_g) to the photooxidation and chemiluminescence of tryptophan. The enthalpy of the exergonic reactions, leading to the formation of luminescing products, was calculated to average -270 kJ-mol.     

FATTY ACID ANALYSIS AS A FUNCTION OF PHOTO-OXIDATION IN EGG YOLK LECITHIN VESICLES

Abstract— The photo-oxidation by O₂(¹Δ_g) of individual lipid components in egg yolk lecithin is examined as a function of time. The rate of oxidation is correlated with the degree of unsaturation in the fatty-acid chains.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

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.     

POLYMER BOUND PYRROLE COMPOUNDS–VI. PHOTOPHYSICAL PROPERTIES OF MONOMERIC MODELS FOR POLYSTYRENE-BOUND PORPHYRINS

Abstract— Several porphyrin esters used as models for polystyrene-bound porphyrins have been prepared and their excited states have been studied by laser flash photolysis, IR phosphorescence of singlet molecular oxygen, O₂(¹Δ_g), and steady-state fluorescence. The photophysical properties of the porphyrin esters in solution are affected by the presence of nitro group(s) in the chain. In this case, an important decrease in φ_f, φ_T and φ_δ (to ca 0.7–0.4 of the value for the parent dimethyl ester) is observed. This is mainly due to intramolecular electron-transfer quenching [by the nitro group(s)] of the first excited singlet state of the porphyrin. The thermodynamic feasibility of this deactivation pathway has been confirmed polarographically. Quenching of the porphyrin triplet state and of O₂(¹Δ_g) by the nitro groups is negligible. The present conclusions explain also the results obtained previously for the photooxidation of bilirubin sensitized by the parent insoluble polystyrene-bound porphyrins. In that case the photooxidation rates were correlated directly with the quantum yield of O₂(¹Δ_g) production by the sensitizer. The consequences of these results for the use of polystyrene-bound porphyrins in sensitized photooxidation processes are 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.     

SENSITIZED PHOTOOXIDATION OF ISOEUGENOL TO VANILLIN

Abstract— The photochemical conversion of isoeugenol to vanillin can proceed via the ¹O₂ (¹Δ_g) state of oxygen and can be effected over a wide range of base and dye concentrations. A typical situation involving methylene blue (0.3 mM), isoeugenol (24 mM) and a base concentration of 0.1 N will give yields of approximately 35% of the theoretical amount.     

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.