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.     

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.     

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.     

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.     

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.     

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.     

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.     

USE OF TRIS (2,2'-BIPYRIDINE) RUTHENIUM(II) DICATION IN A NOVEL METHOD FOR THE DETERMINATION OF SINGLET OXYGEN REACTION RATES: APPLICATION TO STUDIES OF GELATIN

Abstract— A novel method for the determination of singlet oxygen reaction rate constants is described and applied to studies of cyclohexadiene in methanol and gelatins in H₂O and D₂O. The technique uses tris (2,2'-bipyridine) ruthenium(II) dication (Ru(bipy)₃₂₊) as both singlet oxygen sensitizer and in situ oxygen concentration monitor during irradiation of sealed samples. Because of the high efficiency with which the luminescence of Ru(bipy)₃2+* can be detected, and the fact that emission lifetimes are used, the method offers some advantages over those previously described. The advantages and disadvantages of the method are discussed. A rate constant of 2.1 (±0.3) x 10⁶ mol^-1 dm³ s^-1 has been determined for the reaction of ¹O₂ with cyclohexadiene in methanol. For two different photographic gelatins the sums of reaction and quenching rate constants are 2.0 (±0.4) x 10⁶ and 3.1 (±2.0) x 10⁵ mol^-1 dm³ s^-1; for swine skin gelatin this value is 3.9 (±2.4) × 10⁵ mol^-1 dm³ s^-1. Chemical reaction, rather than physical quenching, is the dominant reaction route for gelatins and ¹O₂.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

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.     

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).     

INACTIVATION OF NORMAL AND MUTANT NEUROSPORA CRASSA CONIDIA BY VISIBLE LIGHT AND NEAR-UV: ROLE OF 1O2, CAROTENOID COMPOSITION AND SENSITIZER LOCATION

Abstract— Inactivation of Neurospora crassa conidia from wild-type and mutant strains by visible and near-UV light has been investigated in the presence and absence of photosensitizing dyes. Inactivation by near-UV is virtually unchanged by the presence of deuterium oxide or azide suggesting that, contrary to the situation with visible light and photosensitizing dyes, ¹O₂ is not involved in any substantial way in the formation of lethal lesions. The finding that carotenoid deficient strains are similar to wild-type strains in sensitivity to near-UV inactivation is consistent with ¹O₂ not being involved.
Photodynamic inactivation of conidia by visible light occurs in the presence of methylene blue (MB), toluidine blue O (TB), or acridine orange (AO). Carotenoid deficient strains are more sensitive to such inactivation only when MB and TB are used. These results support the contention that MB and TB mediated damage involves the cell membrane where carotenoids are available for quenching, whereas AO mediated damage occurs in the nucleus sequestered from the protective influence of carotenoids.
A newly isolated, lemon–yellow mutant, mapping to the al -1 locus, exhibits sensitivities to photodynamic inactivation similar to other pure-white mutants at the same locus. The sensitivity of this pigmented mutant is apparently related to insufficient unsaturation (seven to nine double bonds) of the two colored carotenoids, zeta–carotene and neurosporene, produced by the mutant.     

THE ABSOLUTE VALUE OF THE REACTION RATE CONSTANT OF BILIRUBIN WITH SINGLET OXYGEN IN D2O

Abstract— The chemical reaction rate constant of bilirubin with singlet oxygen in basic aqueous solution has been redetermined to be 3.5 × 10⁸ M^-1 s^-1 by a competitive technique using a 1,3-diphenylisobenzofuran in sodium dodecyl sulfate micelles. Bilirubin also physically quenches a singlet oxygen with a rate constant of 9 × 10⁸ M ^-1 s^-1. The lifetime of singlet oxygen in D₂O solution has been determined to be 35 μ s . The absorption cross-section for the molecular oxygen ³∑_g-→¹δ _g + 1 v electronic transition at 1.06μn in aqueous solution is unexpectedly larger than the gas paase cross-section.     

ON THE QUENCHING OF SINGLET OXYGEN BY α-TOCOPHEROL

Abstract —D-α-tocopherol was found to be an effective quencher of ¹O₂ molecules ( k = 2.5 times 10⁸→mol^-1 s^-1 in pyridine) by measuring its effect on the autosensitized photooxidation of rubrene. The quenching process was shown to be almost entirely 'physical', that is, α-tocopherol deactivated about 120 ¹O₂ molecules before being destroyed. The results suggest that this process may be a mechanism for the protective effect of α - tocopherol in photodynamic action.     

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.     

INHIBITION AND ENHANCEMENT OF SINGLET OXYGEN ('Ag) DIMOL CHEMILUMINESCENCE

Abstract— Various nitrogen containing compounds have previously been shown to quench singlet oxygen (10z). When measuring the dimol ¹O₂ light emission arising from the H ₂O₂ /OCI- reaction, we found that certain cyclic diamines increase the emission of light, while other amines were inhibitory. This increase of light emission was seen with both 1, 4diazabicyclo[2.2.2]octane and N, N'-dimethylpi-perazine but not with acyclic analogues. Sodium azide inhibited both the normal and enhanced light emission. The enhanced light emission shows spectral properties characteristic of ^lO₂ dimol emission.     

THE EFFECT OF 1,4-DIAZABICYCLO[2.2.2]OCTANE ON THE RADIOSENSITIVITY OF BACTERIA

Abstract— Hydroxyl radicals ('OH) are scavenged by 1,4-diazabicyclo[2.2.2]octane (DABCO) at a diffusion-controlled rate of 1.25 ± 0.1 × 10⁹ M ^-1s^-1. Unlike other efficient 'OH scavengers which exhibit protection of bacteria against irradiation both in oxic and hypoxic conditions, DABCO has been shown to protect Serratia marcescens and various strains of Escherichia coli only in oxic conditions.
DABCO appears to eliminate a component of the sensitization afforded by oxygen in all strains of E. coli tested. The level of this protection increases from ∼15% in the wild type AB 1157 to ∼100% in the recA uvrA mutant AB 2480. It is suggested that DABCO protects against lethal events that can occur on macromolecules other than DNA such as the cell membrane.
Results with added glycerol, as well as work in D₂O solution, indicate that DABCO is more likely to be acting by scavenging radicals rather than by quenching ¹O₂. If ¹O₂ is a component of the sensitization afforded by oxygen, then it is unlikely to be formed in a hydrophilic environment in the cell.     

pH DEPENDENCE OF SINGLET OXYGEN PRODUCTION IN AQUEOUS SOLUTIONS USING THIAZINE DYES AS PHOTOSENSITIZERS

Abstract— The production of singlet oxygen by thiazine dye photosensitization, as measured by the rate of photooxidation of tryptophan, was found to be very sensitive to changes of pH in the range 5–9. For methylene blue in aerated solutions, the production of ¹O₂* is approximately five times more efficient in basic than in acidic medium. This was shown to be related to the p K 's of the triplet dyes, by evaluating the yields of ¹O₂* from the lifetimes and the quenching rate constants for the two ionic species of sensitizer triplets measured by laser flash photolysis. Changes in the quenching rate constants of the thiazine triplet states can be correlated with the triplet energies.     

THE PHOTOOXIDATION OF DIETHYLHYDROXYLAMINE BY ROSE BENGAL IN MICELLAR AND NONMICELLAR AQUEOUS SOLUTIONS

The photooxidation of N,N -diethylhydroxylamine (DEHA) by Rose Bengal (RB) has been investigated in micellar and nonmicellar aqueous solutions. We measured the quantum yield of oxygen consumption forming H₂O₂ and monitored two intermediates, the superoxide and diethylnitroxide radicals. When the pH was vaned, the quantum yield of oxidation remained constant for 6 < pH < 10.5, decreased in acidic pH, and increased considerably in NaOH solution; these changes could be attributed to the protonation and dissociation processes of the >N-OH moiety of DEHA. The formation of diethylnitroxide radical was enhanced by superoxide dismutase or strong alkaline solution. Around neutral pH, the oxidation proceeded mainly via electron transfer from DEHA to the RB triplet ( k _q = 10⁷ M ^-1 s^-1) with little ¹O₂ participation ( k_q < 10⁵ M ^-1 s^-1). However, when RB was incorporated into micelles in alkaline solution, the contribution of the singlet oxygen pathway increased at the expense of electron transfer, which was inhibited by the less polar micellar environment. Dark autoxidation of DEHA was accelerated by heavy metal impurities and increased very strongly in NaOH solution.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.