PHOTOOXIDATION AND SINGLET OXYGEN SENSITIZATION BY PROTOPORPHYRIN IX AND ITS PHOTOOXIDATION PRODUCTS

Protoporphyrin IX and its various ester derivatives have been previously shown to undergo self-sensitized photooxygenation to yield hydroxyaldehydes (photoprotoporphyrin) and mono- and diformyl deuteroporphyrin derivatives. In the present study the photoreactions of these products in the presence of oxygen have been investigated. All of the photooxidation products are themselves good sensitizers of singlet oxygen. In addition spin trapping experiments indicate these products can produce superoxide in low-to-moderate efficiency by an excited state electron transfer process. The photo-products themselves are somewhat more stable to photooxidation than protoporphyrin IX itself. The two monoformyl-monovinyl deuteroporphyrins have been found to undergo further photooxidation at the vinyl groups to yield primarily monoformyl hydroxyaldehydes in a reaction mainly involving singlet oxygen analogous to the initial reaction of protoporphyrin IX.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—1. THE MOLECULAR MECHANISMS*

Abstract— DNA and RNA syntheses were inhibited immediately after proflavine treated HeLa cells were irradiated with visible light (400–500 nm). The molecular mechanism for this photooxidation may be either a free radical-mediated (Type I) or singlet oxygen-mediated (Type II) reaction. Non-toxic free radical and singlet oxygen quenchers were added to cells and sensitizer before irradiation to quench the appropriate excited state intermediate. Photooxidative damage (the inhibition of incorporation of [¹⁴C]-thymidine) in this system was greatly reduced in the presence of free radical quenchers (glutathione, penicillamine) and not significantly affected by the presence of singlet oxygen quenchers (α-tocopherol, β-carotene, DABCO). This suggests that at least part of the photodynamic damage in HeLa cells is via a Type I mechanism.     

PROBING PHOTODYNAMIC DAMAGE IN NUCLEIC ACIDS WITH A DAMAGE-SPECIFIC DNA BINDING PROTEIN: A COMPARISON OF THE B and Z DNA CONFORMATIONS

—We have employed a damage-specific DNA binding protein from human cells as a probe for base damage in polymers irradiated with white light in the presence of methylene blue. Protein-recognizable damage is introduced only into guanine-containing polymers and quenching of damage introduction by H₂O and sodium azide suggest the involvement of a singlet oxygen mechanism. Using poly d(G-m⁵C), we have demonstrated that the left-handed double helical Z conformation is much less susceptible to guanine photooxidation than is the usual B conformation. We speculate that this difference in reactivity may reflect steric hindrance at the purine C-4 position and could provide some insight into the initial steps of the reaction between singlet oxygen and guanine in nucleic acid polymers.     

Studies of the photo-oxidation of polydienes: Part 1—Photo-stabilising effect of some derivatives of diacetyl-monooxime benzoylhydrazone nickel(II) chelates on cis-1,4-polybutadiene

Three derivatives of diamagnetic diacetyl-monooxime benzoylhydrazone nickel(II) chelates were evaluated as uv stabilisers for cis-1,4-polybutadiene (BR) in solution and in films. In the presence of methylene blue (MB) as singlet oxygen sensitiser, such chelates inhibit the effect of singlet oxygen formed during irradiation by visible light. The photo-stabilising mechanism of the chelates is discussed in terms of their action as singlet oxygen quencher as well as their action as uv absorbers. Further, a stabilising mechanism which involves the trapping of alkyloxy and/or alkylperoxy radicals has also been suggested for two of the chelates. Combination of the most effective chelate and β-carotene as singlet oxygen quencher exhibits synergistic behaviour. The polymer samples which were irradiated in its presence were examined using the Differential Scanning Calorimeter (DSC) technique. The results obtained also demonstrate the photo-stabilising effect of the chelate.     

SPECTROSCOPY AND PHOTOCHEMISTRY OF BILIRUBIN PHOTOPRODUCTS. I. METHYLVINYLMALEIMIDE*

Abstract— Methylvinylmaleimide, a product of the photooxidation of bilirubin, exhibits a broad luminescence band at approximately 450 nm on filter paper at room temperature. In methanol solvent, at 77 K, a broad phosphorescence band centered at 465 nm is observed. The experimental and theoretical spectroscopic properties of this compound are compared to those of the related molecules maleimide and phthalimide. Methylvinylmaleimide is photodegraded at pH 7.6 in a self-sensitized reaction that in part involves singlet oxygen.     

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).     

PHOTOSENSITIZATION OF AMINO ACIDS BY DI-CYAN-HEMIN: KINETIC AND EPR STUDIES

Abstract— The low spin hemin derivative di-cyan-hemin can sensitize the photooxidation of amino acids. The efficiency of this dye as a photosensitizer is generally lower than that of other porphyrins such as hematoporphyrin in aqueous solution, but it is significantly enhanced by addition of ethanol. On the basis of the identified photooxidation end-products of some amino acids, photooxidation kinetic data and EPR measurements, it appears that methionine is quantitatively converted to methionine sulfoxide by a pure Type II (singlet oxygen) mechanism, whereas in the case of cysteine and tryptophan a mixed Type ILType II mechanism is probably operative.     

PHOTOOXIDATION OF AROMATIC SULFIDES

Abstract

Dye sensitized photooxidation of sulfides proceeds by way of singlet oxygen to give sulfoxides and sulfones, and the evidence for several intermediates has been reported. We now report that photooxidation of aromatic sulfides gives the product of an unusual type; and the reaction may proceed by either superoxide or hydroperoxysulfurane. 9-Ethylthiofluorene was photooxidized in dichloromethane or acetone with methylene blue as sensitizer. Three main products were isolated and identified as fluorenone (15%), 9-fluorenyl ethyl sulfoxide (19%) and 1-hydroxyl-9-fluorenone (23%).     

The fate of oxygen in the quenching of excited singlet states

Measurement of the efficiency of rubrene-sensitizied photooxidation of 1,3-diphenylisobenzofuran imply that direct formation of singlet oxygen via oxygen quenching of excited rubrene singlets is inefficient. This contrasts with recent publications based upon studies of self-sensitized rubrene photooxidation. The observed inefficiency can be rationalized in terms of the spin-allowed decay of an initially formed ³(T₁ + ¹Δ) complex state to a lower energy ³(T₁ + ³Σ) state prior to complex dissociation.     

Imaging by sensitized oxygenations of photochromic anthracene films: examination of effects that improve performance and reversibility

The aliphatic anthracene compound 1 and the oligomeric anthracene 2 were synthesized. Thin films of 1 and 2 mixed with the sensitizers tetraphenylporphyrin (TPP) and methylene blue (MB) were irradiated with visible light in air. Upon formation of singlet oxygen, the anthracene units were converted quantitatively to the corresponding endoperoxides. Heating of the irradiated samples afforded the parent anthracenes with high yields. Here, we demonstrate that the kinetics and reversibility of this reaction strongly depend on the microenvironment of the anthracene groups in the two compounds. The photooxidation of thin films of 1 is accompanied by interesting changes in the morphology of the film and allows the first application of 1 as a nondestructive negative-tone photoresist for lithography and as an oxidizing ink. The morphology of 2 remained unchanged after photooxidation as a result of the stabilizing oligomer backbone. This stabilizing effect significantly improves the photochromic performance of 2. The reversibility of the photooxidation is very high (>90 %) for oligomeric films of 2 after several cycles of irradiation and heating. Decomposition of the anthracene and a loss of the activity of the sensitizer diminish slightly the performance of the monomeric species.     

Photosensitized Oxidation of Tetrabromobisphenol A by Humic Acid in Aqueous Solution†

The brominated flame retardant 3,3′,5,5′‐tetrabromobisphenol A (TBBPA) may accumulate in the environment, including surface waters, and degrade there to potentially toxic products. We have previously shown that singlet oxygen (¹O₂), produced by irradiation of rose bengal with visible light, oxidizes Triton X‐100‐solubilized TBBPA to yield the 2,6‐dibromo‐p‐benzosemiquinone anion radical while consuming oxygen (Environ. Sci. Technol. 42 , 166, 2008). Here, we report that a similar ¹ O ₂ ‐induced oxidation can be initiated in aqueous solutions by the irradiation of TBBPA dissolved in a humic acid (HA) solution. HA is a known weak ¹ O ₂ photosensitizer and we indeed detected the infrared ¹ O ₂ phosphorescence from HA preparations in D ₂ O. When an aqueous preparation of HA was irradiated (λ > 400 nm) in the presence of TBBPA, oxygen was consumed, and the 2,6‐dibromo‐ p ‐benzosemiquinone anion radical was generated and detected using electron paramagnetic resonance. Radical formation and oxygen consumption were inhibited by sodium azide, a singlet oxygen quencher. Our results suggest that solar radiation, in the presence of HA, may play an important role in the photodegradation of TBBPA in the aquatic environment.     

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.     

The photodegradation of cis-1,4-polybutadiene, polystyrenebutadiene and polyisoprene

The photodegradation of the synthetic rubbers cis-1,4-polybutadiene, polystyrenebutadiene and polyisoprene was studied. The chemical structure of the polymers as well as the effect of changing the solvent and the additive were particularly taken into consideration. The polychloroprene rubber in this study was irradiated either as a film or in solution. The presence of methylene blue affects the endothermic peaks observed in its differential scanning calorimetry diagrams. Methylene blue acts as a photostabilizer dye in solutions and films of polychloroprene. Mixtures of methylene blue, β-carotene as a singlet oxygen quencher and Fmbanox as a known anti-oxidant showed synergystic behaviour.     

Photodynamic Crosslinking of Proteins. III. Kinetics of the FMN- and Rose Bengal-sensitized Photooxidation and Intermolecular Crosslinking of Model Tyrosine-containing N-(2-Hydroxypropyl)methacrylamide Copolymers

As part of a study on the role of Tyr residues in the photosensitized intermolecular crosslinking of proteins, we have surveyed the kinetics of the rose bengal- and flavin mononucleotide (FMN)-sensitized photooxidation and crosslinking of a water-soluble N-(2-hydroxypropyl)methacrylamide copolymer with attached 6-carbon side chains terminating in tyrosinamide groups (thus the -OH group of the Tyr is free, but both the amino and carboxyl groups are blocked, simulating the situation of a nonterminal Tyr in a protein). The intermolecular photodynamic crosslinking of the Tyr copolymer can result only from the formation of Tyr-Tyr (dityrosine) bonds, because the copolymer itself is not photooxidizable. Rose bengal, primarily a Type II (singlet oxygen) sensitizer, sensitized the rapid photooxidation of the Tyr residue in the Tyr copolymer only at high pH, where the Tyr phenolic group is ionized; crosslinking did not occur with rose bengal under any of the reaction conditions used. In contrast, FMN, which can sensitize by both Type I (free radical) and Type II processes, sensitized the photooxidation of the Tyr copolymer over the pH range 4-9.5. Also, significant photocrosslinking occurred, but only from pH 4 to 8, with a maximum rate at pH 6. Crosslinking required the presence of oxygen. Studies with inhibitors, D2O as solvent, catalase and superoxide dismutase indicated that the photooxidation and photocrosslinking of the Tyr copolymer with FMN at pH 6 were not mediated by singlet oxygen, superoxide or hydrogen peroxide. It appears that crosslinking involves the abstraction of an H atom from the Tyr phenolic group to give Tyr and FMN radicals. The Tyr radical in one Tyr copolymer can then react with a Tyr radical in another Tyr copolymer to give an intermolecular dityrosine crosslink.     

磺化酞菁类染料光敏氧化胆固醇及L-半胱氨酸的反应

本文合成了磺化酞菁、磺化酞菁镓和磺化酞菁铝,研究了它们光敏氧化胆固醇及L-半胱氨酸的反应。染料的聚集态和溶液的pH值对反应速率有不同程度的影响。D₂O加速反应而NaN₃猝灭反应的结果表明,光敏氧化反应主要通过Ⅱ型(涉及¹O₂)机制进行。     

Sensitized Photooxidation of Ortho-Prenyl Phenol: Biomimetic Dihydrobenzofuran Synthesis and Total 1O2 Quenching†

The sensitized photooxidation of ortho-prenyl phenol is described with evidence that solvent aproticity favors the formation of a dihydrobenzofuran [2-(prop-1-en-2-yl)-2,3-dihydrobenzofuran], a moiety commonly found in natural products. Benzene solvent increased the total quenching rate constant (k_T) of singlet oxygen with prenyl phenol by ~10-fold compared to methanol. A mechanism is proposed with preferential addition of singlet oxygen to prenyl site due to hydrogen bonding with the phenol OH group, which causes a divergence away from the singlet oxygen ‘ene’ reaction toward the dihydrobenzofuran as the major product. The reaction is a mixed photooxidized system since an epoxide arises by a type I sensitized photooxidation.     

Studies on the photooxidation mechanism of polymers. I. Photolysis and photooxidation of polystyrene

A new mechanism has been proposed for the photooxidation of polystyrene as film and in benzene. The initial stage of the photooxidative degradation may involve reactions of singlet oxygen with polystyrene molecules. Singlet oxygen may be formed in the reaction between excited benzene ring in polystyrene molecule and molecular oxygen. The addition of singlet oxygen quenchers such as 1,3-cyclohexadiene or β-carotene reduces the rate of polymer degradation in benzene solution. The mechanisms of the photolysis of polystyrene as film and in benzene solution, in vacuo and in the presence of oxygen, are discussed and interpretations proposed. The pronounced yellowing of polystyrene during the photooxidation process is interpreted as a reaction involving benzene ring-opening photooxidation in polystyrene molecule. These results were obtained by comparing ultraviolet and infrared spectra in experiments of photooxidation of pure liquid benzene and polystyrene film.     

PURE SINGLET OXYGEN CYTOTOXICITY FOR BACTERIA

We have modified the separated-surface-sensitizer singlet oxygen generating system previously described (Midden and Wang, 1983) for the efficient exposure to pure singlet oxygen of bacteria collected on membrane filters. Physical separation of the photosensitizer and the bacteria eliminates the possibility of direct interaction between bacteria and photoexcited sensitizers that could lead to Type I (non-singlet oxygen) photooxidation processes. This system was used to examine the bacterial cytotoxicity of singlet oxygen. The role of singlet oxygen was confirmed by measuring the decrease in cytotoxicity as the distance between the singlet oxygen source and the bacteria was increased. The gas phase half-life of the intermediate responsible for cell killing, determined from this distance dependence analysis (24 ± 6 ms), is the same as that calculated from literature data for the gas phase half-life of singlet oxygen (53 ± 37 ms). Killing of various strains of Salmonella lyphimurium and Escherichia coli was compared at the same dose of singlet oxygen. Bacteria were killed by singlet oxygen at levels several orders of magnitude lower than those effective in killing by H₂O₂. Altered DNA repair capacities (uvrB, recA, xth, nth , pKM101) did not affect survival. Incomplete cell wall lipopolysaccharide formation decreased survival following singlet oxygen exposure. Overproduction of the singlet oxygen quencher histidine increased survival, as did accumulation of the dipeptide carnosine (β-alanyl-L-histidine). No evidence for mutagenicity of exogenous singlet oxygen exposure was obtained in a variety of S. typhimurium strains killed to 35% survival.     

Chemiluminescence upon Photooxidation of 4,4"-Diazidodiphenyl with Molecular Oxygen

It was found that irradiation of the solution of 4,4"-diazidodiphenyl in ethyl alcohol with UV-light at a wavelength longer than 280 nm in the presence of dissolved oxygen resulted in appearance of post-luminescence. The luminescence intensity increases with increasing the oxygen concentration; the luminescence virtually disappears after purging the solution with argon. The observed emission of light was attributed to chemiluminescence of the products of photooxidation of 4,4"-diazidodiphenyl. The rate dependence of the afterglow shows two maxima. The first is observed immediately after cessation of the UV irradiation of the solution and its transfer to a photometric cell. The intensity of the corresponding chemiluminescence decreases by an exponential law with _e= 5 s. The second maximum appears after a certain time interval after the first one. Addition of a triplet sensitizer (Michler's ketone) to the reaction mixture results in quenching of the chemiluminescence from the photooxidation products, whereas the addition of substances that stabilize electrophilic species in the singlet state leads to an increase in the chemiluminescence intensity. It was suggested that the chemiluminescence resulted from reactions involving the singlet nitrene adduct with oxygen.     

The Mechanism of Photofrin Photobleaching and Its Consequences for Photodynamic Dosimetry

Abstract— We report experimental results that support a theory of self-sensitized singlet oxygen-mediated bleaching of the porphyrin photosensitizer Photofrin. Microelectrode measurements of photodynamic oxygen consumption were made near the surface of individual, Photofrin-sensitized EMT6 spheroids during laser irradiation. The progressive decrease in photochemical oxygen consumption with sustained irradiation is consistent with a theory in which bleaching occurs via self-sensitized singlet oxygen reaction with the photosensitizer ground state. A bleaching model based solely on absorbed optical energy density is inconsistent with the data. Photobleaching has a significant effect on calculated photodynamic dose distributions in 500 pin diameter spheriods. Dose distributions corrected for the effects of bleaching produce a new estimate (12.1 ± 1.2 m M ) for the threshold dose of reacting singlet oxygen in this system.