Photodegradation of a polyamidehydroxyurethane type polymer in aqueous solution. The role of some dyes in the degradation

The influence of some dyes on the photodegradation of a polyamidehydroxyurethane type polymer in aqueous solution has been studied. It has been found that, among the dyes used, only riboflavin sensitizes and accelerates the degradation of the polymer. It is proposed that under ultraviolet irradiation riboflavin undergoes photoreduction to lumichrome, which sensitizes the photodegradation of the polymer. It is also possible that the mechanism of photodegradation involves the participation of singlet oxygen.     

A STUDY OF THE PHOTODYNAMIC EFFICIENCIES OF SOME EYE LENS CONSTITUENTS

We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR-spin trapping method and the SOD-inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N-formylkynurenine (NFK), 3-hydroxykynurenine (3HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and 3HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and 3HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and 3HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.     

Oxidation of human catalase by singlet oxygen in myeloid leukemia cells

Catalases are oxidized by singlet oxygen giving rise to more acidic conformers detected in zymograms after electrophoresis in polyacrylamide gels. This shift in catalase mobility can be indicative of singlet oxygen production in vivo. Catalase from human cells, as from many organisms, is susceptible to in vitro modification by singlet oxygen. Human myeloid leukemia (U937) cells were treated under different stress conditions and catalase activity and its electrophoretic mobility was monitored. The U937 cells were found to have high levels of catalase activity, as compared to cultured fibroblasts, and to be very resistant to oxidative stress. Hydrogen peroxide did not modify the electrophoretic mobility of catalase, even at doses that produced cell damage. Conditions that primarily generate superoxide, such as treatment with paraquat or heat shock, also failed to modify the enzyme. In contrast, photosensitization reactions using rose Bengal gave rise to a more acidic conformer of catalase. Singlet oxygen quenchers prevented catalase modification by rose Bengal and light. The growth medium had a photosensitizing activity. Catalase was not modified in cells illuminated in phosphate buffer but was modified in cells illuminated in phosphate buffer containing riboflavin. Intense light per se also generated a slight shift in the electrophoretic mobility of catalase. Ultraviolet light (350 or 366 nm) did cause a change in catalase, but to a less acidic catalase conformer, indicating other modifications of the enzyme. The main effect of photosensitization with methylene blue was crosslinking of the enzyme, although some shift to acidic conformers was observed at a low concentration of the photoactive compound. Results indicate that catalase can be modified by singlet oxygen generated intracellularly, even though the enzyme is predominantly inside peroxisomes. Under some photosensitization conditions, catalase modification can be used as a marker to detect intracellular singlet oxygen.     

Recent developments in the detection of singlet oxygen with molecular spectroscopic methods

Singlet oxygen is a unique reactive oxygen species, as its chemical reactivity derives from its characteristic electronically-excited state. The involvement of singlet oxygen in many important atmospheric, physical, chemical, biological, and therapeutic processes has attracted intense research interest in recent years. The detection and the quantification of singlet oxygen are very important for understanding its mechanism of action in various processes.Due to its highly reactive nature, singlet oxygen has very few direct methods of determination. Only molecular phosphorescence at 1270 nm has been utilized. Indirect methods using spectrophotometric, fluorescent or chemiluminescent probes have therefore been extensively studied.This review reflects recent developments in singlet-oxygen detection with molecular spectroscopic methods. We begin with a brief introduction of the basic properties, the production and the applications of singlet oxygen. With this background information, we review the four molecular spectroscopic methods (i.e., emission, spectrophotometry, fluorescence and chemiluminescence). We pay special attention to attractive chemical probes with high selectivity and sensitivity in quantifying singlet oxygen.     

Enhancement of riboflavin-mediated photo-oxidation of glucose 6-phosphate dehydrogenase by urocanic acid

We have investigated the riboflavin (RF)-sensitized inactivation of glucose 6-phosphate dehydrogenase (G6PD) in the presence and absence of trans-urocanic acid (UCA). The inactivation of the enzyme results from its direct oxidation by the excited triplet RF in a Type-I-photosensitized reaction whose efficiency increases at low oxygen concentration. The addition of histidine to the system produced no change in the inactivation rate, discarding the participation of singlet oxygen in the reaction. On the other hand, the presence of UCA results in its bleaching, with a significant enhancement of RF-mediated inactivation of G6PD. Both the consumption of UCA and G6PD are faster at low oxygen concentrations. UCA also produced a decrease in the sensitizer photodecomposition yield. These results indicate that the enhancement of the RF-mediated G6PD inactivation observed in the presence of UCA is not a singlet oxygen-mediated process. It is proposed that UCA consumption and its effect on G6PD inactivation are due to a complex reaction sequence initiated by a direct oxidation of UCA by the excited sensitizer triplet. The oxidation of the semireduced flavin gives rise to reactive oxygen species (ROS) responsible for the increased rate of the process. This is supported by the protection afforded by several additives with ROS removal capacity: benzoate, superoxide dismutase and catalase.     

HEMOLYSIS OF HUMAN ERYTHROCYTES BY ACTIVATED OXYGEN SPECIES

Abstract— The hemolysis of human erythrocytes by irradiation at 254 nm has been studied. Neither superoxide radicals nor singlet oxygen play a significant rôle and it is likely that the major species involved are hydroxyl radicals and, indirectly, carbonate anion or formate radicals. Similarly, when erythrocytes are treated with a system commonly used as source of superoxide radicals (photoreduction of riboflavin) it has been demonstrated that O^-₂ does not participate in lysis, but that singlet oxygen (possibly with hydroxyl radicals) is a major oxygen species involved in destruction of the cell membrane.     

The Relative UV Sensitizer Activity of Purified Advanced Glycation Endproducts

Abstract— The oxidation products of ascorbic acid react with lens proteins to form advanced glycation endproducts (AGE) that are capable of generating reactive oxygen species when irradiated with UVA light. L-Threose, the most active of these oxidation products, was reacted with N -acetyl lysine and six AGE peaks were isolated by RP-HPLC. Each peak exhibited fluorescence and generated superoxide anion and singlet oxygen in response to UV light. Solutions of these AGE peaks (50 μg/mL) generated5–10 nmol/mL of superoxide anion during a 30 min irradiation. This activity was 100-fold less than the superoxide anion generated by kynurenic acid and 400-fold less than riboflavin.
Ultraviolet irradiation generated from 1.2 to 2.7 μmol/mL of singlet oxygen with the purified threose AGE compounds. This activity was similar to that seen with other purified AGE compounds (pentosidine, LM-1 and Ac-FTP) and with kynurenine and 3-OH kynurenine. This considerable singlet oxygen formation, however, was still 40-fold less than that obtained with kynurenic acid and 100-fold less than riboflavin under the same irradiation conditions. In spite of this lower sensitizer efficiency, the purified AGE generated20–60-fold more singlet oxygen on a weight basis than either crude ascorbic acid glycated proteins or a preparation of water-insoluble proteins from aged normal human lenses. On a molar basis, therefore, AGE could account for the sensitizer activity in these protein preparations if they represented less than 1% of the total amino acids.     

The nature of the lowest triplet configuration of flavins

Abstract— The phosphorescence spectrum of riboflavin has been reexamined at 77°K in an ethylene glycol: water matrix. The phosphorescence occurs at approximately the 605 nm (ca. 2-·05 eV) region, in reasonable agreement with the theoretical calculations of 1·6–1·8 eV [21]. The emission was found to be negatively polarized, and to have a relatively long lifetime (0·56 sec). On the basis of this data and additional theoretical results, it is concluded that the lowest triplet of riboflavin is of ³(τ,τ*) type. Upon addition of potassium iodide, the phosphorescence emission is enhanced through spin-orbit perturbation. The presence of oxygen (atmospheric pressure) in the frozen glass apparently has no effect on the emission. The phosphorescence spectra of alloxazine are also presented. Results of the present work have been applied to the photodephosphorylation of menadiol diphosphate in elucidating its mechan ism involving the riboflavin triplet and singlet oxygen.     

Photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen

The photochemical and photobiological properties of 4,8-dimethyl-5'-acetylpsoralen (AcPso), proposed for the photochemotherapy of some skin diseases, were investigated. The photoreaction of AcPso with DNA is weaker in the presence of air than in a nitrogen atmosphere, in terms of total photobinding and DNA cross-linking; when UVA irradiation is performed in air, AcPso behaves as a monofunctional reagent. The quenching effect of oxygen is related to the high capacity of AcPso to produce singlet oxygen. Furthermore, it is demonstrated that AcPso photoadducts are better producers of singlet oxygen than free AcPso in solution. Using DNA sequencing methodology, two modes of DNA photosensitization by AcPso are shown, these lead to the formation of photoadducts mainly at T residues (and at C to a lesser extent) and to photo-oxidized G residues probably via singlet oxygen. Chemical or enzymatic cleavage were used as probes in these experiments. A rapid assay for the detection of the photodynamic effect of a photosensitizer on DNA, involving oxygen, is also described. Finally, the cytotoxicity and genotoxicity of AcPso on E. coli WP2 cells appear to be related to its ability to form photoadducts, in particular cross-links, rather than to its capacity to produce singlet oxygen.     

SINGLET OXYGEN YIELDS AND RADICAL CONTRIBUTIONS IN THE DYE-SENSITISED PHOTO-OXIDATION IN METHANOL OF ESTERS OF POLYUNSATURATED FATTY ACIDS (OLEIC, LINOLEIC, LINOLENIC AND ARACHIDONIC)

Abstract— The triplet state characteristics (spectrum, lifetime and quantum yield) for four dye sensi tisers [methylene blue (MB), erythrosin (ER), haematoporphyrin (HP) and riboflavin (RF)] were determined in methanol by laser flash photolysis and singlet oxygen yields (0.60 to 0.48) from time-resolved measurements of the 1270 nm near infrared emission. The reaction of singlet oxygen with four long chain unsaturated phenyl esters [oleate (18: 1), linoleate (18: 2), linolenate (18: 3) and arachidonate (20: 4)] was followed quantitatively using the singlet oxygen luminescence technique and also, after continuous420–700 nm irradiation, by HPLC and other analysis of the isomeric product monohydroperoxides. The overall quantum yield of photooxidation (∼10^-2) was shown to be consistent with the observed singlet oxygen quenching constants(2–12 times 10⁴ dm³ mol^-1 s^-1) for the four esters studied and the singlet oxygen lifetime in methanol (τ∼ 9 μs). The isomer product distribution was interpreted in terms of a dual singlet oxygen and radical mechanism, the radical contribution increasing with sensitiser in the order ER = MB < HP ≪ RF, but also showing some dependence on substrate unsaturation. Evidence is presented for singlet oxygen quenching by MB and RF ( k_O = 1.6 and 6.0 times 10⁷ dm³ mol^-1 s^-1) and for the accelerated photobleaching of the dye sensitisers in the presence of the unsaturated esters.     

Oregano Essential Oil Interactions with Photogenerated Singlet Molecular Oxygen

Essential oils are a mixture of volatile compounds, products of the secondary metabolism of plants. Once extracted, they can be deteriorated losing their organoleptic and therapeutic properties due to various environmental factors, being light exposure in aerobic conditions the main cause. In this work, the oregano essential oil extraction and characterization from Origanum vulgare plants grown in the experimental field of the FTU-UNSL and its photodegradation in MeOH:H₂O 60:40 v/v solvent were studied. Characterization by EIMS and NIST Mass Spectrometry indicates the main compounds of oregano essential oil, quantified in the extracted oil by GC-MS, are carvacrol (7.14%) and thymol (47.37%). Degradation of essential oil and its two major components can be caused by reactive oxygen species photogenerated from endogenous sensitizers as riboflavin. Our results suggest degradation occurs involving singlet molecular oxygen. Interaction of carvacrol and thymol with singlet oxygen is mainly a physical process, while essential oil has an important reactive component, which indicates there might be other constituents which could contribute to reactive photoprotection. The effect of simultaneous presence of oregano essential oil and tryptophan amino acid—used as a photooxidizable model under riboflavin-photosensitizing conditions—was studied in order to evaluate the possible photoprotection exerted by the essential oil.     

SENSITIZED PHOTOOXYGENATION REACTIONS AND THE ROLE OF SINGLET OXYGEN†,‡

Abstract— In this paper we discuss various theoretical and experimental aspects of the role of singlet oxygen in sensitized photooxygenation reactions. New spectroscopic observations on the photosensitized production of singlet oxygen molecules are presented. The various factors which control the generation and reactions of singlet oxygen molecules are considered in detail. A relatively simple theoretical procedure is developed to predict the relative reactivities of ¹σ, ¹δ and ³σ oxygen toward various organic acceptors, and is used to discuss the chemical and photochemical properties of some of the oxygenation products. Finally, the properties of dioxetanes are examined in connection with the role which they may play in chemi- and bioluminescence. While we have said rather little about photodynamic reactions per se , the results presented in this paper strongly support the suggestion that many of the observed photodynamic effects could be due to reactions of singlet oxygen. Clearly a careful reexamination of various photodynamic effects at the molecular level to establish whether or not reactions of singlet oxygen are involved is now in order.     

New cyclometalated transition-metal based photosensitizers for singlet oxygen generation and photodynamic therapy

The aim of this review article is to introduce recent studies on an emergent class of singlet oxygen photosensitizers of potential applications to the photodynamic therapy,with a primary focus on the cyclometalated transition-metal complexes.Singlet oxygen photosensitization performances of various cyclometalated Ir and Pt scaffolds are reviewed,and the general photophysical properties of relevant systems and the mechanisms of singlet oxygen production via photo-sensitization are also briefly discussed.Thus far,investigations of singlet oxygen sensitization by such Ir and Pt complexes are mainly carried out in organic solvents and under non-physiological conditions,while some research efforts have been made at examining the feasibility of applying pertinent cyclometalated complexes to photodynamic therapy.     

Photosensitizer initiated attacks on DNA under dry conditions and their inhibition: a DNA archiving issue

Long-term aging of dry DNA is thought to be due to the attack of diverse cascades of reactive species with probably, no one single initiator of the cascades explaining all circumstances. Photosensitizer-initiated reactions from methylene blue and riboflavin were used to generate two model systems of reactive species around dry DNA in order to understand such systems and how to block them. Damage was assessed using plasmid DNA as a substrate with an in-situ microgel electrophoretic technique. Photodynamic methylene blue damage to DNA was very oxygen dependent but not that of riboflavin. This indicates that indirect type II pathways, probably via singlet oxygen were important for methylene blue but not for riboflavin. In both the absence and presence of oxygen, the DNA protection offered by dry caffeine and urate to both photodynamic agents indicated that most DNA attack was via electrophilic species. Overall, protection of dry archived DNA from spontaneously reactive species such as free radicals appears to be a real issue and, as expected, the predominant species in air appear to involve oxygen but not exclusively or necessarily so.     

Singlet oxygen and polymer photooxidations. I. Sensitizers,quenchers, and reactants

A study designed to ascertain the role of singlet molecular oxygen in the photodegradation of plastics established that most classes of dye chromophores are sensitizers in polymer films, absorbing light and transferring the absorbed energy to ambient triplet ground state molecular oxygen, generating metastable reactive singlet molecular oxygen. Unsaturated polymers containing polybutadiene, polyisoprene, etc. are highly reactive to singlet oxygen produced through photosensitization, generating hydroxylic and carbonyl derivatives and losing their rubbery properties as consequences of such reactions. Many types of transition metal chelates are singlet oxygen quenchers. The relationships of the structures and spectroscopic properties of these chelates to their efficiency in quenching singlet oxygen are examined and discussed.     

Time-Resolved Study of the Sensitized Formation and Decay of 1O2(1δg) in the Presence of Cyclodextrins*

The effect of β-cyclodextrin and β-hydroxypropyl-cyclodextrin on some properties of the aromatic ketone 1-H-phenalen-1-one and its sulfonate derivative 1-H-phen-alen-1-one-2-sulfonic acid was measured in aqueous solution. From the changes in the UV-visible range of the absorption spectra, the association equilibrium constants for the formation of inclusion complexes were determined. Because these ketones are very efficient sensitizers for the generation of singlet oxygen, time-resolved infrared luminescence was used to measure the lifetime of singlet oxygen in D₂O. Cyclodextrins are weak deactivating agents of singlet oxygen; the upper limits for the bimolecular deactivation constants are 2 times 10⁵M^?1s^?1 and 1 times 10⁵M^?1s^?1 for β-cyclodextrin and β-hydroxypropyl-cyclodextrin, respectively. Besides, they do not affect noticeably the extent of formation of singlet oxygen; this result is explained in terms of relocation of the sensitizer (exit from the cyclodextrin cavity) in the triplet excited state.     

Protective effect of melatonin on photo-damage to lysozyme

The behavior of melatonin in the riboflavin-sensitized photo-oxidation of lysozyme was monitored. Melatonin was found to prevent aggregation of protein and the decrease of enzyme activity induced by photo-oxidation. Electron spin resonance experiments showed that photo-oxidation of lysozyme in the presence of riboflavin resulted in formation of protein radicals, and melatonin was highly effective in reducing the formation of protein radicals. Direct evidence of melatonin’s ability for quenching the triplet state of riboflavin and singlet oxygen was presented. A mechanism of the protective effect of melatonin on photo-oxidation of protein was proposed and the physiological relevance was discussed.     

Antibody-catalyzed oxidation of delta(9)-tetrahydrocannabinol

Marijuana abuse continues to plague society and the lack of effective treatments warrants concern. Catalytic antibodies capable of oxidatively degrading the major psychoactive component of marijuana, Delta9-tetrahydrocannabinol (Delta9-THC), are presented. The antibodies generate reactive oxygen species from singlet oxygen (1O2*), using riboflavin (vitamin B2) and visible light as the 1O2* source. Cannabitriol was identified as the major degradation product of this reaction, demonstrating the ability of an antibody to catalyze a complex chemical transformation with therapeutic implications for treating marijuana abuse.     

New Singlet Oxygen Donors Based on Naphthalenes: Synthesis,Physical Chemical Data,and Improved Stability

Singlet oxygen donors are of current interest for medical applications, but suffer from a short half‐life leading to low singlet oxygen yields and problems with storage. We have synthesized more than 25 new singlet oxygen donors based on differently substituted naphthalenes in only a few steps. The influence of functional groups on the reaction rate of the photooxygenations, thermolysis, half‐life, and singlet oxygen yield has been thoroughly studied. We determined various thermodynamic data and compared them with density functional calculations. Interestingly, remarkable stabilities of functional groups during the photooxygenations and stabilizing effects for some endoperoxides during the thermolysis have been found. Furthermore, we give evidence for a partly concerted and partly stepwise thermolysis mechanism leading to singlet and triplet oxygen, respectively. Our results might be interesting for “dark oxygenations” and future applications in medicine.     

SOME PREVALENT BIOMOLECULES AS DEFENSES AGAINST SINGLET OXYGEN DAMAGE

Abstract— We have compared the relative abilities of some putative biological protectors to block oxidation of 2,5-bis(hydroxymethyl)furan (BHMF)† in illuminated solutions containing the photosensitizer rose bengal and in the separated-surface-sensitizer (S-S-S) system involving pure singlet oxygen (¹ΔA_gO₂). While L-histidine is a well-known quencher of singlet oxygen, free L-histidine is not commonly found in high concentrations in nature. L-Carnosine (β-alanyl-L-histidine), however, is present in the striated muscles of many organisms, most notably mammals, in concentrations up to 40 m M . At neutral pH, carnosine quenched singlet oxygen more effectively than did equimolar histidine, both in solubilized sensitizer studies and in the S-S-S system. In the pure singlet oxygen system, 1 m M carnosine reduced the rate of BHMF oxidation as effectively as 3 m M histidine alone, or a mixture of 3 mM histidine and 3 m M β-alanine. The fungal product L-ergothioneine (2-thiol-L.-histidine betaine) and its synthetic analogue, 2-thiolhistidine, at 1 m M blocked photosensitized BHMF oxidation using solubilized rose bengal, as did urate at 0.5 m M . All three compounds failed to reduce the rate of BHMF oxidation by singlet oxygen in the S-S-S system, however. Homocarnosine (-γ-aminobutyryl-L-histidine) gave levels of protection against BHMF oxidation identical to histidine, but is present in the central nervous system only at micromolar concentrations. Neither 1 m M imidazole nor 5 m M urea reduced BHMF oxidation in either system. We conclude that some prevalent biomolecules may afford protection either by preventing singlet oxygen production (urate, L-ergothioneine) or by intercepting singlet oxygen once formed (L-carnosine). Such protective devices may be of importance in natural systems.