Singlet Oxygen Phosphorescence Research Articles

Phototoxic Phytoalexins. Processes that Compete with the Photosensitized Production of Singlet Oxygen by 9-Phenylphenalenones†

Experiments were performed to elucidate the excited-state behavior of 9-phenylphenalenones, which are phototoxic plant secondary metabolites involved in mechanisms of light-mediated plant defense. Using a combination of time-resolved and steady-state UV/visible spectroscopies, time-resolved IR absorption spectroscopy, time-resolved singlet oxygen phosphorescence measurements and cyclic voltammetry, we provide evidence of an intramolecular charge-transfer process in the excited singlet and the triplet states of 9-phenylphenalenones that modulates the photosensitized production of singlet oxygen.

Chemically Induced Dynamic Electron Polarization Generated through the Interaction between Singlet Molecular Oxygen and Nitroxide Radicals

An absorptive chemically induced dynamic electron polarization (CIDEP) was generated by the quenching of singlet oxygen by nitroxide radicals (TEMPO derivatives). The spin polarization decay time of the nitroxide (measured by time-resolved EPR) correlates with the lifetime of singlet oxygen (measured by singlet oxygen phosphorescence spectroscopy). In addition, a deuterium isotope effect on the spin polarization decay time was observed, a signature of singlet oxygen involvement. With use of isotope labeled nitroxides (15N, 14N), the relative spin polarization efficiencies of TEMPO, 4-oxo-TEMPO, and 4-hydroxy-TEMPO by singlet oxygen were determined. The relative spin polarization efficiencies (per quenching event) decrease in the order 4-hydroxy-TEMPO > TEMPO > 4-oxo-TEMPO, whereas an opposite trend was observed for the total quenching rate constants of singlet oxygen by the nitroxides where the order is 4-hydroxy-TEMPO < TEMPO < 4-oxo-TEMPO.

The Microenvironment Effect on the Generation of Reactive Oxygen Species by Pd−Bacteriopheophorbide

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.

Application of a dithered sampling technique to increase the spatial resolution of singlet oxygen images

Singlet molecular oxygen (a1Δg) is an intermediate in many important oxidative reactions in heterogeneous biological and polymeric systems. By using a custom-made microscope to detect the 1270nm phosphorescence of singlet oxygen (a1Δg→X3Σg−), singlet oxygen images of such systems can be created. The microscope uses an InGaAs linear photodiode array to detect this extremely weak near infrared phosphorescence. In this article, the effects of the microscope’s modulation transfer function and the detector’s spatial sampling frequency on the resolution of the images are examined. It is shown that a dithered sampling technique can increase the accessible resolution. In this approach, data are repeatedly acquired from the sample after it has been systematically moved on the microscope stage.

Protoporphyrin IX and hematoporphyrin derivatives interactions with oxygen studied by time and spectral resolved phosphorescence

In this contribution energy transfer from chemically similar porphyrin-like molecules of hematoporphyrin derivatives and protoporphyrin IX, used in photodynamic therapy as photosensitizers was investigated. The photosensitizers phosphorescence (about 840 nm) kinetics together with singlet oxygen production were measured by means of IR time and spectral resolved phosphorescence (at 1275 nm) in wide range of the photosensitizer concentrations (5–200 μM). The submicrosecond photosensitizers triplet life-times were compared to submicrosecond singlet oxygen phosphorescence rise-times. Moreover, microsecond singlet oxygen phosphorescence life-times and rate constants of singlet oxygen quenching by the photosensitizers were determined. The differences in obtained life-times and concentration trends were discussed and explained in the frame of the photosensitizers aggregation. Hematoporphyrin derivatives behave as non-aggregating photosensitizer, while protoporphyrin IX exhibits an aggregation.

Heteroatom Substitution Induced Changes in Excited-State Photophysics and Singlet Oxygen Generation in Chalcogenoxanthylium Dyes: Effect of Sulfur and Selenium Substitutions

Photophysical properties of a series of tetramethylrosamine dyes in which the xanthylium oxygen atom has been replaced with sulfur or selenium were examined. Dyes with sulfur and, especially, with selenium show decreased fluorescence quantum yield and lifetimes, while at the same time proportionally larger increases in the rates of intersystem crossing lead to higher triplet yields (0.23 and 0.97, correspondingly). The selenium-containing dye (TMR−Se) displays prominent phosphorescence at 77 K and at room temperature when the solution is purged of oxygen. This is reflected in a high yield for singlet oxygen generation as shown by measurements of singlet oxygen phosphorescence. The high singlet oxygen yield for TMR−Se (0.87) suggests its use as an efficient photosensitizer for the generation of singlet oxygen in biology and chemistry applications.

Photooxidation of 4-chlorophenol sensitised by iron meso-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrin in aqueous solution.

The photosensitised degradation of 4-chlorophenol (4-CP) by iron meso-tetrakis(2,6-dichloro-3-sulfophenyl)porphyrin (FeTDCPPS) has been studied in aerated aqueous solution, and is shown to lead to formation of p-benzoquinone (BQ) and p-hydroquinone (HQ) as main photoproducts. In deaerated solution no p-benzoquinone was formed. The photolysis products were identified by high performance liquid chromatography (HPLC) and UV-visible spectroscopy. The photodegradation in aerated solution was also carried out in the presence of sodium azide (NaN(3)) as a singlet oxygen [(1)O(2)((1)[capital Delta](g))] quencher, and showed a significant decrease in the rate of photolysis, suggesting under these conditions, that Type II sensitisation is one of the dominant mechanisms of 4-CP degradation. Support for this comes from laser flash photolysis and time-resolved singlet oxygen phosphorescence measurements. However, these also show direct reaction between the excited porphyrin and 4-CP, indicating that there are two mechanisms involved in the chlorophenol photodegradation.

Luminescence Study of Singlet Oxygen Production by Meso-Tetraphenylporphine

The research in the field of the photodynamic therapy of cancer (PDT) is focused on a development of photosensitizers exhibiting high quantum yield of singlet oxygen production. Direct time-resolved spectroscopic observation of singlet oxygen phosphorescence can provide time constants of its population and depopulation as well as photosensitizer phosphorescence lifetime and relative quantum yields. In our contribution, a study of time and spectral resolved phosphorescence of singlet oxygen photosensitized by meso-tetraphenylporphine in acetone together with the photosensitizer phosphorescence is presented. Time constants of singlet oxygen population and depopulation were determined at wide range of photosensitizer concentrations. The time constant of singlet oxygen generation (0.28 +/- 0.01) micros is slightly shorter then the lifetime of photosensitizer's triplet state (0.32 +/- 0.01) micros. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (approximately 50 micros) decreases with increasing photosensitizer concentration. Therefore, the photosensitizer acts also as a quencher of oxygen singlet state, similarly to the effects observed in [A. A. Krasnovsky, P. Cheng, R. E. Blankenship, T. A. Moore, and D. Gust (1993). Photochem. Photobiol. 57, 324-330; H. Küpper, R. Dedic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj. 1572, 107-113]. Moreover, the increasing concentration of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.

Model studies on the photosensitized isomerization of bixin.

The photosensitized isomerization reaction of the natural cis carotenoid bixin (methyl hydrogen 9'-cis-6, 6'-diapocarotene-6, 6'-dioate) with rose bengal or methylene blue as the sensitizer in acetonitrile/methanol (1:1) solution was studied using UV-vis spectroscopy, high-performance liquid chromatography (HPLC), and time-resolved spectroscopic techniques, such as laser-flash photolysis and singlet oxygen phosphorescence detection. In both N(2)- and air-saturated solutions, the main product formed was all-trans-bixin. The observed isomerization rate constants, k(obs), decreased in the presence of air or with increase in the bixin concentration, suggesting the participation of the excited triplet state of bixin, (3)Bix, as precursor of the cis--> trans process. On the other hand, bixin solutions in the absence of sensitizer and/or light did not degrade, indicating that the ground state of bixin is stable to thermal isomerization at room temperature. Time-resolved spectroscopic experiments confirmed the formation of the excited triplet state of bixin and its deactivation by ground state bixin and molecular oxygen quenching processes. The primary isomerization products only degraded in the presence of air and under prolonged illumination conditions, probably due to the formation of oxidation products by reaction with singlet molecular oxygen. An energy-transfer mechanism was used to explain the observed results for the bixin transformations, and the consequences for food color are discussed.

Oxygen Diffusion in Bilayer Polymer Films

Experiments to quantify oxygen diffusion have been performed on polymer samples in which a film of poly(ethylene-co-norbornene) was cast onto a film of polystyrene which, in turn, was cast onto an oxygen-impermeable substrate. In the technique employed, the time evolution of oxygen transport through the film of poly(ethylene-co-norbornene) and into the polystyrene film was monitored using the phosphorescence of singlet oxygen as a spectroscopic probe. To analyze the data, it was necessary to solve Fick's second law of diffusion for both polymer films. Tractable analytical and numerical solutions were obtained for the problem. Moreover, the numerical solution is sufficiently general that it can be used to simulate oxygen concentration profiles in films consisting of more than two layers. Data obtained from the bilayer films yield a diffusion coefficient for oxygen in poly(ethylene-co-norbornene) that is consistent with diffusion coefficients independently obtained from monolayer films and bulk samples.

Oxygen Diffusion in Copolymers of Ethylene and Norbornene

Oxygen diffusion coefficients, D, have been measured as a function of temperature over the range 5−35 °C in films of poly(ethylene-co-norbornene). In the technique employed, the time evolution of oxygen sorption into the film was monitored using the phosphorescence of singlet oxygen as a spectroscopic probe. Many properties of these amorphous glassy materials depend significantly on the ethylene-to-norbornene ratio in the copolymer. For example, for samples in which the norbornene content in the copolymer was increased from 35 to 62%, the glass transition temperature of the material increased over the range 79−178 °C. Nevertheless, with these same changes in chemical composition, values of D(25 °C) varied only slightly over the range (2−6) × 10-8 cm2 s-1, and values of the apparent diffusion activation energy, Eact, varied slightly over the range 35−40 kJ mol-1. The values of D(25 °C) obtained correlate reasonably well with calculated values of the polymer free volume as well as with the average free volume cavity size determined for these samples using positron annihilation lifetime spectroscopy. These data are consistent with models of diffusion that are based on the dynamic redistribution of local voids of critical volume into which the diffusing molecule can move. This study thus provides the foundation for attempts to regulate oxygen transport in these technologically and industrially important materials.

Singlet Oxygen Images of Heterogeneous Samples: Examining the Effect of Singlet Oxygen Diffusion across the Interfacial Boundary in Phase-Separated Liquids and Polymers

Optical images of glassy, phase-separated polymer films have been generated using the 1270 nm phosphorescence of singlet molecular oxygen. Specifically, upon irradiation of immiscible blends of polystyrene and poly(ethylene-co-norbornene) that contain a singlet oxygen sensitizer, phase-separated droplets as small as ∼10 μm in diameter could be resolved using a microscope designed to detect singlet oxygen phosphorescence. This study demonstrates that it is possible to create singlet oxygen images of systems in which the sensitizer is not mobile (i.e., systems in which the effects of sensitizer bleaching cannot be rectified by diffusion of more dye into a given volume). The effect of singlet oxygen diffusion across the interfacial boundary between phase-separated domains of both liquid and polymer samples has also been examined. For singlet oxygen created in one phase, diffusion into a second phase in which the quantum yield of singlet oxygen phosphorescence is larger and oxygen is more soluble gives rise to a significant change in the intensity of the singlet oxygen signal at the interface. This effect can be pronounced when a long singlet oxygen lifetime facilitates singlet oxygen diffusion over large distances, but can be mitigated when interfacial tension yields a phase boundary with appreciable curvature. Boundary curvature in thin, phase-separated films of polystyrene/poly(ethylene-co-norbornene) is slight. Moreover, the singlet oxygen lifetime in these polymers is sufficiently short that, within its lifetime, singlet oxygen cannot diffuse over an appreciable distance. Under such conditions, singlet oxygen images of interfacial boundaries are sufficiently sharp as to make this optical technique useful for a range of fundamental studies.

Time and spectral resolved phosphorescence of singlet oxygen and pigments in photosystem II particles

Singlet oxygen generated via chlorophyll triplets in photosynthetic systems can destroy basic components of photosynthetic apparatus. In order to study this process a sensitive spectroscopic experimental set-up was built to detect both weak infrared (IR) chlorophyll phosphorescence (between 900 and 1000nm) and singlet oxygen phosphorescence (at 1270nm). The spectral and time resolution of the used monochromator and photon counting multiscaler are sufficient to provide two-dimensional matrix of temporally and spectrally resolved IR luminescence data. We have studied IR emission of photosystem II particles in D2O and H2O buffer including the effect of the sodium dodecyl sulphate (SDS) detergent. Both sub-microsecond lifetime of chlorophyll triplet states and microsecond lifetime of singlet oxygen were determined. The singlet oxygen lifetime in D2O (of 18μs) was approximately five times longer than that in H2O. Quantum efficiency of singlet oxygen generation in photosystem II particles was significantly lower than that in chlorophyll a in acetone, however, it could be substantially increased by addition of SDS.

Phosphorescence of singlet oxygen and meso-tetra(4-sulfonatophenyl)porphin: time and spectral resolved study

Highly sensitive spectroscopic experimental setup was built to detect infrared luminescence with both time and spectral resolution. In this contribution, phosphorescence of meso-tetra(4-sulphonatophenyl)porphin, which is used as a photosensitizer for photodynamic therapy of cancer, and emission of singlet oxygen were studied. Two-dimensional matrices of data (counts as a function of time and wavelength) were obtained. From these matrices, 1.7 μs lifetime of the photosensitizer triplet state and the same rise-time of the singlet oxygen emission were resolved. Further, 3.7 μs lifetime of singlet oxygen phosphorescence was determined.

Characterization of endoperoxide and hydroperoxide intermediates in the reaction of pyridoxine with singlet oxygen.

The photosensitized oxidation of vitamin B6, pyridoxine, is investigated by product and kinetic analysis. Singlet oxygen quenching rates, measured by time-resolved laser flash generation of singlet oxygen followed by monitoring singlet oxygen phosphorescence decay, confirm previous observations that pyridoxine is a moderate quencher. The quenching rate for 3-methoxypyridine is 100 times slower than that for 3-hydroxypyridine, indicating the hydroxy moiety is required for efficient quenching. The chemical quenching rate constant, kr, was estimated by comparison with a known singlet oxygen reaction. Results indicate that the chemical quenching rate of pyridoxine dominates the total quenching. The major reaction product in methanol was isolated and characterized by NMR and MS. The data are consistent with a solvent adduct of the substituted 2,5-pyridinedione. At low temperature, two semistable intermediates were characterized by NMR. The data are consistent with a hydroperoxide and endoperoxide. These intermediates suggest initial attack of singlet oxygen para to the hydroxy group followed by either proton transfer to form the hydroperoxide or addition of the peroxide to the imine to form the endoperoxide. In the presence of protic solvents, the solvent adducts to the imine and elimination of water yield the observed 2,5-pyridinedione product.

New Halogenated Phenylbacteriochlorins and Their Efficiency in Singlet-Oxygen Sensitization

Halogenated phenylbacteriochlorins are synthesized with high yields in a two-step procedure. They have strong absorbances in the red and are very stable to air and light at room temperature. Flash photolysis measurements show that the triplet states of these bacteriochlorins have 30 μs lifetimes in deaerated toluene, that are quenched with diffusion-controlled rate constants by molecular oxygen. Time-resolved photoacoustic measurements, with nanosecond and nanocalorie resolution, show that these bacteriochlorins sensitize the formation of singlet oxygen with nearly unity quantum yield. However, singlet-oxygen phosphorescence measurements indicate that physical quenching occurs before the singlet-oxygen molecules diffuse into solution, and nearly half of the sensitized singlet states are lost.

Quenching of singlet oxygen ( 1Δ g) by cobalt complexes with four nitrogen atoms in the equatorial plane

The quenching of singlet oxygen ( 1Δ g) by cobalt(III)- bis-1,2-benzosemiquinone-diiminato complexes (general structure LCo (III)(BQDI) 2ClO 4) has been studied in different solvents by measuring the singlet oxygen phosphorescence decay in time-resolved experiments. The axial ligand (Ph 3As, Ph 3Sb, N-methyl-imidazole, pyrrolidine) has practically no influence on the quenching; however, the chlorinated benzosemiquinone-diiminato complex has a markedly lower quenching rate constant. The solvent effects can be fully explained by the difference in viscosity, which supports the assumption that the quenching is diffusion controlled. The reactive radius of the encounter pair has been estimated to be 0.3 nm, shorter than the radius of the complex itself, which points to the fact that singlet oxygen must approach the central cobalt atom at the (partially) open axial position. The significance of these results regarding the quenching of singlet oxygen by vitamin B 12 derivatives is discussed.

Photophysical studies on melatonin and its receptor agonists.

Previous work has demonstrated that melatonin inhibits the growth of both dermal and uveal melanoma cells. Recent clinical trials have found that melatonin is an efficacious treatment for metastatic dermal melanoma. The goal of this study was to provide further insight into the oncostatic mechanism(s) of melatonin. The inhibition of the growth of uveal melanoma cells is dose-dependent (0.1-10 nM) within the range of endogenous melatonin concentrations (2 nM) found in the human aqueous humor. We know that this inhibition of growth is receptor-mediated, at least in part, because uveal melanoma cell growth was also blocked by the agonists of melatonin receptors. There are two known membrane receptors for melatonin (Mel(1a) and Mel(1b)) and one known nuclear receptor (Mel2). To determine if singlet oxygen production and/or quenching contributed to the growth inhibition of melatonin, we examined the photophysical properties of melatonin and its agonists. Using flash photolysis, we determined that melatonin and its membrane receptor agonist 6-chloromelatonin (Mel(1a-b), Lilly, Indianapolis, IN) produced very little singlet oxygen (psidelta = 0.073 and psidelta = 0.01, respectively). There was no detectable singlet oxygen phosphorescence at 1,270 nm for the nuclear receptor agonist CG-52608 (Mel2, Novartis, Basel, Switzerland). In contrast, the agonist of the Mel(1b) receptor, S-20098 (Servier, Paris, France), produced singlet oxygen with a quantum efficiency of psidelta = 0.34. Singlet oxygen was quenched by melatonin and 6-chloromelatonin at approximately the same rate (6.1 x 10(7) M(-1)s(-1) and 6.0 x 10(7) M(-1)s(-1) in CD3OD), while the rate of quenching for the nuclear receptor agonist CG-52608 and membrane receptor agonist S-20098 was less (2.2 x 10(7) M(-1)s(-1) and 1.5 x 10(7) M(-1) s(-1), respectively). It appears that the production of singlet oxygen by melatonin would not be sufficient to directly block the proliferation of melanoma cells, but may activate gene products that could contribute to the oncostatic effect.

Oxygen Diffusion in Glassy Polymer Films: Effects of Other Gases and Changes in Pressure

Sensitized singlet oxygen phosphorescence has been used to quantify oxygen diffusion in polystyrene and poly(methyl methacrylate) films. Prompted by unexplained results from previous studies (Macromolecules 1994, 27, 7041−7048; Can. J. Chem. 1995, 73, 1831−1840), the present experiments have been performed to better characterize the behavior of this photophysical probe of oxygen diffusion under a variety of conditions. Specifically, at total pressures less than 500 Torr, the effects of pressure changes and added nitrogen gas on the measured oxygen diffusion coefficient, singlet oxygen phosphorescence intensity, and rate constant for sensitizer quenching by oxygen have been examined. The results indicate that there is a “low” pressure domain in which the oxygen diffusion coefficient depends both on the oxygen pressure and on the pressure of an added copenetrant. Data obtained are discussed within the context of the dual-mode sorption theory.

6] Assay for singlet-oxygen generation by peroxidases using 1270-nm chemiluminescence

Publisher Summary Singlet oxygen ( l Δ g ) represents one possible product of systems composed of a peroxidase, hydrogen peroxide, and a halide. Both heme-containing peroxidases and nonheme vanadium bromoperoxidases can produce singlet oxygen. Peroxidases can also oxidize iodide anion. However, with hypoiodous acid, the reaction is endothermic and singlet oxygen is not produced. To distinguish singlet oxygen from a variety of other reactive products generated by peroxidases, including radicals and various oxidized halide species, a technique with extreme selectivity is required. The measurement of singlet oxygen phosphorescence at 1270 nm is one such technique. Even in complex biochemical systems, chemiluminescence near 1270 nm from other excited species is extremely uncommon. Singlet oxygen ( l Δ g ) is an electronically excited oxygen molecule with 23 kcal per mole more energy than ground-state oxygen. This energy can be released as a 1270-nm photon, but the transition, which requires an electron spin change, is not allowed by the rules of quantum mechanics. Thus, the radiative rate is low. Most deactivating collisions of singlet oxygen with water molecules generate heat via a nonradiative transition. The lifetime of singlet oxygen in water is only 3.1μsec. The modest singlet oxygen production rates found in peroxidase systems and the short singlet oxygen lifetime result in low steady-state singlet oxygen concentrations. The combination of a low steady-state concentration and of a low radiative rate causes the 1270-nm emission from peroxidase systems to be very weak. Thus, measuring the weak 1270-nm emission from singlet oxygen generated by peroxidases would appear to represent a significant technical challenge, but modern solid-state near-infrared detectors have made the measurement of singlet oxygen production by various biochemical systems straightforward.