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.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.     

PHOTOBINDING OF PSORALENS TO BACTERIAL MACROMOLECULES IN SITU AND INDUCTION OF GENETIC EFFECTS IN A BACTERIAL TEST SYSTEM. EFFECTS OF SINGLET OXYGEN DIAGNOSTIC AIDS D2O AND DABCO

The photobinding of radiolabeled psoralen and 8-methoxypsoralen (8-MOP) to biological macromolecules under conditions that affect the lifetime of singlet oxygen (¹O₂) is reported. These conditions are: increase of ¹O₂ lifetime in D₂O and ¹O₂ quenching with DABCO. The photobinding to calf thymus DNA was studied in vitro and the covalent photobinding to DNA and other biological macromolecules (RNA, proteins) was also studied in intact bacteria. The results of the DNA photobinding experiments have been related to the induction of genetic damage in a bacterial test system. In addition, laser flash photolysis has been used to measure the effect of D₂O and DABCO on the psoralen and 8-MOP triplet lifetimes. In general D₂O increases the triplet lifetimes and DABCO quenches the triplet states with the probable formation of radicals. The results suggest that the covalent photobinding of 8-MOP to various biological macromolecules in situ is a basis for cell damage occurring at various cellular targets. Analysis of the results of the mutagenicity test suggests that in the presence of D₂O the mechanism of induction of genetic lesions is not changed and therefore largely seems to be independent of singlet oxygen.     

PHOTODYNAMIC INACTIVATION OF E. COLI BY ROSE BENGAL IMMOBILIZED ON POLYSTYRENE BEADS

Abstract. The photodynamic inactivation of E. coli by visible light and O₂ was found to occur in the presence of the sensitizer rose bengal, immobilized by covalent bonding to polystyrene beads. The demonstrated absence of significant amounts of dissolved rose bengal indicated that an inactivation mechanism based on penetration of sensitizer molecules into the cell's interior could not be operating. Survival curves typically exhibited induction periods followed by rapid exponential death, with 99.99% kill requiring 1–2 h depending on conditions. A mechanism involving the participation of photo-generated singlet excited oxygen O₂(¹δ) in inactivation of E. coli is proposed. The photodynamic inactivation rate increased significantly in H₂O compared with H₂O, which is evidence supporting singlet oxygen as an active intermediate, since O₂(¹δ) has a much longer lifetime in H₂O than in H₂O. H₂O did not act as a short term poison in the absence of sensitizer.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

PHOTOINDUCED LUMINESCENCE AND EPR SIGNALS OF POLYPHENOL AND QUINONE POLYMERS

Abstract— Aqueous basic solutions, pH 9.0 of humic acids and melanin-like, synthetic polymers, obtained with adrenochrome, hydroquinone and purpurogallin, were illuminated with visible light under N₂ or O₂ atmospheres. It has been found that light enhances a singlet electron-paramagnetic-resonance (EPR) signal of polymers both under N₂ and O₂, and induces ultra-weak luminescence in the presence of O₂. Degradative oxidation of polymers, accelerated by light, leads to a decrease of EPR signal intensity and generates weak chemiluminescence.     

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.     

A PULSED LASER AND PULSE RADIOLYSIS STUDY OF AMPHIPHILIC CHLOROPHYLL DERIVATIVES WITH PDT ACTIVITY TOWARD MALIGNANT MELANOMA

Two amphiphilic derivatives of chlorophyll, which have high potential as photodynamic therapy sensitizers for malignant melanoma have been investigated by a combination of laser flash photolysis and pulse radiolysis. It is shown that direct excitation of monomeric forms of these molecules in both hydrophilic and hydrophobic environments produces significant yields of the corresponding triplet states, which have been characterized in terms of spectral and kinetic parameters. In both environments, scavenging of the triplets by oxygen produces singlet oxygen, O₂(^lΔ₈), with essentially unit efficiency as evidenced by time-resolved IR luminescence measurements.     

Photophysical Properties of Tin Ethyl Etiopurpurin I (SnET2) and Tin Octaethylbenzochlorin (SnOEBC) in Solution and Bound to Albumin

Abstract— The photophysical characteristics of two second-generation PDT photosensitizers, tin ethyl etiopurpurin I (SnET₂) and tin octaethylbenzochlorin (SnOEBC), have been measured in homogeneous solution and when bound to bovine serum albumin (BSA). The ground state and triplet state absorption spectra have been characterized, as have triplet lifetimes and quantum yields for intersys-tem crossing, singlet oxygen formation and photobleaching. In total, these parameters provide a complete set of data that can be used to quantitatively compare the photosensitizing efficiencies of these molecules. The photo-bleaching quantum yield of SnET₂ is increased dramatically when it is bound to BSA, thus limiting the production of singlet oxygen at incident fluences above 1 J/cm². In contrast, the quantum yield of photobleaching of SnOEBC is at least an order of magnitude lower than that of SnET₂ under these conditions and does not significantly limit the photosensitization process for typical in vivo or in vitro fluences. This difference is expected to play a significant role in determining the relative photosensitizing ability of these compounds in vivo.     

PHOTOSENSITIZED FORMATION OF ASCORBATE RADICALS BY RIBOFLAVIN: AN ESR STUDY

Abstract— The riboflavin-sensitized photooxidation of ascorbate ion (HA^-) to ascorbate radical (A^-) was followed by electron spin resonance (ESR) spectroscopy in conjunction with oxygen depletion measurements. In air-saturated aqueous media, steady-state amounts of A^- are rapidly established upon irradiation. The ESR signal disappears within a few seconds after the light is extinguished–more slowly under constant irradiation as oxygen is depleted. No photooxidation was observed in deaerated media. Similar results were obtained with other flavins and when ascorbyl palmitate was substituted for HA^-. The effect of added superoxide dismutase, catalase, desferrioxamine, and singlet oxygen scavengers (NaN₃ and tryptophan) was studied, as was replacement of water by D₂O and saturation with O₂. The results are indicative of ascorbate free radical production via direct reaction between ascorbate ion and triplet riboflavin in the presence of O₂. While the presence of superoxide ion tends to reduce the steady-state concentration of A^-, competition from the reaction of HA^- with singlet oxygen is less apparent in this system (at HA^-≥ 1 m M ) than in the previously studied aluminum phthalocyanine tetrasulfonate-photosensitized reaction.     

SEARCH FOR SINGLET OXYGEN LUMINESCENCE IN THE DISPROPORTIONATION OF HO2/O2

Abstract— During the reaction HO₂+ HO₂ (or O₂^-) = H₂O₂+ O₂ in aqueous solution, no luminescence in the region 620–720 nm, expected if the product O₂ were formed in a singlet state, could be detected. If any singlet O₂ is formed, its yield must be less than 10%. Faint luminescence, sometimes found at shorter wavelengths, was shown to arise from reaction of HO₂ with impurities in the reagents present.     

TRIPLET STATES OF CAROTENOIDS FROM PHOTOSYNTHETIC BACTERIA STUDIED BY NANOSECOND ULTRAVIOLET AND ELECTRON PULSE IRRADIATION

Abstract— Absorptions of the triplet excited states of five carotenoids (15,15'-ds phytoene, all- trans phytoene, C-carotene, spheroidene and spirilloxanthin), extracted from the photosynthetic bacteria Rhodopseudomonas spheroides and Rhodospirillum rubrum, have been detected in solution using pulse radiolysis and laser flash photolysis. Triplet lifetimes, extinction coefficients, lowest energy levels and quantum efficiencies of formation have been determined. Comparison of the carotenoid triplet energy levels with that of O₂('Δ_g) suggests that spirilloxanthin, spheroidene and possibly alsoζ-carotene, would be expected to protect against photodynamic action caused by O₂ ('Δ_g), but not cis or trans phytoene. The S → T intersystem crossing efficiences of all five polyenes were found to be low, being a few per cent or less. In their protective role these triplet states can only therefore be effectively reached via energy transfer from another triplet, except in the case of O₂ ('Δ_g). The low crossover efficiencies also mean that light absorbed by such carotenoids in their possible role as accessory pigments would not be wasted in crossing over to the triplet state.     

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.     

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.     

ON THE MECHANISM OF QUENCHING OF SINGLET OXYGEN IN SOLUTION

Abstract— Bimolecular rate constants for the quenching of singlet oxygen O*₂(¹Δ_g), have been obtained for several transition-metal complexes and for β-carotene. Laser photolysis experiments of aerated solutions, in which triplet anthracene is produced and quenched by oxygen, yielding singlet oxygen which then sensitizes absorption due to triplet carotene, firmly establishes diffusion-controlled energy transfer from singlet oxygen as the quenching mechanism in the case of β-carotene. The efficient quenching of singlet oxygen by two trans-planar Schiff-base Ni(II) complexes, which have low-lying triplet ligand-field states, most probably also occurs as a result of electronic energy transfer, since an analogous Pd(II) complex and ferrocene, which both have lowest-lying triplet states at higher energies than the O*₂(¹Δ_g), state, quench much less effectively.     

RATE PARAMETERS FOR THE QUENCHING OF SINGLET OXYGEN BY WATER-SOLUBLE AND LIPID-SOLUBLE SUBSTRATES IN AQUEOUS AND MICELLAR SYSTEMS

Abstract— The laser flash photolysis method has been used to determine the bimolecular rate constants for the reaction between O₂(¹Δ₉) and several lipid-soluble and water-soluble substrates. Values for lipid-soluble substrates have been obtained using aqueous dispersions of surfactants above the critical micelle concentration with 1,3 diphenylisobenzofuran as monitor of singlet oxygen. Under these conditions the hydrophobic substances are solubilized by the micellar phase. For substrates which are water-soluble, 9,10-anthracene dipropionic acid disodium salt was used as singlet oxygen monitor. For several substances, the values obtained are comparable to the values found in homogeneous nonaqueous solutions. In cases where significant differences have been found these have been rationalized according to the individual case. The only major unexpected result concerned β-carotene which, in micellar dispersion, failed to react at all with O₂(¹Δ₉) This may be due to multi-molecular aggregations occurring in the polar medium. The work described herein shows clearly that, under appropriate conditions, singlet oxygen kinetics can be effectively followed in aqueous solutions by time resolved methods. The indiscriminate use of β-carotene as a quencher of O₂(¹Δ₉)in mainly aqueous media is questioned.     

ENHANCING EFFECT OF ASCORBATE ON TOLUIDINE BLUE-PHOTOSENSITIZATION OF YEAST CELLS

Abstract— Using toluidine blue, a potent photosensitizer with a ₁O₂ dominated mechanism in yeast cell inactivation, it was found that addition of ascorbate to the sensitizer-cell mixture during illumination enhanced the inactivation. The enhancement required the presence of oxygen in the reaction mixture. The same enhancement was observed with methylene blue and thionine but not with xanthenes (Rose Bengal and eosin Y). The consumption of O₂ and ascorbate seemed coupled in the enhancement. From the observation that the presence of ascorbate for a very short time (1 s) in the reaction mixture was enough to exhibit the same enhancement, it was concluded that the ascorbate enhancement processes are probably initiated in bulk medium, not intracellularly. The ascorbate enhancement may be a combined consequence of the high electron-accepting property of triplet toluidine blue and the strong tendency of ascorbate to act as an electron donor. The role of oxygen was not specified whether it was directly involved in the photoinactivation of cells. Addition of N J appeared to suppress the photoinacti-vation only in the higher fluence region where ascorbate had been consumed. Thus the ascorbate enhancement seems to occur under low fluence conditions and may probably be independent of the singlet oxygen mechanism.     

Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibiotics can induce skin photosensitivity in some patients and this has been ascribed to the generation of reactive oxygen species, such as singlet oxygen (O₂[¹Δ_g]). We have studied the photochemical properties of the different ionized forms of the fluoroquinolone norfloxacin upon complexation with Mg²+ and Ca²+ ions, as it is proposed that the antibiotic exists mainly as a complex in the blood plasma. We found that the norfloxacin cation (pH < 6) shows no photodegradation after UVA irradiation and has a low quantum yield of O₂(¹Δ_g) generation. The norfloxacin cation does not complex. Ca²+ or Mg²+ ions; when these ions are added to the solution, we observed no changes in the fluorescence quantum yields (φ_flu) and singlet oxygen yields (φ_Δ). In contrast, the neutral (6 ± pH > 8.5) and anionic (pH > 9) forms of norfloxacin are able to complex calcium and magnesium, and their generation of O₂ (¹Δ_g) is decreased by complexation. The neutral zwitterionic form and the anionic form also quench singlet oxygen by both chemical and physical pathways regardless of complex formation, while physical quenching is observed for the cation. At pH > 7.4, norfloxacin photobleaches and complexation to Ca²+ and Mg²+ increases the rate at which photobleaching occurs. Thus, both the pH of the medium and complexation with metal cations may affect the phototoxic potential of this antibiotic.     

A LASER FLASH KINETIC SPECTROPHOTOMETRIC EXAMINATION OF THE DYNAMICS OF SINGLET OXYGEN IN UNILAMMELLAR VESICLES

Abstract— The kinetic properties of O₂(₁Δ_g) have been examined in unilamellar vesicle dispersions composed of didodecyldimethylammonium bromide, di- n -octadecyl phosphate and egg lecithin. Light absorbing sensitizers 2-acetonaphthone, methylene blue and a methylene blue derivative of enhanced water-solubility were used. The rate parameters for singlet oxygen were monitored by observing the time profile of the bleaching of the reactive substrates diphenylisobenzofuran and anthracene dipropionate. The natural lifetime of O₂(₁Δ_g) in D₂O-based suspensions was shown to be 46/JS in good agreement with that found earlier for D₂O alone and D₂O-based micelle systems. The bimolecular rate constants for reaction with diphenylisobenzofuran and dimethylindole (both lipid-bound) and histidine (water-bound) were also in close conformity with the values found earlier in micellar media. Kinetic spectrophotometry has been shown to be a useful technique for examining rate parameters in these heterogeneous media.     

Oxygen Uptake in the Vitamin B2-sensitized Photo-oxidation of Tyrosine and Tryptophan in the Presence of Uracil: Kinetics and Mechanism

Considering the significance of visible light-promoted reactions in complex biological media, the photo-oxidation of the amino acids (AAs) tyrosine (tyr) and tryptophan (trp) was studied in the presence of the naturally occurring oxidative scavenger uracil (ur). The involved photoprocesses, studied at pH 7 and 9, are driven through the reactive oxygen species (ROS) singlet molecular oxygen (O₂(¹Δ_g)), superoxide radical anion (O₂^•−) and hydrogen peroxide (H₂O₂). The effect on the effectiveness of the overall photo-oxidation process due to the presence of an added electron-donating substrate such as ur is not straightforwardly predictable. The addition of the pyrimidine compound, a much lesser photo-oxidizable substrate than the AAs themselves, produced different results: (1) antioxidative for tyr at pH 9, decreasing the overall rate of oxygen uptake; (2) synergistic for tyr at pH 7, increasing the oxidation rate more than the corresponding addition value of the respective individual rates and (3) no effect for trp at both pH values. The final result depends on the respective abilities of the substrates as quenchers of both the long-lived riboflavin triplet excited state and the generated ROS and the pH of the medium. An interpretation for the different cases is attempted through a kinetic and mechanistic analysis.