SINGLET OXYGEN GENERATION BY PORPHYRINS AND THE KINETICS OF 9,10-DIMETHYLANTHRACENE PHOTOSENSITIZATION IN LIPOSOMES

Abstract— Two new sensitizers are introduced for a potential use in photodynamic therapy: Zn²⁺- and MG²⁺-tetrabenzoporphyrin (ZnTBP and MgTBP). A comparative study of the quantum yields of singlet oxygen generation (Φ_Δ) of hematoporphyrin derivative (HpD), Photofrin II (PF-II), Zn²⁺-phthalocyanine tetrahydroxyl [ZnPC(OH)₄] and the newly introduced sensitizers ZnTBP and MgTBP in liposomes, as well as the kinetics of a photochemical reaction sensitized by them, was made by employing the fluorescent membrane probe 9,10-dimethylanthracene (DMA). We followed the photosensitization of DMA in real time by monitoring its fluorescence decrease at 457 nm and found that DMA's photosensitization is oxygen mediated. The kinetic traces of the photosensitization reactions were fitted to an analytical function, and the Φ_Δ values were evaluated. At 10 μ M sensitizer in an aqueous suspension of 2 mg/mL egg phosphatidylcholine (EPC), HpD was found to have the largest value of Φ_Δ (0.215), followed by PF-II (0.191), ZnTBP (0.023), MgTBP (0.019) and ZnPC(OH)₄ (0.005). As a test of the method, Φ_Δ for methylene blue in ethanol was measured and found to be 0.45 as compared to 0.52 reported in the literature. Due to difference in the sensitizers' absorbances at the laser's wavelength, the reaction photosensitized by ZnTBP was the fastest with a time constant of 6.7 min, followed by MgTBP (8.7), PF-II (11.9), HpD (17.1) and ZnPC(OH)₄ (31.2), all at equal sensitizers' concentrations and laser intensities. The binding constants of the sensitizers to EPC liposomes are also reported.     

DETERMINATION OF THE KINETICS OF THE BACK REACTION OF PHOTOSYSTEM II IN THE PRESENCE OF 3–(3,4-DICHLORPHENYL)-1,1-DIMETHYLUREA FROM LUMINESCENCE MEASUREMENTS

Abstract— An alternative method to that used by Mar and Roy (1974) for the determination of the kinetics of the back reaction of photosystem II from the luminescence decay curve in the presence of 3–(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) has been suggested. The new theory relies upon two hypotheses: the well-known recombination hypothesis of luminescence and the assumption that the luminescence yield in the seconds region is given by the variable part of the live fluorescence yield. The second hypothesis was introduced since assuming a constant luminescence yield results in kinetic data that are not consistent with measurements of the kinetics of the back reaction by the restoration of the area over the fluorescence rise curve. The dependence of the live fluorescence yield from the concentration of closed PS II traps was assumed to be represented by Delosme's expression originally derived for the rise curve of the fluorescence yield in the presence of DCMU.
The theory is based on the fact that then the partial and total light sums of luminescence are simple functions of the concentration of the primary electron acceptor Q^- of PS II. Thus, after integrating the luminescence decay curve the theory permits a convenient evaluation of the kinetics of the back reaction [Q^-]( t ) in terms of the partial and total light sum.
This method was applied in order to determine the kinetics of the back reaction in Chlorella fusca in the presence of DCMU. It is shown that the kinetics of deactivation of the S, state can be described using the expression for the kinetics of the back reaction derived by Mar and Roy. As an alternative explanation, a biphasic first order decay of S₂ is proposed.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

Photosensitizing properties of triplet beta-lapachones in acetonitrile solution

The photochemical reactivity of β-lapachone ( 1 ), nor -β-lapachone ( 2 ) and 1,2-naphthoquinone ( 3 ) towards amino acids and nucleobases or nucleosides has been examined employing the nanosecond laser flash photolysis technique. Excitation (λ = 355 nm) of degassed solutions of 1 – 3 , in acetonitrile, resulted in the formation of their corresponding triplet excited states. These transients were efficiently quenched by l -tryptophan, l -tryptophan methyl ester, l -tyrosine, l -tyrosine methyl ester and l -cysteine ( k _q  ≈ 10⁹ L mol⁻¹ s⁻¹). For l -tryptophan, l -tyrosine and their methyl esters new transients were formed in the quenching process, which were assigned to the corresponding radical pair resulting from an initial electron transfer from the amino acids or their esters to the excited quinone, followed by a fast proton transfer. No measurable quenching rate constants could be observed in the presence of thymine and thymidine. On the other hand, efficient rate constants were obtained when 1 – 3 were quenched by 2'-deoxyguanosine ( k _q  ≈ 10⁹ L mol⁻¹ s⁻¹). The quantum efficiency of singlet oxygen (¹O₂) formation from 1 to 3 was determined employing time-resolved near-IR emission studies upon laser excitation and showed considerably high values in all cases (Φ_Δ = 0.6), which are fully in accord with the ππ* character of these triplets in acetonitrile.     

Alternation in Photosynthetic Characteristics of Anabaena doliolum Following Exposure to UVB and Pb

Abstract— Anabaena doliolum , when exposed to either ultraviolet-B (UVB) radiation or Pb, showed reduced growth rate, carbon fixation, O₂-evolution, photosynthetic electron transport activity and ATP pool size. The rate of respiration was found to increase in UVB-treated cells; this increase was more pronounced in the cells exposed to UVB and Pb simultaneously. The UVB-induced inhibition of 2,6-dichlorophenol indophenol (DCPIP) photoreduction and lowering of chlorophyll a fluorescence could not be reversed by artificial electron donors (diphenyl carbazide, NH₂OH and MnCl₂). These electron donors, however, substantially reversed the inhibition caused by Pb, thereby suggesting that UVB primarily inhibits the photosys-tem II (PS II) reaction center and Pb arrests the electron flow at the water splitting site. Nevertheless, the suppressed fluorescence intensity and the reduced emission and excitation peaks of phycobilisomes indicate the involvement of Pb in inhibition of PS II. All combinations of UVB and Pb inhibited the different metabolic processes in a synergistic manner.     

EXCITED STATES OF ANTHOCYANINS: THE CHALCONE ISOMERS OF MALVIDIN 3,5-DIGLUCOSIDE

Abstract The excited state properties of the chalcone isomers of malvidin 3,5-diglucoside (malvin) in acidic aqueous solution (0 < pH < 4) were investigated using steady-state and time-resolved fluorescence spectroscopy. The two chalcone isomers of malvin were first isolated by high-performance liquid chromatography and then characterized by UV/visible absorption and fluorescence spectroscopy. The results were supported by molecular orbital calculations. The rate constants for photodeprotonation ( k ₁= 1 × 10⁹ s^−l) and protonation ( k ₋₁= 1.3 × 10¹⁰ L mol⁻¹ s^−l) were determined, both from the multiexponential fluorescence decays and the fluorescence intensities measured at the emission wavelengths of the neutral and ionized chalcone forms. The results here obtained are relevant for the understanding of the photoreactivity of anthocyanins in acidic medium.     

LASER PHOTOLYSIS OF 3-METHYLLUMIFLAVIN

Abstract— Using high-intensity actinic light, the chlorophyll a fluorescence transient from HCO^-₃-depleted chloroplasts shows a rapid initial rise (O → I) followed by a slow phase (I → P). In the presence of HCO^-₃, the O → I rise is delayed but the I → P phase is much more rapid. Using low-intensity actinic light, the chlorophyll a fluorescence transient from 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU)-treated chloroplasts is delayed in the presence of HCO^-₃. Bicarbonate increases the amount of delayed light emission from chloroplasts given 10 s illumination with weak blue light (0·4 W/m²). DCMU greatly increases the amount of delayed light seen in the presence of HCO^-₃ under these conditions but decreases the amount seen in the absence of HCO^-₃. It is suggested that HCO^-₃ may somehow form or stabilize, in the dark, a number of reaction centers corresponding to the S₁ state in the model of B. Forbush, B. Kok and M. McGloin ( Photochem. Photobiol. 14, 307–321, 1971).     

QUANTIFICATION OF THE SOLVENT EFFECTS ON THE TRIPLET QUANTUM YIELD OF PSORALEN BY THE "LINEAR SOLVATION ENERGY RELATIONSHIP"

Triplet formation quantum yields (Φ_τ) of psoralen in a set of 17 pure solvents ranging from n -hexane to water and in dioxane: water mixtures were obtained by nanosecond laser flash photolysis. The triplet yield increases with solvent polarity. The extremum values are 0.009 and 0.545 in n -hexane and water, respectively. Good correlations of the experimental Φ_τ values with empirical "polarity" scales (Dimroth/Reichardt's E_T [30], Kamlet/Taft's solva-tochromic parameters β, and α, and Swains acity/basity A_S/B_S) were obtained: Ln(φ_T^-1 - 1) = 8.86 - 0.143E_T(30) Ln(φ_T^-1 - 1) = 4.40 - 2.34_τ - 1.70α Ln(φ_T^-1 - 1) = 4.65 - 3.72A_s - 1.12B_s The results are discussed in terms of the sensitivity of psoralen triplet quantum yield to solvent polarity and hydrogen-bonding abilities.     

CHEMICAL CHANGES LEADING TO CHEMILUMINESCENCE OF LUCIGENINE (DIMETHYLBIACRIDYLIUM NITRATE) AND OF SOME PHTHALAZINEDIONE DERIVATIVES

Abstract— Measurements of the redox potential of the chemiluminescent compound 10,10' dimethyl-9,9' biacridylium nitrate (-0.093 V) show that it is thermodynamically possible to reduce it with hydrogen peroxide or with ammonium hydroxide in alkaline solutions at equilibrium concentrations sufficiently high to account for the observed chemiluminescence. Reduction of the compound with ammonium hydroxide takes place much more slowly than the corresponding reaction with hydrogen peroxide so that when both redox couples (O₂/H₂O₂ and N₂H₄/NH₄OH) are present the hydrogen peroxide couple predominates if oxygen is supplied. It was shown that interference with the oxygen supply or its partial removal with nitrogen brings about an increase in chemiluminescence intensity in NH₄OH while increasing the concentration of oxygen diminished the intensity.
5-amino 2,3 phthalazine 1,4 dione (luminol) also appears to undergo a reduction following a two step oxidation. This is shown by the fact that when oxygen was supplied the chemiluminescence intensity was found to be directly proportional to the OH^- concentration while a typical titration curve with p K 11.7 is exhibited by the intensity when the oxygen supply is limited in mixtures of luminol and peroxydisulfate. The peroxide presumably arises in the first oxidation step. Amino peroxyphthalic anhydride is suggested as an intermediate which is reduced to the aminophthalate ion, the presumed emitter in the chemiluminescence.     

Singlet Oxygen Generation by Metallotexaphyrins

Abstract. Metallotexaphyrins have clinical applications as photo-sensitizers of photodynamic therapy (PDT). The singlet oxygen quantum yield (φ>_Δ) was determined for a series of metallotexaphyrin derivatives (Lu [III], Y [III], Cd [II], In [III] and Gd [in]) under conditions where the agents are believed to exist in monomeric form. The results show φ_Δ of metallotexaphyrins vary with the medium and the metal cation. Measurements on the Lu (III) texaphyrin led to φ_Δ= 0.38 in unbuffered 5% Tween 20 and φ_Δ= 0.58 in pH 7.4 phosphate buffer plus 1% Triton X-100 (±10%). The in vitro photodynamic efficiency calculated from φ_Δ is compared to in vivo PDT efficacy in an animal tumor model.     

PHOTOSYNTHETIC OXYGEN EVOLUTION UNDER VARYING REDOX CONDITIONS: NEW EXPERIMENTAL AND THEORETICAL RESULTS

Abstract. –Steady state and transient effects of two electron transport inhibitors (NH₂OH˙HCl and DCMU) and one uncoupler (FCCP) upon the emission of delayed fluorescence have been studied in whole cells of Chlorella pyrenoidosa. Observations were made on changes in the delayed fluorescence emitted 0.5 ms after a brief flash and 60 ms after the extinction of steady illumination and on changes in the induction effects caused by preillumination. Patterns in the time course of events after inhibitor addition occur which have different characteristics for each of the additives. The observations are consistent with a model of delayed fluorescence production based on the recombination of charges.     

RESONANCE FLUORESCENCE IN CHLOROPHYLL a SOLUTIONS

Abstract— Chlorophyll a in aqueous hexane solutions has two fluorescence peaks, two fluorescence excitation peaks in the blue, and two fluorescence excitation peaks in the red. One of these set of peaks (excitation 2.34_μ^-1 and 1.52_μ-1, fluorescence 1–50_μ^-1) is identical with those found in methanol solutions of chlorophyll a and is assigned to an unaggregated species, while the other set of peaks (excitation 2.44_μ-1 and 1.475_μ^-l, fluorescence 1.48_μ^-l) is assigned to a species aggregated in the presence of water. The spectral shifts occurring in the aggregate follow the selection rules obtained by McRae and Kasha for a linear stack of parallel dye molecules. The sharply increased overlap of absorption and fluorescence leads to resonance fluorescence in the aggregate. The resonance fluorescence is demonstrated experimentally.     

COPPER QUENCHING OF THE VARIABLE FLUORESCENCE IN Dunaliella tertiolecta. NEW EVIDENCE FOR A COPPER INHIBITION EFFECT ON PSII PHOTOCHEMISTRY

Abstract— The copper quenching effect on fluorescence in Dunaliella tertiolecta was studied. 30% of variable fluorescence was quenched in the presence of 70 μ,M CuS0₄. We confirmed that the copper inhibitory effect on photosystem II (PSII) activity is located on its oxidizing side. Further, we indicate that the complementary area is decreased by copper. Since the quantum yield of PSII photochemistry was lowered and the rate of PSII primary electron acceptor Q_A remained unaffected, we can conclude that some PSII reaction centers were inactivated by copper.     

Algorithm for analysis of OJDIP fluorescence induction curves in terms of photo- and electrochemical events in photosystems of plant cells: derivation and application

The algorithm for simulation of the OJDIP fluorescence induction curve in chloroplasts under variable conditions is presented. It is derived from analyzes of chlorophyll a fluorescence kinetics upon excitation with single- (STF), twin- (TTF) and repetitive STF excitations, and from the rate equations that describe the sequence of transfer steps associated with the reduction of the primary quinone acceptor Q_A and the release of photochemical fluorescence quenching of photosystem II (PSII) in multi-turnover excitation (MTF). The fluorescence induction algorithm (FIA) considers a photochemical O–J–D, a photo-electrochemical J–I and an I–P component (phase) which probably is associated with a photo-electric interaction between PSI and PSII. The photochemical phase incorporates the kinetics associated with the double reduction of the acceptor pair [PheQ_A] in Q_B–nonreducing reaction centers (RCs) and the associated doubling of the variable fluorescence, in agreement with the three-state trapping model (TSTM) of PSII. Application of and results with the algorithm are illustrated for MTF-induced OJDIP curves, measured in dark-adapted, in STF pre-excited and in DCMU inhibited thylakoids.     

MEROCYANINE DYES: EFFECT OF STRUCTURAL MODIFICATIONS ON PHOTOPHYSICAL PROPERTIES AND BIOLOGICAL ACTIVITY

Abstract Merocyanine derivatives were prepared by structural alterations at the barbituric acid or chalcogenazole moieties. The photophysical properties of the dyes were markedly influenced by the presence of selenium rather than sulfur as a substituent at position 2 of the barbiturate. In methanol, quantum yields of both triplet state (φ_τ) and singlet oxygen sensitization (φ_Δ) were increased by over an order of magnitude, with a concomitant decrease in fluorescence, when selenium was present in the molecule. Photoisomerization, one of the dominant deactivation pathways in the sulfur- or oxygen-containing analogues, was completely absent in the selenium-containing derivatives. Efficient triplet state formation was observed for selenium-containing derivatives incorporated into L1210 cells by diffuse reflectance laser flash photolysis. Cytotoxicity studies, camed out using clonogenic assays on L1210 leukemia cells, showed a good correlation with φ_τ and φ_Δ, measured in solution. Experimental evidence provided by this paper supports a triplet state-, and probably singlet oxygen-, mediated phototoxic mechanism. Photoisomerization or singlet state mechanisms can be discounted.     

BLEACHING OF PURPLE MEMBRANE WITH O-SUBSTITUTED HYDROXYLAMINES

Abstract The retinal Schiff base of bacteriorhodopsin, in the purple membrane from Halobacterium halobium , can be cleaved by hydroxylamine in the presence of light. We have further investigated this reaction with a series of O -substituted hydroxylamines, RONH₂, where R = -H (HA), -CH₃ (MHA), -SO₃− (HAS), benzyl- (BHA), p -nitrobenzyl- (NBHA), and pentafluorobenzyl- (FBHA). All except MHA caused light-induced bleaching of the purple membrane and the chromophore could be regenerated from apomembrane and all- trans retinal. Relative bleaching rate constants were obtained from V = QI _a k ₀ X /( k _r+ k ₀ X ), where V = bleaching rate, Q = quantum yield, I a = absorbed light intensity, X = hydroxylamine concentration, k ₀= rate constant for bleaching and k _r= rate constant for return of photoexcited bacteriorhodopsin to the initial state. This equation fits the time-, concentration- and intensity-dependences of the bleaching reactions in 0.02 M phosphate, pH 7.0. The rate constants k ₀ relative to HA were: MHA: 0; HAS: 0.3; HA: 1.0; BHA: 1.8; FBHA: 10.1; NBHA: 10.8. The relative rate constants do not correlate with the basicity of the derivatives. Instead, the results suggest that the retinal Schiff base is near a non-polar cavity into which an aromatic group can be inserted.     

Interaction of the excited singlet state of neutral red with aromatic amines

Quenching of neutral red (NR; neutral form of the dye) fluorescence by a number of aromatic amines has been investigated in acetonitrile solutions. The bimolecular quenching constants ( k _q) obtained from steady-state and time-resolved measurements for a particular donor–acceptor pair are seen to be the same within experimental error. Correlation of the changes in the k _q values with the oxidation potentials of the donors (amines) indicates that electron transfer (ET) is the mechanism operative in the present systems. Direct evidence for ET has been obtained from picosecond transient absorption studies on a suitable amine–NR pair. Experimentally determined k _q values are seen to correlate well with the free energy changes (Δ G ⁰) for the ET reactions, within the framework of the Marcus outer sphere ET theory. From the correlation between the experimentally determined and theoretically calculated k _q values, it appears that solvent reorganization plays a major role in governing ET dynamics in the systems investigated.     

UVA self-photosensitized oxygenation of beta-ionone

The steady-state UVA (350 nm) photolysis of ( E )-β-ionone ( 1 ) in aerated toluene solutions was studied by ¹H NMR spectroscopy. The formation of the 1,2,4-trioxane ( 2 ) and 5,8-endoperoxide ( 5 ) derivatives in the ratio of 4:1 was observed. Time-resolved laser induced experiments at 355 nm, such as laser-flash photolysis, photoacoustic and singlet oxygen ¹O₂ phosphorescence detection, confirmed the formation of the excited triplet state of 1 with a quantum yield Φ _T = 0.50 as the precursor for the generation of singlet oxygen ¹O₂ ( Φ _Δ = 0.16) and the isomeric α-pyran derivative ( 3 ), which was a reaction intermediate detected by NMR. In turn, the reaction of ¹O₂ with 1 and 3 occurred with rate constants of 1.0 × 10⁶ and 2.5 × 10⁸  m ⁻¹s⁻¹ to yield the oxygenated products 5 and 2 , respectively, indicating the relevance of the fixed s-cis configuration in the α-pyran ring in the concerted [2+4] cycloaddition of ¹O₂.     

SOLID PHASE PHOTOREDUCTION OF METHYLVIOLOGEN ON CELLULOSE SENSITIZED BY TRIS(2,2'-BIPYRIDYL)RUTHENIUM COMPLEX

–Methylviologen (MV²⁺) adsorbed on cellulose could be reduced photochemically in the solid phase sensitized by tris(2,2'-bipyridyl)ruthenium(II) complex, [Ru(II)(bpy)₃], using disodium ethylene-diaminetetraacetic acid (Na₂EDTA) as a reducing agent. Formation of the cation radical (MV ⁺.) was confirmed by visible and EPR spectra. The MV⁺. formed on cellulose was remarkably stable against air oxidation and rapidly accumulated even by the irradiation under air. Water adsorbed on the cellulose greatly retarded the photoreaction. Action spectrum showed that the excitation of Ru(II)(byp)₃ is responsible for the photochemical reaction. The quenching of the emission from Ru(II)(bpy)*₃ by MV²⁺. indicated that a primary photochemical reaction occurs between Ru(II)(bpy)*₃ and MV²⁺. The main reaction path is the reduction of MV²⁺ by Ru(II)(bpy)₃, giving MV⁺. and Ru(III)(bpy)₃, followed by the reduction of Ru(III)(bpy)₃ to Ru(II)(bpy)₃ with Na₂EDTA, which in turn is oxidized to give carbon dioxide.     

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.