QUENCHING OF SINGLET MOLECULAR OXYGEN BY PHTHALOCYANINES AND NAPHTHALOCYANINES

Using the direct measurement of the photosensitized luminescence of singlet molecular oxygen (1O2) the rate constants (kq) have been determined for 1O2 quenching by the monomeric molecules of the following phthalocyanines and naphthalocyanines in chloroform: tetra-(4-tert-butyl) phthalocyanine (I); octa-(3,6-butoxy) phthalocyanine (II), tetra-(6-tert-butyl)-2,3 naphthalocyanine (III), aluminium tetra-(1-tert-phenyl)-2,3 naphthalocyanine (IV), tri-(n-hexyl-siloxy) derivatives of silicon- (V), tin- (VI), aluminium- (VII) and gallium- (VIII) 2,3 naphthalocyanine. The following kq values were obtained (kq x 10(-8) M-1 s-1): 2.9 (I), 59 (II), 100 (III), 20 (IV), 3.9 (V), 53 (VI), 33 (VII), 110 (VIII). As most of the quenchers have the low-lying triplet levels, a contribution of the quenching mechanism based on the energy transfer from 1O2 to these levels has been analysed. A formula is proposed describing the relation between kq values caused by this mechanism, and photophysical constants of the quencher triplet state. This formula was applied to phthalocyanines, naphthalocyanines, beta-carotene and bacterochlorophyll a. The data suggest that the energy transfer can fully explain the activity of V and strongly contributes into the activities of II, III and VI-VIII. A charge transfer interaction might be an additional mechanism involved in 1O2 quenching by compounds studied. As some phthalocyanines and naphthalocyanines are strong physical quenchers of singlet oxygen they can be used as efficient inhibitors for photodestructive processes in photochemical systems.     

REACTION AND QUENCHING OF SINGLET MOLECULAR OXYGEN WITH ESTERS OF POLYUNSATURATED FATTY ACIDS

Abstract The rate constants for the reactive (k_R) and unreactive (k_Q) interaction of singlet molecular oxygen with three esters of polyunsaturated fatty acids (PUFA: cis-methyl oleate, MO; cis-methyl linoleate, MLA and cis-ethyl linolenate, ELN) are determined. The values of the ratio k_Q/k_R are 0.51, 0.26 and 0.20 for MO, MLA and ELN, respectively. This variation results principally from that of k_R because the values of k_Q are only slightly different (1.24 × 10⁴M^?1 s^?1 for MO and ～1.0 × 10⁴M^?1 s^?1 for MLA and ELN). It is shown that the rate constant k_Q characterizes mainly an interaction with the unreactive part of the molecule rather than with the double bonds (solventlike quenching). Contrary to the already reported case of 1,5-polyenes for which k_Q <<k_R, the present results and those obtained from a number of literature data show that for PUFA and their esters, neither k_R+ k_Q nor k_R are proportional to the total number of double bonds or of methylene groups adjacent to the double bonds. Instead, a linear correlation is observed by plotting k_Rvs the number of methylene groups adjacent to two double bonds. It is deduced that contrary to a common assumption, biallylic hydrogens have a reactivity higher than that of singly allylic hydrogens (reactivity ratio 1.19). The consequence of this result on the estimation of relative contributions of singlet oxygen and radical mechanisms to oxidation processes is discussed. Moreover, the whole of these results allows prediction of the values of k_R and k_Q for all unsaturated fatty acids (and their esters) of similar structure.     

CUTANEOUS PHOTOTOXICITY OF TETRACYCLINE ANTIBIOTICS: GENERATION OF FREE RADICALS and SINGLET OXYGEN DURING PHOTOLYSIS AS MEASURED BY SPIN-TRAPPING and THE PHOSPHORESCENCE OF SINGLET MOLECULAR OXYGEN

Chlortetracycline (CTC) generated an aryl radical in aqueous buffer (pH 7.4) during near UV irradiation, as evidenced by the formation of 2-methyl-2-nitrosopropane spin adducts. The radical was produced via dechlorination, a photoprocess not previously reported for tetracyclines. Demeclocyc-line (DEM), another chlorinated tetracycline, did not produce detectable aryl radicals. Relative ¹O₂ yields obtained by direct luminescence measurements at 1268 nm for five tetracyclines in alkaline ethanol (demeclocycline · tetracycline · chlortetracycline · doxycycline · minocycline) showed that DEM produced approximately three times as much singlet oxygen as CTC. This constitutes direct evidence that tetracyclines sensitize both Type I and Type II photoreactions.     

FORMATION OF SINGLET MOLECULAR OXYGEN BY 8-METHOXYPSORALEN*

Abstract— The formation of singlet molecular oxygen (^lO₂) by energy transfer from the excited 8-meth-oxypsoralen (8-MOP) molecule was investigated. This was done in several ways: (a) In the reaction of irradiated 8-MOP with the ¹O₂ acceptor 2-methyl-2-pentene, the characteristic oxidation products were identified. (b) The rate of the 8-MOP sensitized photooxidation of 3, 4-dihydroxy phenylalanine (dopa), which appeared to be also a useful ¹O₂ acceptor, was larger in D₂O than in H₂O. (c) The β-values for reaction of ¹O₂with dopa in the presence of 8-MOP or of methylene blue as ¹O₂ generators were in accordance with each other. The consequences of ¹O₂ formation by 8-MOP sensitization is discussed for the clinical use of this compound.     

PHOTOBLEACHING OF MEROCYANINE 540: INVOLVEMENT OF SINGLET MOLECULAR OXYGEN

The purpose of this study was to assess the mechanism of merocyanine 540 (MC540) photobleaching in a liposomal system. Broad based visible irradiation of MC540 in unilamellar dilauroylphosphatidylcholine (DLPC) vesicles resulted in dye bleaching that was strictly O2 dependent. The rate of self-sensitized photobleaching was enhanced in D2O and inhibited by both azide and histidine, consistent with 1O2 intermediacy (Type II chemistry). Supportive evidence for this mechanism was obtained by using a Type II sensitizer, aluminum phthalocyanine tetrasulfonate (AlPcS lambda max = 678 nm). Irradiation of AlPcS and MC540 in DLPC with lambda greater than 630 nm (absorbed only by AlPcS) light resulted in rapid bleaching of MC540, which was stimulated by D2O and inhibited by azide. A rate constant of 10(7) M-1 s-1 was determined for the chemical quenching of 1O2 by MC540. The rate constant for physical quenching of 1O2 by MC540 was estimated to be ca 10(9) M-1 s-1.     

PRODUCTS AND RELATIVE REACTION RATES OF THE OXIDATION OF TOCOPHEROLS WITH SINGLET MOLECULAR OXYGEN

Abstract— The relative reactivity of singlet molecular oxygen, 0₂(¹Δ_g), α-,β-,Γ-and δ with -tocopherol (vitamin E) was investigated using microwave discharge generation as a uniquely clean source of singlet oxygen and using a hydrocarbon solvent to approximate the membrane environment. The relative efficiencies of the tocopherols for O₂(¹Δg) were found to decrease in the order: D-α-tocopherol > D-β-tocopheroI > D–Γ-tocopherol > D-δ-tocopherol. The reaction products in all cases were found to be mixtures of quinone and quinone epoxides apparently resulting from decomposition of the primary product, the hydroperoxydienone.     

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.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

DEACTIVATION OF SINGLET MOLECULAR OXYGEN BY THIOLS AND RELATED COMPOUNDS, POSSIBLE PROTECTORS AGAINST SKIN PHOTOSENSITIVITY

Abstract— From time-resolved measurements of the decay of singlet molecular oxygen phosphorescence at 1270 nm in D₂O, direct estimates have been gained for the rate constants of the singlet oxygen reactions with a group of sulphur compounds in the pD range 5 to 13. In the case of most of the thiols, the results are consistent with singlet oxygen reacting exclusively with the thiolate anions. At the normal physiological pH 7, the apparent rate constants (in units of M^-1 s^-1) were 8.9 times 10⁶ (cysteine), 2.5 times 10⁶ (N-acetyl cysteine), 2.9 times 10⁶ (glutathione), 3.0 times 10⁵ (2-mercaptoethanol), 2.3 times 10⁷ (ergothioneine) and 2.7 times 10⁶ (2-mercaptopropionyl glycine). For methionine the rate constant, 1.4 times 10⁷, was independent of pD in the range studied. These sulphur compounds, in particular N-acetyl cysteine and ergothioneine, or related compounds, might be considered as possible candidates for protection against skin photosensitivity side effects associated with the photodynamic therapy of solid tumours and as observed in the disease erythropoietic protoporphyria.     

SINGLET OXYGEN GENERATION OF PORPHYRINS, CHLORINS, AND PHTHALOCYANINES

The production of singlet oxygen was measured indirectly for three classes of photosensitizers: porphyrins (Photofrin II, TPPS4), chlorins (MACE, DACE), and a phthalocyanine (CASPc). Buffered solutions of sensitizers and singlet oxygen acceptors were irradiated with a CW dye laser and the oxygen depletion was monitored electrochemically with a Clark-type microelectrode. A comparison of oxygen-depletion rate constants and quantum efficiencies yields the order of efficiency of the sensitizers: TPPS4 greater than MACE greater than PII greater than DACE greater than CASPc. For singlet oxygen acceptors the order was: furfuryl alcohol greater than imidazole greater than tryptophan. CHO cell suspensions were also used as acceptors. Here the order of efficiency (per absorbed photon) was PII greater than MACE approximately CASPc. Expressed in terms of oxygen depletion per cell the order was CASPc approximately PII greater than MACE. When performing cell clonogenicity studies the order of efficiencies, expressed as percentage cell kill per unit weight of sensitizer, was CASPc greater than PII greater than MACE approximately DACE. The discrepancy between the efficiencies of sensitizers to generate singlet oxygen and their cytotoxicity was explained in terms of photodegradation (for the chlorins), intracellular localization (for PII), and contributions from a Type I mechanism (for CASPc).     

1H-INDENYLFURAN AND THIOPHENE DERIVATIVES– A NEW CLASS OF SINGLET OXYGEN SENSITIZERS

Abstract— Photophysical and photochemical properties of 1H-indenylfuran and thiophene derivatives were studied. UV spectra showed that all the compounds tested absorbed UV light in the region 350–380 nm. The fluorescence spectra of 4a-e showed bands in the region 410–470 nm and quantum yields (φ_F) in the range 0.25–0.88. Attempts to calculate the triplet energy of 4a-e failed except for 4c and 4d , which showed E_T= 43–44 kcal mol^-1. These compounds are a new class of singlet oxygen sensitizers. The sensitized reaction of 2,5-dimethylfuran with singlet oxygen was followed showing that 4a and 4e are more reactive than α-terthiophene (4.0 and 2.5 times respectively), while the other compounds show the same reactivity as 1 . This behavior can be explained considering the different intersystem crossing quantum yields of compounds 4a-e . Diazabicyclo[2. 2. 2]octane is a quencher of singlet oxygen in this reaction, while we can exclude superoxide ion formation using the photooxidation of α, α-dimethylstilbene.     

EFFECT OF SINGLET OXYGEN QUENCHERS ON OXIDATIVE DAMAGE TO LIPOSOMES INITIATED BY PHOTOSENSITIZATION OR BY RADIOFREQUENCY DISCHARGE

Abstract —Lipid model membrane systems, liposomes, may be oxidized by both toluidine blue Osensitized photo-oxidation and radiofrequency-discharge-generated singiet oxygen. Oxidation can be followed by the appearance of lipid peroxides, malondialdehyde formation, and ultimately by the lysis of the liposomes. Inhibition of oxidation is observed in both systems using either β-carotene or DABCO. These observations are consistent with the view that type II (singlet oxygen) reactions are involved in this photodynamic system.     

REACTION OF RETINOL AND RETINAL WITH SINGLET OXYGEN

Abstract— The rate constants for the reactions of all- trans retinol and retinal with singlet oxygen were measured in a variety of solvents of different polarities. The rate constants increased with increasing solvent dielectric constant, which suggests that a charge transfer mechanism plays a part in the reaction. Further, the rate constant of reaction of singlet oxygen with retinal is greater than that with retinol. Since retinal has a lower ionization potential than retinol, these relative rates also support the hypothesis of charge transfer involvement in the reaction.     

9-CIS, 11-CIS-RETINAL* FROM DIRECT IRRADIATION OF ALL-TRANS-RETINAL. NEW GEOMETRIC ISOMERS OF VITAMIN A AND CAROTENOIDS 9

Abstract The irradiation mixture of all-trans-retinal in acetonitrile was shown to contain four cis,cis isomers as well as the four cis isomers reported earlier. The relatively new 9-cis, 11-cis-retinal was isolated and characterized.     

PHOTOCHEMISTRY OF cis-POLYISOPRENE AND ITS SINGLET OXYGEN ADDUCT

Abstract— Reaction of singlet oxygen (¹Δ_g, ¹O₂) with cis -polyisoprene yields an allylic hydroperoxide with an olefinic double bond shifted in the polymer chain. The photochemical decomposition of the resultant hydro-peroxide and the subsequent polymer chain scission kinetics have been studied in the absence of oxygen. Quantum yields of hydroperoxide decomposition range from 3.1 to 8.4 in cyclohexane, depending on the initial amount of hydroperoxide in the polymer. On the other hand, the quantum yields for polymer chain scission are low, and vary with the frequency of the incident light. The ratio for number of polymer scissions per number of hydroperoxy groups decomposed is of the order of 10^-2. The polymer chain degradation is sensitized by the addition of ketones. Based on these data, a reaction mechanism for the overall photodegradation of the cis -polyisoprene initiated by singlet oxygen is proposed.     

NEW POLYMER SYNTHESES. XIII. SYNTHESIS AND CHARACTERIZATION OF NOVEL POLYKETONES VIA FRIEDEL-CRAFTS REACTION AND BASED ON DIARYLIDENE DERIVATIVES

ABSTRACT

An interesting new class of polyketones based on diarylidene derivatives were synthesized by Friedel-Crafts reaction of 2,7-dibenzylidenecycloheptanone (I) and dibenzylideneacetone (II) monomers, with different diacid chlorids including: isophthalic, terephthalic, 3,3′-azodibenzoic, 4,4′-azodibenzoic, biphenic, adipic, sebacic and oxalic diacids in dry dichloromethane solvent and AlCl₃ catalyst. The model compounds were synthesized by reacting I and II with benzoyl chloride and characterized by ¹H-NMR, IR and elemental analyses. The polyketones were insoluble in most organic solvents but dissolved easily in concentrated sulfuric acid. The thermal properties of these polymers were evaluated and correlated to their structural units by TGA, DTG measurements and had inherent viscosity up to 0.36–0.84 dI/g. The crystallinity of some polymers were tested by X-ray analysis. The electronic spectra of selected examples were measured in DMSO solution and the morphological properties were detected by SEM.     

REACTIVITY OF SINGLET OXYGEN TOWARD AMINO ACIDS AND PEPTIDES

Quenching of singlet oxygen (¹O₂) in D₂O-ethanol by the amino acids tryptophan, tyrosine, histidine, methionine, cysteine and their derivatives was measured by exciting the sensitizers rose bengal or meso-tetra (N-methyl-4-pyridyl)porphyrin tetratosylate in the presence of oxygen and the above quenchers in solution. In our polar solvent, containing 75% D₂O on a molar basis it was found that (1) substitution of the aromatic ring in indole, phenol and imidazole by the electron-donating methyl group increases the total (i.e. nonreactive and reactive) quenching rate constant by a factor of five to eight. Free or blocked amino and carboxyl groups removed by two methylene groups from the ring counteract the above increase in the rate constant. The reactive quenching of singlet oxygen, which leads to oxidative destruction of the aromatic ring, correlates with the above substitution effects. It has been proposed that the quenching process takes place by formation of an exciplex between ¹O₂ and the quencher. Thus our results indicate that the better an electron donor the amino acid residue is the more pronounced is the charge transfer contribution in the exciplex formed with ¹O₂ and the more likely it is to lead to charge separation and hence to a chemical reaction. (2) Oligopeptides in solution or peptide bonds linked to the amino acid residue have only a minor effect on singlet oxygen. It can therefore be expected that the polypeptide chains per se in the protein network will not interact significantly with the single oxygen molecules present. The quenching of the latter should, to a first approximation, depend only on the presence of the above reactive amino acid residues and to their accessibility to ¹O₂ as well as on the effective dielectric constant within the protein structure.     

PHOTOOXYGENOLYSIS OF VITAMIN B12 and RELATED CORRINS: COBALT(III)CORRINS AS SUBSTRATES and QUENCHERS FOR SINGLET OXYGEN (1Δg)*

Under photooxygenolytic conditions, vitamin B₁₂ can be degraded to two isomeric dioxosec-ocorrins by a regioselective oxygenolytic cleavage of the corrin macrocycle (preliminary report in Krautler and Stepanek (1985), Angew. Chem. 97 ,71–73; Angew. Chem. Intl. Ed. Engl. 24 ,62–64): irradiation of an oxygen saturated solution of vitamin B₁₂ ( 1 ), of KCN and of methylene blue (molar ratio (1:1:0.005) in CD₃OD at ca 200 K with visible light led to a selective oxygenolysis of the vitamin. The two products, potassium Co_αCo_β-dicyano-5,6-dioxo-5,6-seco-5′6′-dimethylbenzimidazolyl-cobam-idate ( 3 , 10% yield) and potassium Co_αCo_β-dicyano-14,15-dioxo-14,15-seco-5′6′-dimetliylbenzirnida-zolyl-cobamidate (4, 25% yield), and 1 (31% yield) were chromatographically separated and isolated. The structures were established spectroanalytically, and by the help of an acid catalyzed methanolysis of 3 and 4 to the related Co_αCo_β -dicyano-heptamethyl- dioxosecocobyrinates (6 and 7). When irradiated with visible light in any oxygen saturated methanolic solution containing methylene blue, vitamin B₁₂ itself exhibited considerable inertness. Only upon addition of a stoichiometric amount of KCN to such a solution (to afford the adduct 2) did the photolysis lead to oxygenation of the cobaltcorrin within a useful time. The regioselectivity of the oxygenolysis of 2 and the ratio of products formed are comparable to the outcome of the photooxygenolysis of the structurally related heptamethylcobyrinate 5 (Kräutler (1982) Helv. Chim. Acta 65 ,1941–1948). The reaction at the methyl-substituted meso-positions of the corrin macrocycle is indicated to involve singlet oxygen (¹Δ_g). In support of this interpretation, the preparative photolysis of 2 at 200 K also turned out to be about 7 times slower when CH₃OH was used instead of CD₃OD.     

SINGLET OXYGEN INDUCES FRANK STRAND BREAKS AS WELL AS ALKALI- AND PIPERIDINE-LABILE SITES IN SUPERCOILED PLASMID DNA

A covalently closed, circular, supercoiled plasmid was exposed to singlet oxygen by a separated-surface sensitizer. For each exposure, the quantity of single oxygen entering the DNA target solution was estimated by its oxidation of histidine. After singlet oxygen exposure, some DNA samples were treated to disclose occult lesions. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed circular and linear species, and all bands were quantitated fluorometrically. Exposure of supercoiled plasmid DNA to singlet oxygen induced frank DNA strand breaks, alkali-labile sites (pH 12.5, 90 degrees C, 30 min), and piperidine-labile sites (0.4 M, 60 degrees C, 30 min), all in a dose-dependent manner. Yields of alkali-labile and piperidine-labile sites ranged from one to four times the frank strand break yield. Replacement of buffered H2O by buffered D2O as the DNA solvent for singlet oxygen exposures increased DNA lesion yields by a factor of 2.6 (averaged over lesion classes). Our data for the detection of frank strand breaks is at variance with published results from studies in which singlet oxygen was derived from a thermolabile endoperoxide dissolved in the DNA solution.