Synthesis of a novel hypocrellin B derivative and its photogeneration of semiquinone anion radical

In order to improve the photosensitizing activity of HB further, the complex of 5,8-di-Br-HB with Al(3+) was first designed and synthesized in high yield. 5,8-di-Br-HB forms a 2:1 type (metal-ligand ratio) complex with Al(3+) measured by molar ratio and continuous variation methods. The new photosensitizer was characterized by UV-Vis, IR, 1H NMR and elemental analysis measurements. Based on the above experimental results, we first proposed a polymer-like structural model of the complex. The water-solubility and red absorption of the [Al(2)(5,8-di-Br-HB)Cl(4)](n) complex are both enhanced over hypocrellin B. In addition, the EPR and spectrophotometric measurements demonstrate that semiquinone anion radical of [Al(2)(5,8-di-Br-HB)Cl(4)](n) can be produced by [Al(2)(5,8-di-Br-HB)Cl(4)](n) photosensitization. The generation efficiency of ([Al(2)(5,8-di-Br-HB)Cl(4)](n))(.-) is almost equal to that of HB(.-). These results obtained indicated that [Al(2)(5,8-di-Br-HB)Cl(4)](n)was at least a favorable Type I phototherapeutic agent.     

Photodynamic action of amino substituted hypocrellins: EPR studies on the photogenerations of active oxygen and free radical species

A novel method has been employed to prepare 2-butylamino-2-demethoxy hypocrellin A (BADMHA) and 2-butylamino-2-demethoxy hypocrellin B (BADMHB). Both compounds exhibit stronger absorption at the phototherapeutic window (600-900 nm). The spin trapping and spin counteraction studies have shown that they are both efficient generators of the active oxygen (1O2, O2*-) in the aerobic condition. Under the anaerobic condition they generate non-oxygen free radical (semiquinone radical anion), and the active oxygen mechanism of photosensitization can be converted into non-oxygen free radical mechanism with the depletion of oxygen. The quantum yields of 1O2 generation of BADMHA and BADMHB are 0.46 and 0.44, respectively. Both are lower than those of their parent compounds HA and HB. But the productions of superoxide anion are enhanced significantly compared with HA and HB, indicating they are both favorable Type I phototherapeutic agents.     

Electric magnetic resonance and spectrophotometry evidence on the photodynamic activity of a new perylenequinonoid pigment

Di-cysteine substituted hypocrellin B (DCHB) is a new water-soluble photosensitizer with significantly enhanced red absorption at wavelengths longer than 600 nm over the parent compound hypocrellin B (HB). The photosensitizing properties (Type I and/or Type II mechanisms) of DCHB have been investigated in dimethylsulfoxide (DMSO) and aqueous solution (pH 7.4) using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic DMSO solution, the semiquinone anion radical of DCHB (DCHB^•−) is predominantly photoproduced via self-electron transfer between excited- and ground-state DCHB species. The presence of an electron donor significantly promotes the formation of the reduced form of DCHB. When a deoxygenated aqueous solution of DCHB and an electron donor are irradiated with 532 nm light, the hydroquinone of DCHB (DCHBH₂) is formed via the disproportionation of the first-formed DCHB^•− and second electron transfer to DCHB^•− from the electron donor. When oxygen is present, singlet oxygen (¹O₂), superoxide anion radical (O₂^•−) and hydroxyl radical (^•OH) are produced. The quantum yield of ¹O₂ generation by DCHB photosensitization is estimated to be 0.54 using Rose Bengal as a reference, a little lower than that of HB (0.76). The superoxide anion radical is also significantly enhanced by the presence of electron donors. Moreover, (O₂^•−) upon disproportionation generated H₂O₂ and ultimately the highly reactive ^•OH via the Haber-Weiss reaction pathway. The efficiency of (O₂^•−) generation by DCHB is obviously enhanced over that of HB. These findings suggest that the photodynamic actions of DCHB may proceed via Type I and Type II mechanisms and that this new photosensitizer retains photosensitizing activity after photodynamic therapy-oriented chemical modification.     

4,5-dimethylthio-4'-[2-(9-anthryloxy)ethylthio]tetrathiafulvalene, a highly selective and sensitive chemiluminescence probe for singlet oxygen

4,5-Dimethylthio-4'-[2-(9-anthryloxy)ethylthio]tetrathiafulvalene has been designed and synthesized as a highly selective and sensitive chemiluminescence (CL) probe for singlet oxygen ((1)O(2)). The design strategy for the probe is directed by the idea of photoinduced electron-transfer process and carried out through the incorporation of electron-rich tetrathiafulvalene unit into a reactive luminophore of anthracene specific for (1)O(2). Upon reaction with reactive oxygen species (ROS), such as hydrogen peroxide, hypochlorite, superoxide, hydroxyl radical, or (1)O(2), the probe exhibits both strong CL response to and high selectivity for (1)O(2) only, rather than the other ROS. This remarkable CL property permits (1)O(2) to be distinguished easily from the other ROS and makes the probe possible to be used widely for (1)O(2) detection in many chemical and biological systems and even in light water (H(2)O) environments. This applicability has been demonstrated by monitoring the (1)O(2) generation in a metal-catalyzed decomposition system of tert-butyl hydroperoxide. Moreover, the CL reaction mechanism of the present system is also discussed, clearly confirming that the introduction of electron-rich tetrathiafulvalene into the 9-position of anthracene can greatly activate its reactivity toward (1)O(2).     

Reactive oxygen species produced upon photoexcitation of sunscreens containing titanium dioxide (an EPR study)

Commercial sunscreen products containing titanium dioxide were irradiated with lambda>300 nm and the formation of oxygen- (.OH, O2.-/.OOH) and carbon-centered radicals was monitored by EPR spectroscopy and spin trapping technique using 5,5-dimethyl-1-pyrroline N-oxide, alpha-phenyl-N-tert-butylnitrone (PBN), alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin traps, and free nitroxide radical 4-hydroxy-2,2,6,6-tetramethylpiperidine N-oxyl. The photoinduced production of singlet oxygen was shown by 4-hydroxy-2,2,6,6-piperidine. The generation of reactive oxygen radical species upon irradiation of sunscreens significantly depends on their composition, as the additives present (antioxidants, radical-scavengers, solvents) can transform the reactive radicals formed to less harmful products. The continuous in situ irradiation of titanium dioxide powder, recommended for cosmetic application, investigated in different solvents (water, dimethyl sulfoxide, isopropyl myristate) resulted in the generation of oxygen-centered reactive radical species (superoxide anion radical, hydroxyl and alkoxyl radicals).     

Active oxygen species generated from photoexcited fullerene (C60) as potential medicines: O2-* versus 1O2

To characterize fullerenes (C(60) and C(70)) as photosensitizers in biological systems, the generation of active oxygen species, through energy transfer (singlet oxygen (1)O(2)) and electron transfer (reduced active oxygen radicals such as superoxide anion radical O(2)(-)* and hydroxyl radical *OH), was studied by a combination of methods, including biochemical (DNA-cleavage assay in the presence of various scavengers of active oxygen species), physicochemical (EPR radical trapping and near-infrared spectrometry), and chemical methods (nitro blue tetrazolium (NBT) method). Whereas (1)O(2) was generated effectively by photoexcited C(60) in nonpolar solvents such as benzene and benzonitrile, we found that O(2)(-)* and *OH were produced instead of (1)O(2) in polar solvents such as water, especially in the presence of a physiological concentration of reductants including NADH. The above results, together with those of a DNA cleavage assay in the presence of various scavengers of specific active oxygen species, indicate that the active oxygen species primarily responsible for photoinduced DNA cleavage by C(60) under physiological conditions are reduced species such as O(2)(-)* and *OH.     

A High Sensitivity Detection Method of Singlet Oxygen and Superoxide Anion

This paper, for the first time, reports a method that can be used as a highly sensitive probe for singlet oxygen (^1O2) and superoxide anion (O2^-) in vitro or in vivo. FCLA(3,7-dihydro-6-{4-[2-(N‘-(5-fluoresceinyl)thioureido)ethoxy]phenyl}-2-methylimidazo{1,2-a}pyrazin-3-one sodium salt), a chemiluminescence (CL) analysis reagent, has been reported to sensitively react with ^1O2 and O2^- to emit photons with a spectral peak of 525nm. In this work,when human serum albumin (HSA) was added into FCLA solution to enhance the CL intensity,approximately 20 times, compared to that without HSA. The enhanced CL had the same 525 nm spectral peak, identical to that without HSA. By gradually reducing the molecular oxygen content in the solution, we find that the auto-oxidation of oxygen molecules dissolved in the solution plays an important role in the CL process. Based on these experimental evidences, we propose a novel and highly sensitive detection method of ^1O2 and O2^- which may have a great potential in chemical and medical applications.     

Spectroscopic studies and photodynamic actions of hypocrellin B in liposomes

Hypocrellin B (HB), a lipid-soluble natural pigment of perylenequinone derivative, is considered as potential photosensitizer for photodynamic therapy. Liposomes loaded with HB can constitute a simple model system, appropriate for better understanding the photodynamic action of HB in vivo. The steady-state absorption and emission spectra, quantum yield and lifetime of fluorescence of HB incorporated into egg L-a-phosphatidyl-choline (EPC) liposome were examined. The photochemical properties (Type I and/or Type II) of HB have also been studied in aqueous dispersions of small unilamellar liposomes of EPC using electron paramagnetic resonance and spectrophotometric methods, respectively. The quantum yield of 1O2 generated by HB is ca 0.76 in chloroform solution and it did not change upon the incorporation of HB into liposomes of EPC. The superoxide anion radical was generated by the electron transfer from the anion radical of HB (HB.-) to oxygen. The disproportionation of O2.- can generate H2O2 and ultimately the highly reactive .OH via the Fenton reaction. It could be that the disproportionation proceeded too fast, so we could not detect O2.- directly in aqueous dispersions of liposome EPC. Moreover, the self-sensitized photooxygenation of HB embedded in liposomes was studied, and almost fully (87%) inhibiting this reaction of HB by p-benzoquinone (as the quencher of O2.-) in aqueous dispersion of liposome EPC indicated that the radical mechanism (Type I) might be mainly involved in this oxygenation. All these findings suggested that the photodynamic action of HB proceeded via both Type-I and -II mechanisms, but Type-I mechanism might play a more important role in the aqueous dispersion.     

可见光/H2O2体系中的协同敏化

分析了可见光/H₂O₂体系中产生协同敏化效应的原因. 通过分析苯酚红、 甲基橙、 天青Ⅰ及三者混合物的紫外-可见光谱发现, 三者混合后拓宽了在可见光区的响应范围, 提高了对可见光的利用率. 采用邻菲罗啉法、 二苯基卡巴肼法和磷钼酸铵分光光度法分别测试了上述有色物在可见光/H₂O₂体系中产生光生电子e、 单线态氧和超氧阴离子自由基的量, 结果表明, 三者混合物在可见光下超氧阴离子自由基的产量明显比单独有色物的高, 光生电子和单线态氧的产量相差不大, 说明超氧阴离子自由基产量的提高是产生协同敏化的主要原因. 测试结果还证实光生电子不能催化过氧化氢产生羟自由基.     

Photodynamic properties of hypocrellin A, complexes with rare Earth trivalent ions: role of the excited state energies of the metal ions

Fifteen complexes of hypocrellin A (HA) with rare earth trivalent ions (except Pm3+) along with the complex of HA with Sc3+ were prepared, and their photodynamic activities, including absorption in the phototherapeutic window (600-900 nm); water-solubility; triplet lifetime; generation of reactive oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion radical (O2-*), and hydroxyl radical (OH*); generation of semiquinone anion radical; and affinity to DNA, as well as photosensitized damage on calf thymus DNA (CT DNA), were compared in detail using the UV-visible spectrum, fluorescence spectrum, spin-trapping EPR technique, and laser photolysis technique. All complexes exhibit a red-shifted absorption spectrum, an increased absorbance above 600 nm, improved water solubility, and an enhanced affinity to CT DNA over the parent HA. For ions that possess low-energy excited states, including Ce3+, Pr3+, Nd3+, Sm3+, Eu3+, Tb3+, Dy3+, Ho3+, Er3+, Tm3+, and Yb3+, the corresponding complexes show undetectable or nearly undetectable fluorescence, a triplet excited-state lifetime, generation of ROS, and photodamage in CT DNA. In contrast, for ions that do not possess low-energy excited states, including Sc3+, Y3+, La3+, Gd3+, and Lu3+, the corresponding complexes exhibit higher photodamage abilities with CT DNA with respect to HA, benefitting from both their comparable or even higher 1O2 quantum yields and an electrostatic affinity that is higher for DNA than HA.     

Photodynamic action of a water-soluble hypocrellin derivative with enhanced absorptivity in the phototherapeutic window II. Photosensitized generation of active oxygen species

Cysteine-substituted hypocrellin B (Cys-HB) is a water-soluble perylenequinonoid derivative with significantly enhanced absorptivity in the range of wavelength longer than 600 nm. Electron paramagnetic resonance (EPR) measurements, quenching experiments and 9,10-diphenyl-anthracene bleaching studies were used to investigate the photodynamic action of Cys-HB in the presence of oxygen. Illumination of Cys-HB solution, in the presence of oxygen, generated singlet oxygen, superoxide anion radical, hydroxyl radical and hydrogen peroxide. The accumulation of active oxygen species was transformed into that of the semiquinone anion radical with the depletion of oxygen, detected by the spin counteraction of TEMPO radical formed via the reaction of TEMP with singlet oxygen produced by Cys-HB photosensitization. Oxygen content, Cys-HB concentration and reaction environment affected the transformation and the competition between the Type I and Type II reactions. Compared with hypocrellin B (HB), Cys-HB primarily remained similar and slightly lower capability of active oxygen-generation, confirmed to be a favorable phototherapeutic agent.     

Synthesis, characterization and photodynamic activity of phenmethylamino-demethoxy-hypocrellin B

Two phenmethylamino hypocrellin B derivatives are novel photodynamic agents synthesized by a mild reaction between hypocrellin B and phenmethylamine. The red absorption of the photosensitizers is enlarged distinctly and the peri-hydroxylated perylenequinone structure of the parent HB is preserved. 9,10-diphenyl-anthracene (DPA) bleaching and electron paramagnetic resonance (EPR) spin trapping techniques were used to study the photodynamic activities of the phenmethylamino hypocrellin B derivatives in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2*-) generated in the process of illumination of the phenmethylamino hypocrellin B in aerobic solution were observed. The photodamage of PMAHBs to MGC803 cancer cells was investigated in vitro. The results in vitro reveal that the phenmethylamino hypocrellin B derivatives show a much less significant decrease in cytotoxicity than that of their parent HB. It exhibits higher selectivity of light-orientation, which can decrease the damage to normal tissues by irradiating the tumor tissues, and so increases the drug safety.     

SPECTROSCOPIC STUDIES OF CUTANEOUS PHOTOSENSITIZING AGENTS. XVII. BENZANTHRONE

The photochemistry of benzanthrone (7H-benz[de]-anthracene-7-one) has been studied using electron paramagnetic resonance (EPR) in conjunction with the spin trapping technique and the direct detection of singlet molecular oxygen luminescence. Irradiation (lambda ex = 394 nm) of benzanthrone (BA) in aerated ethanol, dimethylsulfoxide or benzene resulted in the generation of superoxide (O2-.) which was trapped by 5,5-dimethyl-1-pyrroline-N-oxide. The ethoxy radical was also detected in ethanol. Photolysis of BA in deaerated basic ethanol led to the formation of BA anion radical, BA-., which was detected directly by ESR. This radical anion decayed back to BA with a unimolecular rate constant of 1.5 x 10(-3) s-1. The 1O2 quantum yields (lambda ex greater than 345 nm) for BA in ethanol, 90% ethanol and basic ethanol (0.1N NaOH) were 0.89, 0.88 and 0.28 respectively relative to Rose Bengal. The lower yield of 1O2 in basic ethanol may be attributable to the reaction of oxygen with BA-. (which is generated in higher yield at alkaline pH) to give O2-.. These findings suggest that on exposure to light BA can generate active oxygen species which may be responsible for the photocontact dermatitis caused by BA in industrial workers exposed to this chemical.     

New trends in photobiology. The interaction of UVA radiation with cultured cells

Recent work concerning the interaction of UVA radiation (320-380 nm) with cultured cells is reviewed with particular emphasis on the involvement of cellular oxidative stress in the biological effects of this radiation on eucaryotic cells. Possible chromophores are considered and their role in generation of various oxidant species including hydrogen peroxide, superoxide anion, singlet oxygen and hydroxyl radical. DNA and membranes are discussed as possible targets for the lethal action of long wavelength radiation. Four mechanisms of cellular defence are proposed: (1) DNA repair; (2) antioxidant enzymes; (3) endogenous free radical quenchers; (4) inducible protection.     

Diastereoselective synthesis and ESR study of 4-phenylDEPMPO spin traps

[reaction: see text] The cis and trans diastereoisomers of 5-(diethoxyphosphoryl)-5-methyl-4-phenylpyrroline N-oxide (4-PhDEPMPOt 8 and 4-PhDEPMPOc 9) were prepared stereoselectively and used as spin traps for hydroxyl and superoxide radicals. The spin adduct formed by reaction of the cis stereoisomer 9with superoxide radical anion exhibited an 8-line ESR spectrum, showing only a reduced alternating line width phenomenon. This spectrum is simpler than the 12-line spectrum of DEPMPO-OOH, which exhibits a strong alternating line width phenomenon. The half-life times of the 4-PhDEPMPOc-OOH and DEPMPO-OOH adducts were of the same order: 14.5 and 15.5 min, respectively.     

Comparison of the aerobic photoreactivity of A2E with its precursor retinal

A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E, 3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]pyridinium) is a blue-absorbing molecular constituent of human ocular lipofuscin and contributes to the golden-yellow emission of this pigment. Lipofuscin photoproduces toxic reactive oxygen intermediates (ROI), but the specific molecular components responsible for this phototoxicity remain unidentified. In this article the aerobic photoreactivity of A2E is quantified by comparison with its biosynthetic precursor, all-trans-retinal, and with other appropriate standards. Under blue-light exposure the efficacies for formation of cholesterol (Ch) hydroperoxides and the superoxide radical anion (O2*-) were determined using high-pressure liquid chromatography with electrochemical detection and electron spin resonance oximetry and spin trapping, respectively. Photogeneration of singlet oxygen after blue-light excitation of A2E was demonstrated unambiguously by the Ch peroxidation assay. After blue-light irradiation of A2E, O2*- were detected, but the concentration was insufficient to account for the measured production of O2*- by the solvent extract of lipofuscin granules. The collective data support the conclusion that A2E does not produce sufficient concentrations of ROI to be the primary phototoxic constituent of lipofuscin.     

Activation of electron-transfer reduction of oxygen by hydrogen bond formation of superoxide anion with ammonium ion

A hydrogen bond formed between the superoxide anion and the ammonium ion (NH4+) accelerates electron transfer from the C60 radical anion to oxygen significantly, whereas the tetra-n-butylammonium ion has no ability to form a hydrogen bond with the superoxidie anion, exhibiting no acceleration of the electron-transfer reduction of oxygen. The second-order rate constant of electron transfer from C60*- to O2 increases linearly with increasing concentration of NH4+. This indicates that O2*- produced in the electron transfer from C60 to O2 is stabilized by 1:1 complex formation between O2*- and NH4+. The 1:1 complex formed between O2*- and NH4+ was detected by ESR. The binding of O2*- with NH4+ results in a positive shift of the reduction potential of O2 with increasing concentration of NH4+, leading to the acceleration of electron transfer from C60*- to O2.     

Liquid chromatography/tandem mass spectrometry analysis of long-chain oxidation products of cardiolipin induced by the hydroxyl radical

The anionic phospholipid cardiolipin (CL) is found almost exclusively in the inner membrane of mitochondria, playing an important role in energy metabolism. Oxidation of CL has been associated with apoptotic events and various pathologies. In this study, electrospray ionization mass spectrometry coupled with liquid chromatography (LC/ESI-MS) was used to identify tetralinoleoyl-cardiolipin (TLCL) modifications induced by the OH(·) radical generated under Fenton reaction conditions (H(2)O(2) and Fe(2+)). The identified oxidation products of TLCL contained 2, 4, 6 and 8 additional oxygen atoms. These long-chain oxidation products were characterized by LC/ESI-MS/MS as doubly [M-2H](2-) and singly charged [M-H](-) ions. A detailed analysis of the fragmentation pathways of these precursor ions allowed the identification of hydroperoxy derivatives of CL. MS/MS analysis indicated that CL oxidation products with 4, 6 and 8 oxygen atoms have one fatty acyl chain bearing 4 oxygen atoms ([RCOO+4O](-)). Even when the TLCL molecule was oxidized by the addition of eight oxygen atoms, one of the acyl chains remained non-modified and one fatty acyl chain contained three or four oxygen atoms. This led us to conclude that under oxidative conditions by the OH(·) radical, the distribution of oxygens/peroxy groups in the CL molecule is not random, even when CL has the same fatty acyl chains in all the positions. Using mass spectrometry, the oxidation products have been unequivocally assigned, which may be useful for their detection in biological samples.     

Active oxygen species (1O2, O2*-) generation in the system of TiO2 colloid sensitized by hypocrellin B

TiO2 semiconductor colloids have been successfully employed in environmental clean-up, antibacterial and bactericidal action under ultraviolet light due to its strong redox ability and high yield of active oxygen species (1O2, O2*-), *OOH) generation. Hypocrellin B, isolated from Hypocrella bambusae (B.et.Br) Sacc, a natural pigment with strong and broad absorption over the visible light region, was used in our work in an attempt to extend the photoresponse of TiO2 to visible light and maintain the high generation of active oxygen under visible light illumination. The formation of the HB-TiO2 chelate was characterized by UV-Vis and surface enhanced raman spectroscopy (SERS) and it was found that the chelate still had high efficiency of active oxygen generation. The possible generation mechanism was explored by Electron Paramagnetic Resonance (EPR) and time-resolved transient spectra techniques, showing that singlet oxygen (1O2) and superoxide radical anion (O2*-)) were produced via energy transfer and electron transfer, respectively. The application of HB-TiO2 chelate in environment protection and bacteria sterilization was implied.     

Scavenging effects of metal complexes of water-soluble thiacalix[4]arenetetrasulfonate on superoxide anion radicals

The scavenging effects of metal complexes of thiacalix[4]arenetetrasulfonate (Me-TCAS[4], Me=H?, Fe3(+), Mn3(+), Mn2(+), Cu2(+), and Zn2(+)) on superoxide anion radicals (O??) generated from the xanthine-xanthine oxidase system were investigated by the nitroblue tetrazolium (NBT) method and electron spin resonance (ESR) spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide as a trapping reagent. As a reference, calix[4]arenetetrasulfonate (H?)-CAS[4]), calix[6]arenehexasulfonate (H?-CAS[6]) and calix[8]areneoctasulfonate (H?-CAS[8]) were also examined. The results by the NBT method indicated that Fe3(+)- and Mn3(+)-TCAS[4] exhibited the highest O?? scavenging activity among Me-TCAS[4] and H?-CAS[n] (n = 4, 6, 8) in this study. The IC?? values of Fe3(+)- and Mn3(+)-TCAS[4] for O?? scavenging activity were estimated to be 5.3 and 7.8 μM, respectively, and were almost the same as those of tannin acid, catechin and their derivatives, which are known as very effective scavengers of O??. Scavenging activities were in the order of Fe3(+)- and Mn3(+)-TCAS[4]>Mn2(+)-, Cu2(+)-, and Zn(2+)-TCAS[4]>H(2)-TCAS[4] and H?-CAS[n] (n=4, 6, 8). Each activity of Me-TCAS[4] (Me=Fe3(+), Mn3(+), Mn2(+), Cu2(+), and Zn2(+)) was higher than that of the corresponding metal ion, indicating that H?-TCAS[4] has the ability to raise the activity of the metal ion itself by forming a complex. Also, the ESR spin-trapping method revealed that Fe3(+)- and Mn3(+)-TCAS[4] showed high O?? scavenging activities, similarly to the results by the NBT method.