ESR evidence of the photogeneration of free radicals (GDHB*-, O2*-) and singlet oxygen ((1)O2) by 15-deacetyl-13-glycine-substituted hypocrellin B

15-Deacetyl-13-glycine-substituted hypocrellin B (GDHB) is a new type of hypocrellin derivative with an enhanced red absorption longer than 600 nm and water solubility. Visible light (> 470 nm) irradiation of an anaerobic aqueous solution of GDHB, the formation of GDHB*- was detected by an ESR method in the absence or presence of electron donor. When exposed to oxygen, superoxide anion radical and singlet oxygen were formed. The superoxide anion radical was generated by GDHB*- via electron transfer to oxygen and this process was significantly enhanced by the presence of electron donors. Singlet oxygen ((1)O2) was also formed in the photosensitization of GDHB in aerobic solution and 1,4-diazabicyclo [2,2,2] octane (DABCO), sodium azide (NaN3) and histidine inhibited the generation of (1)O2. A 9,10-diphenyl antracene (DPA)-bleaching method was used to determine the quantum yield of (1)O2 generated from GDHB photosensitization. The (1)O2 quantum yield was estimated to be 0.65. With the depletion of oxygen, the accumulation of GDHB*- would replace that of (1)O2. Evidence accumulated that the photodynamic action of GDHB may proceed via both type I and type II mechanisms and that a type II mechanism will be transformed into a type I mechanism as oxygen gets depleted.     

血清胆红素的光敏氧化

竹红菌乙素是一种芘醌类的光敏剂.实验结果表明在可见光的照射下.它能加速血清胆红素的光氧化,氧化速率提高5倍以上.比较在不同溶剂中各种活性氧淬灭剂对胆红素光氧化的抑制作用,指出血清胆红素光敏氧化反应包括自由基氧化反应(Ⅰ型反应)和单态氧氧化反应(Ⅱ型反应)等多重机制.     

Effect of structural modifications on photosensitizing activities of hypocrellin dyes: EPR and spectrophotometric studies.

Mono-substituted hypocrellin B (MHB) and di-substituted hypocrellin B (DHB) by mercaptoacetic acid are new photosensitizers synthesized to improve the red absorption and water solubility of the parent hypocrellin B (HB). The photochemistries (Type I and/or Type II) of MHB and DHB have been studied in homogeneous solutions using electron paramagnetic resonance (EPR) and spectrophotometric methods. In anaerobic homogeneous DMSO solution, DHB*- (or MHB*-) was predominantly photoproduced via self-electron transfer between the excited- and ground-state species. The presence of an electron donor significantly promotes the formation of the reduced form of DHB (or MHB). As compared with hypocrellin B, the efficiencies of DHB*- and MHB*- generation was enhanced obviously. When oxygen-saturated solutions of DHB (or MHB) were illuminated with 532 nm light, singlet oxygen (1O2), superoxide anion radical (O2*-), hydroxyl radical (*OH) and hydrogen peroxide (H2O2) were formed. DHB and MHB generate 1O2 with quantum yields of 0.18 and 0.22, respectively, which are much lower than that of HB (0.76) in chloroform. The superoxide anion radical was significantly enhanced by the presence of electron donors. The rate of O2*- production was also dependent on the concentration of DHB or MHB. Moreover, O2*- upon disproportionation can generate H2O2 and ultimately the highly reactive *OH via the Fenton reaction and other pathway with the involvement of DHB*- (or MHB*-). As in the case of DHB*- (or MHB*-), the efficiencies of O2*- and *OH generation by DHB and MHB were also enhanced obviously, consistent with the fact that DHB*- (or MHB*-) acts as the precursor of O2* and thus *OH. These findings suggest that the photodynamic actions of DHB and MHB may proceed via enhanced Type I mechanism and reduced Type II mechanism as compared with that of HB.     

Glycoconjugated hypocrellin: photosensitized generation of free radicals (O2*-, *OH, and GHB*-) and singlet oxygen (1O2).

To improve water solubility and specific affinity for malignant tumors, glycoconjugated hypocrellin B (GHB) has been synthesized. Illumination of deoxygenated DMSO solution containing GHB generates a strong electron paramagnetic resonance (EPR) signal. The EPR signal is assigned to the semiquinone anion radical of GHB (GHB*-) based on a series of experimental results. Spectrophotometric measurements show that the absorption bands at 645 nm and 502 nm (pH 8.0) or 505 nm (pH 11.0) arise from the semiquinone anion radical (GHB*-) and hydroquinone (GHBH2) of GHB, respectively. GHBH2 is readily formed via the decay of GHB*- in water-contained solution. The increase of pH value of the reaction media promotes this process. When oxygen is present, superoxide anion radical (O2*-) is formed, via the electron transfer from GHB*-, the precursor, to ground state molecular oxygen. Hydroxyl radical can be readily detected by DMPO spin trapping when aerobic aqueous solution containing GHB is irradiated. As compared with the parent compound, hypocrellin B (HB), the efficiency of O2* and *OH generation by GHB photosensitization is enhanced significantly. Singlet oxygen (1O2) can be produced via the energy transfer from triplet GHB to ground state oxygen molecules, with a decreased quantum yield, i.e., 0.19. These findings suggest that the new GHB possesses an enhanced type I process and a decreased type II process as compared with hypocrellin B.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

Combined effects of singlet oxygen and hydroxyl radical in photodynamic therapy with photostable bacteriochlorins: evidence from intracellular fluorescence and increased photodynamic efficacy in vitro

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λ(max) ≈ 750 nm, ε ≈ 10(5) M(-1) cm(-1)), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.     

Butylamino-demethoxy-hypocrellins and photodynamic therapy decreases human cancer in vitro and in vivo.

2-Butylamino-2-demethoxy-hypocrellin A (BAHA) and B (BAHB) are new photosensitizers synthesized by a mild reaction of hypocrellins and butylamine. In BAHA and BAHB, the peri-hydroxylated perylenequinone structure of the parent hypocrellins is preserved and the red absorption is enhanced distinctly. Electron paramagnetic resonance spin trapping measurements and 9,10-diphenylanthracene bleaching studies were used to investigate the photodynamic action of BAHA and BAHB in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O2(*-)) produced by illuminating BAHA and BAHB in aerobic solution have been observed. Compared with hypocrellin A and B, BAHA and BAHB primarily remained able to generate 1O2 and enhanced distinctly the O2(*-)-generating abilities. The photodynamic action of BAHA and BAHB in the therapy of cancer was investigated in vitro and in vivo. Both in vitro and in vivo results revealed a significant decrease in cancer cell growth. Laser or dye alone had no effect, indicating that intratumor BAHA and laser therapy may prove useful in unresectable cancer.     

Lipid peroxidation induced by cercosporin as a possible determinant of its toxicity

The photodynamic action of cercosporin was assayed in various kinds of natural and artificial membranes. Cerosporin induces lipoperoxidation of liposomes, rat liver and pea internode mitochondria and microsomes, estimated both as malondialdehyde (MDA) formation and O2 consumption. Cercosporin-induced lipoperoxidation is inhibited by either singlet oxygen quenchers, free radical trapping agents or EDTA. Superoxide anion (O2-), hydrogen peroxide and hydroxyl radicals (.OH) are not involved in the activity of cercosporin. In addition cercosporin, by chelating iron, lowers the lipoperoxidation induced by such a metal. Therefore cercosporin stimulates, through singlet oxygen production, the hydroperoxide formation but, at the same time, it inhibits the continuation of the iron-mediated free radical chain. The present results suggest that cellular lipid peroxidation has a certain relevance to toxic activity of cercosporin.     

Photodynamic activity and singlet oxygen

The primary mechanisms for the photodynamic action of pigments and dyes, the principles of their division into mechanisms of type I and type II, and the role of these processes in biological systems are reviewed. Singlet oxygen is considered to be an indicator of the mechanisms of photodynamic reactions. The methods of its detection are described, which are based on the use of chemical traps, measurements of infrared phosphorescance at 1270 nm, and the registration singlet oxygen-sensitized delayed fluorescence caused by the summation of the energy of two singlet oxygen molecules by one dye molecule.     

Photodynamic effects of two hydroxyanthraquinones

The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2-* is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2-* and *OH. Both optical and ESR measurements indicate that O2-* (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.     

Anion of hypericin is crucial to understanding the photosensitive features of the pigment

Photosensitive behaviors of hypericin (HYP) have attracted much attention, because of HYP's great potential in photodynamic therapy. It has been found that HYP differs from homologous pigments, such as hypocrellin A (HA), in photosensitive features. For instance, despite the comparable triplet state quantum yields, HYP holds a much lower singlet oxygen yield than HA. To understand the unique photosensitive behaviors of HYP, time-dependent density functional theory is employed to calculate a series of excited-state properties of HYP and its anion (dominant in polar solvents), which are then compared with excited-state properties of HA. It is revealed that the stronger electron-donating power of HYP anion than that of HA is responsible for the HYP's photosensitive features.     

Photophysical properties and photodynamic activity in vivo of some tetrapyrroles

Some of the photophysical properties (stationary absorbance and fluorescence, fluorescence decay times and singlet oxygen quantum yields) of pheophorbide a, metal-free, ClAl-, Cu- and Mg-t-butyl-substituted phthalocyanines, metal-free, ClAl- and Cu-t-butyl-substituted naphthalocyanines and of a number of tetraphenylporphyrins (5,10,15,20-tetraphenylporphyrin, 5,10,15,20-tetra(m-hydroxyphenyl)porphyrin, 5,10,15,20-tetra(p-hydroxyphenyl)porphyrin) have been studied in comparison with hematoporphyrin IX in order to select potent photosensitizers for the photodynamic treatment of cancer. The photodynamic activity of these compounds was investigated using Lewis lung carcinoma in mice. As a consequence of the photophysical parameters (relatively short singlet state lifetimes, and high singlet oxygen quantum yields) the photodynamic activities of pheophorbide a, t-butyl-substituted ClAl-phthalocyanine and ClAl-naphthalocyanine were selected for study in greater detail. Under the conditions employed in the present study, pheophorbide a was found to be the most effective sensitizer, as judged from its strong absorption at the excitation wavelength as compared with the hematoporphyrin derivative and greater singlet oxygen quantum yield relative to the phthalocyanines and naphthalocyanines. The photodynamic activity was observed to be strongly dependent on the photophysical parameters of the compounds. The primary mechanism underlying the photodynamic activity of these sensitizers probably consists of energy transfer from the lowest triplet state of the dyes to molecular oxygen, resulting in the formation of singlet oxygen (type II of photosensitization).     

Photogeneration of free radicals (*OH and HB*-) and singlet oxygen (1O2) by hypocrellin B in TX-100 micelles microsurroundings

To solve the problems faced in clinical use of hypocrellins, a water-soluble preparation of Hypocrellin B (HB), HB-Triton X-100 (TX-100) micelles, was prepared. To evaluate the photodynamic activity, the free radicals (^•OH and HB^•¯) and singlet oxygen (

1

O

2

) generated via photosensitization of the preparation in aqueous solution were detected by using electron paramagnetic resonance (EPR) and spectrophotometric methods. It was observed that

1

O

2

was formed with a quantum yield of 0.72, similar to that for HB in organic solvents, further, hydroxyl radicals (

•

OH) could also be efficiently produced by the new preparation, which have never before been detected following HB photoactivities. In addition, the semiquinone anion radicals (HB^•-) could also be generated via the self-electron transfer between an excited triplet state and a ground state molecule. The accumulation of HB^•- would replace that of

•

OH or

1

O

2

with the depletion of oxygen in the system. All these findings suggested that the HB-TX-100 micelles could play the photodynamic action through not only the type I mechanism by free radicals (^•OH, O₂^•- and HB^•-) but also the type II mechanism by singlet oxygen (

1

O

2

). It can be concluded further that the new preparation basically maintains the inherent photodynamic activity of HB, or even higher.     

Photogeneration of singlet oxygen (1O2) and free radicals (Sen·-, O·-2) by tetra-brominated hypocrellin B derivative

To improve photodynamic activity of the parent hypocrellin B (HB), a tetra-brominated HB derivative (compound 1) was synthesized in high yield. Compared with HB, compound 1 has enhanced red absorption and high molar extinction coefficients. The photodynamic action of compound 1, especially the generation mechanism and efficiencies of active species (Sen^·-, O^·-₂ and ¹O₂) were studied using electron paramagnetic resonance (EPR) and spectrophotometric methods. In the deoxygenated DMSO solution of compound 1, the semiquinone anion radical of compound 1 is photogenerated via the self-electron transfer between the excited and ground state species. The presence of electron donor significantly promotes the reduction of compound 1. When oxygen is present, superoxide anion radical (O^·-₂) is formed via the electron transfer from Sens^·- to the ground state molecular oxygen. The efficiencies of Sens^·- and O^·-₂ generation by compound 1 are about three and two times as much as that of HB, respectively. Singlet oxygen (¹O₂) can be produced via the energy transfer from triplet compound 1 to ground state oxygen molecules. The quantum yield of singlet oxygen (¹O₂) is 0.54 in CHCl₃ similar to that of HB. Furthermore, it was found that the accumulation of Sens^·- would replace that of O^·-₂ or ¹O₂ with the depletion of oxygen in the sealed system.     

New long-wavelength perylenequinones: synthesis and phototoxicity of hypocrellin B derivatives

Five new derivatives of hypocrellin B were obtained from the reactions of hypocrellin B with ammonia and ethanolamine. Their photophysical and photochemical properties were investigated. The phototoxicity of one compound on AH cells irradiated with red light (lambda = 600-700 nm) was also studied. Their significantly enhanced red absorptivities at wavelengths longer than 600 nm and singlet oxygen-generating function qualify them as promising photodynamic therapy agents.     

Photodynamic properties of meta-tetra(hydroxyphenyl)chlorin in human tumor cells.

The photodynamic properties of a second-generation photodynamic sensitizer, meta-tetra(hydroxyphenyl)chlorin (mTHPC) were studied by dye-sensitized photoinactivation (650 nm) of HT29 human adenocarcinoma cells in culture. The photocytotoxicity of mTHPC in vitro depended on the presence of molecular oxygen. A strong inhibition of the photocytotoxicity of mTHPC was observed upon addition of sodium azide, a known singlet oxygen quencher. Photocytotoxicity was not inhibited by scavengers of superoxide anion radical, hydrogen peroxide and hydroxyl radicals. We suggest that mTHPC photosensitizes cell killing predominantly by type II, singlet oxygen-mediated photodynamic reactions. Illumination of cells preloaded with mTHPC induced peroxidation of membrane lipids. Inhibition of photoperoxidation by alpha-tocopherol (0.1 mM) present during illumination did not result in any decrease in toxicity, suggesting that reactions of lipid peroxidation play only a minor role in the overall photocytotoxic effect of mTHPC.     

In vitro models for photosynthetic energy conversion

A summary is presented of recent work on the photochemistry of chlorophyll in solution. It is shown that reactions occur which are close counterparts ofin vivo photoprocesses. These are (a) photoproduction of chlorophyll cation radical (analog of photosystem I reaction centre primary photoprocess), (b) one-electron phototransfer from bacterio-chlorophyll to quinone (analog of bacterial reaction centre primary photoprocess), (c) chlorophyll photosensitized one-electron transfer from hydroxylic compounds to quinone (analog of photosystem II reaction centre photoprocess). The mechanisms of these reactions and their implications for photosynthetic energy conversion are discussed.     

The enzymatic one-electron reduction of porphyrins to their anion free radicals

The anaerobic enzymatic one-electron reduction of uroporphyrin I (in the absence of light) by the ferredoxin/ferredoxin:NADP+ oxidoreductase system was investigated using NADPH as the source of reducing equivalents. The porphyrin anion free radical metabolite formed by one-electron reduction of the parent molecule was detected with ESR spectroscopy. The ESR spectrum exhibited a singlet (g = 2.0021) with a 5.4-G peak-to-peak linewidth. The reduction process was also investigated under aerobic conditions. The reduction of molecular oxygen to superoxide anion radical by the porphyrin anion radical was demonstrated by using the ESR technique of spin trapping. The ESR spectra of the spin-trapped oxygen-derived radicals were superoxide dismutase-sensitive and catalase-insensitive, supporting the assignment of the trapped radical to the superoxide anion radical. These aerobic experiments demonstrate electron transfer from the porphyrin anion radical to molecular oxygen. The anaerobic reduction of Photofrin II by hepatic microsomes and the ferredoxin/ferredoxin:NADP+ oxidoreductase system to a porphyrin anion radical was also investigated. Free radical formation by ferredoxin: NADP+ oxidoreductase is totally dependent upon ferredoxin. The ESR spectrum of this porphyrin free radical also exhibited a singlet (g = 2.0026) with a 15-G peak-to-peak linewidth.     

Interactive image analysis system to determine the motility and velocity of cyanobacterial filaments

An interactive image analysis system has been developed to analyse and quantify the percentage of motile filaments and the individual linear velocities of organisms. The technique is based on the "difference" image between two digitized images taken from a time-lapse video recording 80 s apart which is overlaid on the first image. The bright lines in the difference image represent the paths along which the filaments have moved and are measured using a crosshair cursor controlled by the mouse. Even short exposure to solar ultraviolet radiation strongly impairs the motility of the gliding cyanobacterium Phormidium uncinatum, while its velocity is not likewise affected. These effects are not due to either type I (free radical formation) or type II (singlet oxygen production) photodynamic reactions, since specific quenchers and scavengers, indicative of these reactions, failed to be effective.     

Photosensitized damage to calf thymus DNA by a hypocrellin derivative: mechanisms under aerobic and anaerobic conditions

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB*- radical), followed by the second electron transfer from DNA to DCHB*- or the disproportionation of DCHB*- to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.