Photochemistry and Photobiology

Published by Wiley
The absorption spectra of bacteriochlorophyll (BChl) c solutions in two mixtures of two solvents (acetonitrile with pyridine and dimethylsulfoxide with methanol) exhibiting different refractive indices were measured and deconvoluted into Gaussian components. The refractive index of mixed solvents was changed by the change in the ratio of the volumes of the liquids used in the mixture. Using the Qy(0,0) band half widths and absorption coefficient, based on the simplified formula proposed by Knox, the dipole strengths of the Qy(0,0) BChl c transition for various values of solvent refractive index were calculated and compared with values given by Knox and Spring. For both investigated combinations of two liquids, the dependence of Qy(0,0) transition dipole strength of the BChl c on solvent refractive index was almost linear. The slopes of the lines obtained from the experimental absorption bands were different for two investigated solvent mixtures. More accurate linear dependence and similar slopes of lines for both solvent mixtures were obtained using half widths and absorption coefficients of the Gaussian components of Qy(0,0) transition. It is explained by the superposition of the contributions from other electronic and vibronic transitions of BChl c monomers or possibly also from transitions of the pigments involved in some complexes with solvent molecules in the absorption region investigated. The results show that the formula proposed by Knox can be successfully applied also for BChl c, after elimination of the overlapped contributions from the other transitions, by applying Gaussian analysis to select only contribution from Qy(0,0) pigment transition.
Optical properties are important parameters in port wine stain laser treatment models. In this study we investigated whether changes in blood optical properties occur during a 0.5 ms laser pulse. Blood from three volunteers was irradiated in vitro with laser pulses (radiant exposure 2-12 J cm-2, wavelength 586 nm, pulse length 0.5 ms). Reflection and transmission coefficients, measured using double integrating spheres, decreased slightly during the first part of the pulse. At 2.9 J cm-2 radiant exposure, the reflectance increased, independent of total radiant exposure of the pulse. This was caused by blood coagulation. A second sudden increase in reflection and a significant increase in transmission occurred near 6.3 J cm-2 and was accompanied by a "popping" sound, indicating rapid expansion of bubbles due to blood vaporization. A multilayered model of blood was used to fit calculated transmission coefficient curves to the measurements and determine temperature-dependent optical blood absorption. Heat diffusion was shown to be of minor importance. A 2.5-fold increase in absorption for temperatures increasing from 20 to 100 degrees C, accurately describes transmission coefficients measured up to 2.9 J cm-2.
Lutetium texaphyrin, PCI-0123, is a pure, water-soluble photosensitizer with a large broad absorption band centered at 732 nm. The compound was tested for photodynamic therapy (PDT) effectiveness in a murine mammary cancer model. The texaphyrin macrocycle as illustrated by magnetic resonance imaging and 14C-radiolabeled texaphyrin studies was shown to be tumor selective; a tumor-to-muscle ratio of 10.55 was seen after 5 h. Lutetium texaphyrin, at a drug dose of 20 mumol/kg with irradiation 5 h postinjection at 150 J/cm2 and 150 mW/cm2, had significant efficacy (P < 0.0001) in treating neoplasms of moderate size (40 +/- 14 mm3) and also had significant efficacy (P < 0.0001) in treating larger neoplasms (147 +/- 68 mm3). The PDT efficacy was correlated with the time interval between PCI-0123 administration and light exposure. A 100% cure rate was achieved when photoirradiation took place 3 h postinjection compared to 50% for 5 h using 10 mumol/kg and 150 J/cm2 at 150 mW/cm2. The PDT efficacy was attributable to the selective uptake/retention of the texaphyrin photosensitizer in addition to the depth of light penetration achievable at the 732 nm laser irradiation.
Lutetium texaphyrin (PCI-0123) is a pure, water-soluble photodynamic therapy (PDT) agent that is activated by tissue-penetrating far red light. The sensitizer is highly fluorescent and exhibits a strong, broad emission signal at 750 nm. In vitro cellular uptake studies revealed an increase in sensitizer retention with incubation time. Confocal laser scanning microscopy demonstrated that the intracellular localization site of PCI-0123 is the lysosomes. Ensuing illumination of the EMT6 cells led to lysosomal breakup, extensive cytoplasmic blebbing and subsequent cell death. Noninvasive spectral imaging analysis of PCI-0123 fluorescence depicted selective drug uptake, compared to surrounding normal tissue, in EMT6 mammary sarcomas syngeneic to BALB/c mice. The PCI-0123 PDT was shown to effectively treat the EMT6 murine sarcoma. Irradiation (732 nm light) 3 h postintravenous injection of 10 mumol PCI-0123 per kg gave 100% cures (no evidence of cancer), whereas light exposure at 5 h resulted in 75% cures. Hematoxylin and eosin histologic examination of photoirradiated tumors indicated apoptosis of the EMT6 neoplasms at early times post-PDT progressing, with time, to extensive necrotic areas. Gel electrophoresis of extracted photoirradiated tumors showed the typical apoptotic DNA ladder pattern that increased in intensity following PDT treatment.
UV radiation suppresses delayed-type hypersensitivity responses to intradermally injected tuberculin purified protein derivative in Mantoux-positive individuals. The effect of the topically administered isoflavonoid NV-07alpha, a synthetic derivative of the isoflavonoid equol, on UV-induced suppression of Mantoux reactions was assessed in 18 healthy Mantoux-positive volunteers. A single, fixed dose of solar-simulated UV radiation was delivered to the volunteers' lower backs. Different concentrations of NV-07alpha or its vehicle were applied to different sites within the irradiated field immediately after UV exposure and again 24 h later. Forty-eight hours after irradiation, Mantoux testing was performed at both the irradiated sites and adjacent, unirradiated sites. The intensity of Mantoux reactions was measured 72 h later with a reflectance erythema meter and by measuring the diameter of each reaction. Although lower concentrations of NV-07alpha (0.5 and 2 mM) did not prevent UV immunosuppression, 4 mM NV-07alpha partially but significantly attenuated UV-induced suppression of Mantoux-induced erythema. Minimal erythema doses were also determined for sites treated with NV-07alpha or its vehicle immediately after UV exposure. NV-07alpha had no significant effects on UV erythema. We conclude that 4 mM NV-07alpha prevented the suppressive effects of UV radiation on Mantoux responses in humans but did not affect UV-induced erythema at the concentrations used.
Photodynamic therapy (PDT) has been used for many years for both palliative and curative treatment of bronchial carcinomas. However, prolonged skin phototoxicity and reduced depth of penetration has limited the widespread use of PDT. We studied the endobronchial phototoxicity of a novel photosensitizer, WST 09 (Tookad). Fourteen pairs of Large White-Landrace male piglets were given intravenous WST 09 followed by laser light illumination of the left mainstem bronchus. Different settings for light dose (fluence), fluence rate (FR), drug dose (D) and drug-light interval (DLI) were applied to each pair. Bronchial toxicity was assessed with repeat bronchoscopic photographic evaluation as well as by pathologic examination following autopsy. Animals developed no toxicity, moderate toxicity or severe toxicity. Increased toxicity was seen with increasing D and fluence and decreasing DLI, whereas no increased toxicity was seen with higher FR. PDT-related histological changes in the normal bronchus confirm the vascular effect of WST 09. Depending on the parameter settings for fluence, D and DLI, the lesions ranged from focal intramucosal ischemia to transmural infarction with subsequent acute inflammation and fibrosis. Clinically feasible parameters for drug and light dosimetry were documented. These data will be important in determining safe starting doses for human phase I/II studies.
Methylated analogues of cis-dichlorobis(1,10-phenanthroline)rhodium(III)chloride (BISPHEN) have been prepared in order to increase the hydrophobicity of the parent compound, and thus create octahedral rhodium (III) complexes suitable for use as anticancer and antiviral agents that can be photoactivated. The parent complex has been shown in earlier work to be unable to cross through cell membranes. Octamethylation, as in the case of cis-dichlorobis(3,4,7,8-tetramethyl-1,10-phenanthroline)rhodium(III)chloride (OCTBP), provides enough hydrophobicity to be taken up by KB tumor cells. It also provides a higher level of ground-state association with double-stranded DNA and increases the quantum efficiency of photoaquation by greater than 10-fold, relative to BISPHEN. OCTBP forms covalent bonds to deoxyguanosine when irradiated with the nucleoside, as has been seen with the parent complex. Irradiation of OCTBP in the presence of the KB or M109 tumor cell lines using narrow-band UVB (lambda = 311 nm) irradiation initiates a considerable amount of phototoxicity. There is evidence that OCTBP acts as a prodrug (i.e. after passing through the cell membrane the metal complex is photolyzed to cis-chloro aquo OCTBP, which may be the active phototoxic agent). OCTBP and the tetramethyl analogue cis-dichlorobis(4,7-dimethyl-1,10-phenanthroline)rhodium(III)chloride (47TMBP) also show photoaquation upon excitation with visible light (lambda > 500 nm), and indeed, some phototoxicity of KB cells is observed at these wavelengths as well. This is attributed to direct population of photoactive triplet-excited states. These results, together with our earlier studies of cis-dichloro[dipyrido(3,2-a: 2',3'-c)phenazine (1,10-phenanthroline)rhodium(III)chloride (DPPZPHEN) demonstrate that such octahedral rhodium complexes are viable "photo-cisplatin" reagents.
Rapidly proliferating transformed mammalian cells can be photodestroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM delta-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,10-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.
The octahedral rhodium complex, cis-dichloro bis(1,10 phenanthroline)rhodium(III) chloride (BISPHEN), is known to form covalent linkages with DNA involving the attachment of the metal to a base. In order to determine the sequence selectivity of this chemistry, solutions of the complex containing one of the double-stranded DNA plasmids, pBSSK.c-raf (eco) or pBSKS+.XE.LTR-F (a construct that contains sequences derived from the long terminal repeat [LTR] region of the human immunodeficiency virus) have been irradiated using UVA light. The DNA samples were denatured after irradiation, a primer was annealed to one of the strands, and a complementary strand was constructed using a polymerase enzyme. Polyacrylamide gel sequencing analysis was used to reveal stops created in the complementary strand caused by the polymerase encountering a metal-bound base. The data indicate that "hot spots" primarily occur at, or adjacent to, guanines (G), with a particularly strong preference for strings of G. In the latter case, the hottest spot is at the 5'G. These results are consistent with our previously postulated mechanism for the covalent binding chemistry which involves photooxidation of deoxyguanosine by the excited state of the metal complex as the primary photochemical step.
The ability of the DNA duplex to behave as an efficient organized medium for cis-trans isomerization induced by electron transfer (ET) has been explored. Isomerization studies, luminescence quenching and DNA photocleavage assays show that photoexcited Ru(1,10-phenanthroline [phen])3(2+) transfers an electron to E,Z1,4-bis[2-(1-methylpyridinium-4-yl)vinyl]benzene (E,Z pMPVB), which subsequently undergoes one-way isomerization to E,E pMPVB. The unusual feature of the system is manifested by the lack of friction that is usually imposed on the photoisomerizable ligand by highly organized media. The apparent rate of ET in DNA increases when compared with the homogeneous solution. However, after correction for the local concentration of the reagents onto the biopolymer, the rate constant becomes independent of the DNA concentration and is at least 4 x 10(2) times smaller than that in the homogeneous aqueous solution. Using the photoinduced isomerization system, a large enhancement in the efficiency of single-strand break formation was found in plasmid DNA over that for Ru(phen)3(+2) alone using irradiation at lambda > 480 nm.
1,2-Diaryloxyethene has recently been proposed as a linker in singlet oxygen-mediated drug release. Even though 1,2-diaryloxyethenes look very simple, their synthesis was not an easy task. Previous methods are limited to symmetric molecules, lengthy step and low yield. We report on a facile synthetic method not only for 1,2-diaryloxyethenes but also their sulfur and nitrogen analogs in yields ranging from 40 to 90% with more than 90% purity at the vinylation reaction. Most recently, light-controlled drug release systems were proposed, where singlet oxygen generated from low energy light and a photosensitizer cleaves electron-rich alkenes, e.g. 1,2-diaryloxyethene. However, the synthesis of 1,2-diaryloxyethene was not well established. Herein, we report a facile synthesis of 1,2-diaryloxyethenes and its sulfur and nitrogen analogs. Model compounds were prepared in four steps from 4-phenylphenol via Ullman-type coupling reaction.
The interaction of biological carotenoids with 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), a thermodissociable source of electronically excited ketones, was investigated using reversed-phase high-performance liquid chromatography. Incubation of the all-trans isomers of beta-carotene, lycopene and canthaxanthin with HTMD led to significant trans-to-cis isomerization, with cis isomers accounting for 20-50% of products formed (the balance assigned as oxidation products). The isomers forming from all-trans-beta-carotene were identified as 9-cis-, 13-cis- and 15-cis-beta-carotene by cochromatography of cis isomer standards and by on-line diode array absorbance spectroscopy. An HTMD-dependent cis-to-trans isomerization was observed in incubations started with 15-cis-beta-carotene, and it occurred more rapidly and to a greater extent than the isomerization of all-trans-beta-carotene. The isomer patterns generated from lycopene and beta-carotene are generally similar to those reported recently for various human tissues (Stahl et al., 1992, Arch. Biochem. Biophys. 294, 173-177).
Isolated calf thymus DNA was treated with the 1,2-dioxetanes 3-acetoxymethyl-3,4,4-tri-methyl-1,2-dioxetane, 2,3-dimethylbenzofuran dioxetane, 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD), 3,3,4,4-tetramethyl-1,2-dioxetane and 3,4,4-trimethyl-1,2-dioxetane (TrMD), which on thermal decomposition generate triplet-excited carbonyl products. To monitor quantitatively the formation of the mutagenic oxidation product 7,8-dihydro-8-oxoguanine (8-oxoGua), a sensitive and selective HPLC electrochemical assay was used after acidic hydrolysis (HF/pyridine) of the dioxetane-treated DNA. High yields of 8-oxoGua (up to ca 4% of the available guanine) were obtained for HTMD and TrMD. Both were investigated in detail with respect to effects of concentration, time and temperature. The oxidative reactivity of 1,2-dioxetanes was compared with several type I (benzophenone and riboflavin) and type II (methylene blue and rose bengal) photooxidants and disodium 1,4-etheno-2,3-benzodioxin-1,4-dipropionate as a chemical source of singlet oxygen. The persistence of 8-oxoGua towards oxidation by HTMD was examined in the reaction with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodGuo) and with oxidized DNA. It was shown that, indeed, 8-oxoGua is consumed in the oxidized DNA on prolonged exposure to an excess of HTMD. The reaction of 8-oxodGuo with HTMD afforded the two 4R* and 4S* diastereomers of 9-(2-deoxy-beta-D-erythropentofuranosyl)-4, 8-dihydro-4-hydroxy-8-oxoguanine as main oxidation products. Trapping experiments with tert-butanol confirmed that hydroxyl radicals are not involved, whereas the use of the triplet quenchers sodium 9,10-dibromo-anthracene-2-sulfonate and 2,3-diazabicyclo[2.2.1]hept-2-ene established that triplet-excited states are mainly responsible for the observed DNA oxidation through type I action (electron transfer chemistry). The role of singlet oxygen was tested by means of deuterium isotope effects in D2O versus H2O, but no definitive conclusion could be reached in regard to the involvement of 1O2 in these oxidations.(ABSTRACT TRUNCATED AT 250 WORDS)
Recent increase and wider use of ionic liquids (ILs) for various applications has drawn attention to their toxicological consequence on human health. The present study explores effects of three different kinds of widely used ILs, such as 1-methyl-3-octylimidazolium chloride, 1-buytl-3-methyl imadazolium tetrafluoroborate and 1-benzyl-3-methyl imidazolium tetrafluoroborate, on liposome properties of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) by applying curcumin as molecular probe. Fluorescence intensity of curcumin is reported as a novel rotor which is sensitive to viscosity and thus the fluidity of the solvent. It follows a linear relationship of log fluorescence vs viscosity as proposed by Förster-Hoffmann equation. Curcumin binds strongly to liposome. At low concentration, the lipophilic drug curcumin does not appreciably influence the phase transition temperature of DPPC but as concentration reaches high levels significantly depresses the phase transition temperature. ILs diminish membrane fluidity. 1-methyl-3-octylimidazolium chloride disorders membrane properties by lowering the phase transition as is observed for higher concentration of curcumin, but 1-buytl-3-methyl imidazolium tetrafluoroborate and 1-benzyl-3-methyl imidazolium tetrafluoroborate do not modify phase transition temperature perceptibly; rather they broaden the phase transition at low molar concentration ratio. The three different kinds of ILs under study behave similarly at a high IL:DPPC ratio (1:2), while they behave differently at lower ratios (1:10-1:5).
The kinetics of the microsecond phototransformation intermediates of 124 kDa Avena phytochrome (I700(1,2) were studied in the presence of bound monoclonal antibodies at various temperatures. A global analysis was applied to the decays at all wavelengths at each temperature in order to derive the rate constants and the decay-associated spectra of the three decay components. Monoclonal antibodies bound to specific epitopes altered the Arrhenius parameters of both I700(1,2) decay components. The strongest influence on these parameters was observed with OAT 8 (epitope between residues 624 and 686), which decreased by more than 50% the activation parameters of both components. This decrease is interpreted to result from an increased flexibility induced by this antibody in the ground state or in the transition state of bonds changing during the decay of both I700 transients. Thus, the OAT 8 epitope appears to be functionally important during the decay of the I700(1,2) intermediates. For the case of I700(1), bound OAT 23 and OAT 25 (epitopes between residues 1 and 66) reduced even further the relatively small flexibility of these bonds in the red light-absorbing form of phytochrome (Pr) without antibodies, as reflected by the high preexponential factors for its decay. This resulted also in higher activation energies for this decay in the presence of the antibodies. Thus, the amino-terminus should act as a rigid spacer of the chromophore cavity without affecting it during the microsecond transformation, because the Arrhenius parameters for these decays are similar to those for small phytochrome. The possible implications of the influence of the various antibodies on the bleaching remaining after the decay of I700(1,2) are discussed.
1,2-Dioxetanes are efficient sources of triplet excited carbonyl compounds on thermal decomposition. They cause photochemical and photobiological transformations in the dark. In order to study the genotoxicity and mutagenicity of 1,2-dioxetanes, the replicating shuttle vector pZ189 was damaged with 3,3,4-trimethyl-1,2-dioxetane (TrMD) or 3-hydroxymethyl-3,4,4-trimethyl-1,2-dioxetane (HTMD) in vitro and subsequently transfected into normal human lymphoblasts. We found a dose-dependent increase of genotoxicity (decrease of plasmid survival) and increase of mutation frequency with both dioxetanes. However, TrMD was less mutagenic than HTMD at similar genotoxicity. Sequence analysis of the supF gene revealed more point mutations with TrMD and 100% with HTMD were G:C to T:A and G:C to C:G transversions. These are the typical mutations following 7,8-dihydro-8-oxoguanine (8-oxo-G) formation, the main DNA lesion induced by TrMD and HTMD. Only with TrMD we found 5.4% G:C to A:T transitions, probably reflecting the more pronounced ability of TrMD to form some pyrimidine dimers. Our results indicate that 8-oxo-G is also the most relevant modification in in vivo mutagenesis.
In this article we have examined the very low-temperature photochemistry of three acyclic 1,3-dienes. We have used high-temperature deposition techniques combined with matrix isolation to create samples enriched with the thermally meta-stable s-cis form. This technique has allowed us to examine the separate photochemistry of the s-cis and s-trans conformers. Our results suggest the presence and the absence of barriers on the excited-state surface. In particular, we have found that the electrocyclic closure and s-cis-s-trans photochemical isomerization stops at 15 K for 2,3-dimethyl-1,3-butadiene-d10. The closure occurs at higher temperatures in solution but is slowed by a deuterium isotope effect. The s-trans conformer of EE-2,4-hexadiene shows almost no photoreactivity in a matrix under 254 nm irradiation, but the s-cis conformer is rapidly converted to ZE-2,4-hexadiene (ZE-HXD). The photoreactivity of ZE-HXD is similar in that there is a relatively quick conversion of the s-cis conformer under these conditions, with only a very slow conversion of the s-trans to photoproducts.
— Theanthraquinone–2-sulfonate photo-sensitized splitting of cis-syn 1,3-dimethylthymine dimer gives rise to large CIDNP effects in the reaction product 1,3-dimethylthymine. The polarization originates from a radical ion pair formed by electron transfer from the dimer to the triplet state sensitizer. In a deoxygenated solution the sign of the polarization of theC–6 proton is reversed compared to the predicted one on account of the CIDNP sign rules. In an aerated solution the correct sign is observed. This can be accounted for by assuming reduction of the lifetime of the radical pair in the presence of oxygen. The time-resolved photo-CIDNP technique was used to study the time dependence of the 1,3-dimethylthymine signal. To account for this time-dependence a cation radical disproportionation reaction is proposed.
The emission properties of a series of substituted 1,3-diarylisobenzofurans have been studied. Most compounds exhibit very intense emission in the nanosecond timescale at room temperature as well as at 77 K. The room temperature emission is attributed to the deactivation of a twisted intramolecular charge transfer excited state, based on its energy, shape and solvent dependence. The experimental results are strongly supported by a theoretical study on one representative compound. The DFT/TD-DFT calculations demonstrate that the initial excited state relaxes toward a twisted structure.
The 5 microM hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) was studied in homogeneous ethanolic solutions and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine; both the porphyrin and DPBF are embedded in the phospholipid bilayer. The rate and quantum yield of DPBF photooxidation were found to increase upon increasing the substrate concentration and were higher in the liposome system, while they were unaffected by the fluidity of the phospholipid bilayer. Time-resolved spectroscopic measurements showed that the photooxidation of DPBF in ethanol solution proceeds by a type II O2(1 delta g)-involving mechanism. In the liposomal vesicles the high local concentration of hematoporphyrin (Hp) and DPBF in the phospholipid bilayer (ca 2000-fold higher than the stoichiometric concentration) enhances the probability of energy transfer from triplet Hp to DPBF with generation of triplet DPBF; hence O2 (1 delta g) formation can be promoted by both triplet Hp and triplet DPBF. A minor fraction of triplet DPBF quenchings appears to generate radical species which propagate DPBF damage by chain reaction.
Intramolecular processes of deactivation of 1,3-dimethyl-4-thiouracil (DMTU) from the second excited singlet (S2) (pi, pi*) and the lowest excited triplet (T1) (pi, pi*) states have been studied using perfluoro-1,3-dimethylcyclohexane (PFDMCH) as a solvent. The spectral and photophysical (PP) properties of DMTU in CCl4, hexane and water have also been described. For the first time, the fluorescence from S2 state DMTU has been observed. The picosecond lifetime of DMTU in the S2 state (tau(S2)) in PFDMCH has been proposed to be determined by a very fast intramolecular reversible process of hydrogen abstraction from the ortho methyl group by the thiocarbonyl group. The shortening of tau(S2) in CCl4 is interpreted to be caused by the intermolecular interactions between DMTU (S2) and the solvent. Results of the phosphorescence decay as a function of DMTU concentration were analyzed using the Stern-Volmer formalism, which enabled determination of the intrinsic lifetime of the T1 state (tau0(T1)) and rate constants of self-quenching (k(sq)). The lifetimes, tau0(T1), of DMTU in PFDMCH and CCl4 are much longer than the values hitherto obtained in more reactive solvents. The PP properties of DMTU both in the S2 and T1 states have been shown to be determined by the thiocarbonyl group.
— A photochemically induced reaction of1,3-dimethylthymine (DMT) with isopropanol leads to the formation of four alcohol adducts. The products have been identified as the cis and trans isomers of5,6-dihydro-1,3-dimethyll-6-(2-hydroxy-2-propyl) thymine (I and II), 2.4-diaza-8-hydroxy-2.4,6.8-tetramethylbicyclo[4.2.0]octan-1,3-dione (III), and5,6-dihydro-1,3-dimethyl-6-(2-oxo-l-propyl)-thymine (IV). An acetone photosensitized reaction of DMT with isopropanol gives the same products in a similar relative yield distribution. In both of these reactions, cyclobutane dimers of DMT are produced as well. Free radical reactions of 2-hydroxyisopropyl radicals with DMT, initiated by decomposition of di-t-butyl peroxide, leads to formation of only one of the cis and trans isomers described above. along with1,3-dimethyl-5-(2-hydroxy-2-methyl-1-propyl)uracil (V).
This article reports further elaboration of the authors' investigations on the acid-catalyzed photorearrangements undergone by derivatives of 1,3-dimethylcyclooctapyrimidine-2,4-dione. In this case the results are represented for compounds containing a simple methyl or fluorine substituent at different positions in the eight-membered ring. Upon photolysis in frozen benzene containing trifluoroacetic acid, the monomethylated compounds give varying yields of pentalenopyrimidinedione products that are not generated from the fluorinated compounds, which are either inert or undergo hydration accompanied by dehyrofluorination. Plausible mechanisms are advanced to account for the formation of the observed photoproduct species. The work represents an interesting extension and uncovered new aspects of the photorearrangement process.
A derivative of all-trans-retinal (RAL) and ethanolamine, A2-E, is the main fluorescent component of human retinal lipofuscin. The accumulation of lipofuscin has been correlated with exposure to ambient radiation and loss of photoreceptors. A possible precursor to A2-E is the imine formed from RAL and ethanolamine. This compound, (E,E,E,E)-2-[9-(2-hydroxyethyl)imino-3,7-dimethyl-1,3,5,7- decatrien-1-yl]-1,3,3-trimethylcyclohexene (HIDD), has been synthesized and structurally characterized. The photophysical and photochemical properties of HIDD and its protonated form, HIDD-H+, have been investigated using steady-state and time-resolved methods. Both HIDD and HIDD-H+ are weakly fluorescent, and the fluorescence lifetime and quantum yield for HIDD are ca 0.6 ns and 4.0 +/- 0.5 x 10(-4), respectively. HIDD forms a triplet excited state on direct excitation that decays with kd = 3.4 x 10(4) s-1. The extinction coefficient and quantum yield of intersystem crossing for the HIDD triplet are measured as 7.6 +/- 1.3 x 10(4) M-1 cm-1 and 0.055 +/- 0.006, respectively. The triplet excited state of HIDD-H+ can be sensitized by triplet energy transfer and has a decay rate constant of 1.6 x 10(4) s-1. The lifetime of the HIDD triplet excited state is observed to decrease with increasing concentration of the well-known electron or hydrogen atom donors, 2,3,5,6-tetramethyl-1,4-phenylenediamine and 2,3,5-trimethylhydroquinone, and the bimolecular rate constants for these reactions are approximately 5.4 x 10(6) M-1 s-1 and 1.7 x 10(8) M-1 s-1, respectively. These types of reactions may model photooxidative mechanisms of damage in the retina.
The interaction between aromatic hydrocarbons and purine or its analogues has been widely noted in connection with the possibility of complex formation between carcinogens and DNA or its precursors. Weil-Malherbe[1], Boyland and Green[2] reported that 1,3,7,9-tetramethyluric acid (TMUA) solubilizes aromatic hydrocarbons in aqueous solution and exerts a strong quenching action on fluorescence of the hydrocarbons. The solubilization has been attributed to formation of a molecular complex [2]. Complex formation is now thought to be mainly due to van der Waal's force (polarization and dispersion force) rather than charge transfer (CT) one[2,3–5]. Van Duuren [3,5] examined fluorescence spectra of TMUA-hydrocarbon complexes both in solution and in potassium bromide pellets. In solution, the fluorescence spectrum of the complex showed no change in the position of the maximum and in intensity, when compared with that of the free hydrocarbon. In the solid state, however, a blue shift was observed for the fluorescence of the complex. From these results, it was concluded that the complex is entirely dissociated in solution and the fluorescence shift observed in the solid state is the result of the interaction in the excited state. The present note describes a new observation which is useful for understanding the mechanism of fluorescence quenching in the hydrocarbons-TMUA systems.
Photoirradiation of nitrogen-saturated aqueous solutions containing aluminum phthalocyanine tetrasulfonate (AlPcS4) at 675 nm in the presence of 2,5-dichloro-diaziridinyl-1,4-benzoquinone (AZDClQ) and hypoxanthine (HX) produces the oxidized HX derivatives, xanthine (X) and uric acid (UA). Concentrations of the AZDClQ semiquinone, X and UA increase at the expense of HX with an increase in irradiation time. Almost negligible decomposition of HX, as well as very low amounts of X, are detected if photolysis occurs under identical conditions but in the absence of AZDClQ. Addition of calf-thymus DNA produces quinone-DNA covalent adducts after photolysis of anaerobic samples containing quinone, DNA and AlPcS4, in the presence or absence of HX and at pH 5.5. However, larger amounts of quinone-DNA adducts are detected if HX is present. The results presented here could have applications in the photodynamic treatment of hypoxic tissues such as solid tumors, under conditions of high HX concentration, where Type-I pathways could be more important than singlet oxygen generation.
—1,4-Diphenylbutadiyne (DPB), the first member of a new chemical class of phototoxic compounds, was found to be active against E. coli (strain B) under nitrogen but not in the presence of air. It was also phototoxic against both Candida utilis and Saccharomyces cerevisiae aerobically, and against the latter anerobically. The maximum activity against C. utilis occurred at 360 nm, and the uv light was definitely required in the phototoxic act. Circular dichroism spectroscopy disclosed that DPB underwent photoreaction with DNA. This technique, as well as melting temperature profiles, hyperchromicity studies, and the elution profile from hydroxyapatite chromatography indicated that no cross-linking to double strands had occurred, and that DPB behaved as a monofunctional reagent, interacting only with single strands of DNA photochemically. Finally, 2,5-diphenylthiophene was synthesized from DPB but was not phototoxic.
Abstract— The photobiological activity of nitrazepam, the active ingredient of the drug Mogadon®. which is known to induce phototoxic effects, that of clonazepam, the active ingredient of the drug Rivotril® and that of flunitrazepam, the active ingredient of the drug Rohypnol®, has been investigated. Using Salmonella typhimurium strain TA-100 as the test organism for cytotoxicity, it was demonstrated that not only nitrazepam, but also the structurally related compounds clonazepam and flunitrazepam were phototoxic. Furthermore, it was shown that this phototoxic effect was due to a photodynamic action of the compounds. Investigations of the (photo)biological activity of the decomposition products (7-nitroso, 7-hydroxylamino, 7-amino analogues and the azoxy dimer) of the parent 7-nitro compounds revealed that they were not involved in the phototoxic effect. Investigations on structurally related benzodiazepines have established that a 7-nitro substituent in the benzodiazepine nucleus is the structural characteristic responsible for the appearance of phototoxicity and that the magnitude of this phototoxic effect is strongly determined by the presence or absence of a methyl-group at the N-1 position. Finally, photomutagenicity tests performed with the same bacterial indicator indicated that under the present experimental conditions the oxygen dependent reactive species formed upon irradiation of nitrazepam, clonazepam and flunitrazepam with longwave UV light did not exert mutagenic effects.
The absorption and fluorescence properties of nifedipine (NPDHP), felodipine (CPDHP) and a series of structurally related 1,4-dihydropyridines were studied in aqueous solution and organic solvents of different properties. The absorption and fluorescence spectra were found to depend on the chemical nature of the substituents at the position 4 of the 1,4-dihydropyridine ring (DHP) and on solvent properties. In aqueous solution, the fluorescence spectra of 4-phenyl substituted compounds are blue-shifted with respect to the alkyl substituted compounds. The more fluorescent compound is CPDHP. Nifedipine is not fluorescent. All compounds, with the exception of CPDHP, present monoexponential fluorescence decay with very short lifetime (0.2-0.4 ns). CPDHP showed a biexponential emission decay with a long-lived component of 1.7 ns; this behavior is explained in terms of different conformers because of the hindered rotation of the phenyl group by the ortho-substitution. Analysis of the solvent effect on the maximum of the absorption spectrum by using the linear solvent-energy relation solvato-chromic equation indicates the redshifts are influenced by the polarizability, hydrogen bonding ability and the hydrogen bond acceptance of the solvent. Whereas, the fluorescence characteristics (spectra, quantum yields and lifetimes) are sensitive to the polarizabilty and hydrogen bond ability of the solvents. Photo-decomposition of nifedipine is dependent on the solvent properties. Faster decomposition rates were obtained in nonprotic solvents. The 4-carboxylic derivative goes to decarboxylation. Under similar conditions, the other DHP compounds did not show appreciable photodecomposition.
The electronic nature of substituents attached to the 4-aryl moiety of 1,4-dihydropyridines strongly affect the photophysical and photochemical behavior of these family of compounds. The presence of an electron-donor substituent on the 4-aryl moiety (or the absence of electron-withdrawing ones), modifies the luminescence lifetimes (τ < 100 ps) and diminishes the photodecomposition quantum yields. For electron-withdrawing substituents, the photodegradation quantum yield is affected by the media, changing more than two orders of magnitude as the polarity is increased. Studies in micellar media allow us to conclude that 4-aryl-1,4-dihydropyridines are located near to the interface, however the surface charge of micelles has no effect on the photodegradation rate constant or the photoproducts profile. The main conclusion of this work is that the photolability of 4-aryl-1,4-dihydropyridines can be significantly reduced by the incorporation of antioxidant moieties. This article is protected by copyright. All rights reserved.
Abstract— Hydroxyl radicals ('OH) are scavenged by 1,4-diazabicyclo[2.2.2]octane (DABCO) at a diffusion-controlled rate of 1.25 ± 0.1 × 109M-1s-1. Unlike other efficient 'OH scavengers which exhibit protection of bacteria against irradiation both in oxic and hypoxic conditions, DABCO has been shown to protect Serratia marcescens and various strains of Escherichia coli only in oxic conditions. DABCO appears to eliminate a component of the sensitization afforded by oxygen in all strains of E. coli tested. The level of this protection increases from ∼15% in the wild type AB 1157 to ∼100% in the recA uvrA mutant AB 2480. It is suggested that DABCO protects against lethal events that can occur on macromolecules other than DNA such as the cell membrane. Results with added glycerol, as well as work in D2O solution, indicate that DABCO is more likely to be acting by scavenging radicals rather than by quenching 1O2. If 1O2 is a component of the sensitization afforded by oxygen, then it is unlikely to be formed in a hydrophilic environment in the cell.
The photochemistry of lapachol and other 1,4-naphthoquinone (NQ) derivatives, e.g. 2-methoxy-1,4-naphthoquinone (MeONQ), 2-hydroxy-1,4-naphthoquinone (2-HONQ) or 5-hydroxy-1,4-naphthoquinone (5-HONQ) and 2-methyl-5-hydroxy-1,4-naphthoquinone (P-NQ) in solution at room temperature was studied by ultraviolet-visible spectroscopy after nanosecond laser pulses at 248 nm. The triplet state and semiquinone radicals were observed for MeONQ, HONQ and P-NQ, whereas for lapachol, intramolecular H-atom and charge transfer processes take place, as in the case of vitamin K1. The photoinduced reaction of NQ into HONQ is initiated by nucleophilic water addition to the triplet state, and for the secondary reactions, a modified mechanism is proposed.
The photoreaction of 2-methyl-1,4-naphthoquinone (MQ, menadione) with DNA and polynucleotides in argon-saturated aqueous solution (pH 7) was studied. Results from laser flash photolysis experiments indicate that triplet quinone reacts with DNA and polyA but not detectably with polyU by one-electron oxidation of the bases of the nucleic acid with formation of the radical anion of the quinone. Irradiation of argon-saturated solutions containing MQ and DNA or polynucleotides (polyU, polyA, polyG or polyC) with 334 nm light leads to an increase in molecular weight for single-stranded DNA, polyA and to a much less extent for polyU. This finding indicates crosslink formation with quantum yields in the range of 10(-5)-10(-3).
A careful study of the linoleic acid hydroperoxide (LOOH) profile obtained upon peroxidation of linoleic acid (LA) photosensitized by tiaprofenic acid (TPA) and analogous ketones has been undertaken to distinguish between type-I and type-II photoperoxidation mechanisms. 1,4-Cyclohexadiene and 1,2-dimethylcyclohexa-2,5-dienecarboxylic acid (CHDCA) have also been used as models for LA since they also have double allylic systems. Coirradiation of LA with TPA and decarboxytiaprofenic acid (DTPA) in acetonitrile and micellar media produced significant amounts of conjugated dienic LOOH. The cis,trans to trans,trans ratio depended on the irradiation time; thus, this parameter is an ambiguous tool for mechanistic assignment. An interesting finding was the decrease of the LOOH level after long irradiation times in mixtures photooxidized by DTPA, which is attributed to quenching of the DTPA triplet by the generated dienic LOOH. High-performance liquid chromatography analyses confirmed that the main pathway operating in photodynamic lipid peroxidation sensitized by (D)TPA is a type-I mechanism. However, product studies using CHDCA have clearly shown that a type-II mechanism is also operating and might contribute to the overall photooxidation process in a significant way.
New evidence about the path followed in the photochemical reaction of 4-(2-nitrophenyl)-1,4-dihydropyridines such as the drugs nifedipine (Compound 1) and nisoldipine (Compound 2) to give the corresponding nitrosophenylpyridines has been found through determination of the steady-state photochemical parameters and a comparison of the photoreactions in solution and in matrix at 90 K. Additional support is given by comparison with the isomeric 4-(3-nitrophenyl)dihydropyridine as well as with simpler derivatives, such as the corresponding 4-methyldihydropyridine. In Compounds 1 and 2, the lowest lying singlet, localized on the dihydropyridine chromophore, is deactivated by (largely exothermic) electron transfer to the nitrobenzene moiety, as evidenced by the complete quenching of the blue fluorescence observed in analogues not containing the electron-accepting group. Intramolecular proton transfer ensues in the 2-nitrophenyl derivatives with a relatively medium-independent quantum yield of approximately 0.3 and leads to an aromatic zwitterion, which is detected in matrix at 90 K (photoionization of this intermediate takes place in 2-methyltetrahydrofuran secondary). The intermediate is smoothly converted into the end product upon melting the glass. The 3-nitrophenyl analog, for which such a path is not available, is less reactive by about three orders of magnitude at 366 nm, although the quantum yield arrives at approximately 0.01 by irradiation at 254 nm in MeOH, reasonably via the nitrophenyl localized triplet.
Steady-state UV irradiation of aqueous solutions containing cytochrome c (cyt c) and N,N'-bis(2-phosphonoethyl)-1,4,5,8-naphthalenediimide (BPNDI), a water-soluble aromatic imide, resulted in the reduction of the heme iron from the Fe(III) to the Fe(II) oxidation state. The reaction kinetics were followed by the increase of the ferrocytochrome c absorbance band at 549 nm. The rate of the photochemical reaction was pH dependent, reaching its maximum values over the pH range 4-7. Addition of electrolyte (NaCl) at pH 5 resulted in a decrease in the reaction rate, as expected for reactions between oppositely charged species. Flash photolysis studies revealed that the actual reductant in the reaction was a photogenerated BPNDI radical anion, which transferred an electron to the cyt c heme iron. The participation of imide radicals in the process was confirmed by the ready reduction of cyt c by BPNDI radicals chemically generated with sodium dithionite.
The pyrimidine nucleobases contained in DNA undergo a variety of photoinduced reactions in which two moieties become joined to form a product (e.g. formation of cyclobutane dimers and [6-4] adducts). Herein, we describe a new type of photoconjugation reaction that has been shown to occur for 5-methylcytosine (5-MeC), 1,5-dimethylcytosine (1,5-diMeC), 1-methylthymine and thymidine; in this reaction the 5-methyl group of one nucleobase (or nucleoside) becomes attached to the 4-position of the second moiety. For example, 5-MeC forms α-4'-(5'-methylpyrimidin-2'-one)-5-methylcytosine. The various (α-4) conjugates are produced upon irradiation of the parent compound in frozen aqueous solution at -78.5°C. The UV spectra of these compounds display a characteristic "double humped" profile, similar to that expected from overlaying the spectrum of parent nucleobase with that of a 2'-pyrimidone moiety. Preliminary results suggest that thymine and 5-methyl-2'-deoxycytidine (5-MedCyd) form analogous photoproducts. A variety of other previously unreported photoproducts are described as well for the 5-MeC, 1,5-diMeC and 5-MedCyd systems.
Eight single-stranded oligodeoxyribonucleotides 32P-labeled at the 5'-end were synthesized; they were annealed with the complementary oligodeoxyribonucleotides to form the corresponding double-stranded helices. These duplexes possessed standard Watson-Crick base pairs, locally perturbed sites of a base mismatch, or a bulge. Further, 5'-32P-labeled oligodeoxyribonucleotides with a hairpin loop were also synthesized. Cleavage of these single- and double-stranded oligodexyribonucleotides selectively at the deoxyguanosine residue was accomplished by use of 3-(p-tolylamino)-1,5-azulenequinone 1 upon irradiation with 350 nm UV light. The single strands were cleaved more efficiently than the double-helices. For the helices containing a deoxyguanosine residue at a bulge, at a hairpin loop or toward the end, the cleaving efficiency was increased. Computation results indicate that two possibilities exist for agent 1 to form two "Watson-Crick type" hydrogen bonds with guanine in single-stranded oligodeoxyribonucleotides; yet, only one possibility exists in duplexes.
In etiolated rye seedlings transferred to light the expression of chlorophyll a/b binding protein mRNA varies when the seedlings are grown in a day/night cycle. The fluctuation pattern follows a circadian rhythm. Exposure of 4-day old etiolated seedlings to continuous white light revealed two maxima within the first 24 h before the 24 h cycle period appeared. These first two maxima are also observable after a pulse of white light or after a pulse of red light. These results indicate a possible involvement of phytochrome in the endogenous regulation of the rhythm.
A variety of nucleic acid components and related compounds undergo photoreaction with water to form so-called "photohydrates" (e.g. uracil forms 6-hydroxy-5,6-dihydrouracil). However, the corresponding hydrates of 5-methylcytosine (a minor nucleobase in eukaryotic DNA) and related compounds have not been characterized. We report the preparation of opened-ring forms of such products for 5-methylcytosine (m5C) and 1,5-dimethylcytosine (DMC). This was accomplished via thermal reaction of ring-opened amine adducts (e.g. N-carbamoyl-3-amino-2-methylacrylamidine (IVa) or N-(N'-methylcarbamoyl)-3-amino-2-methylacrylamidine (IVb)) produced by photo-induced reactions of m5C with ammonia or methylamine. When these adducts were treated with dilute trifluoroacetic acid, the amino group at the 3-position was replaced with a hydroxyl group; with IVa, N-carbamoyl-3-hydroxy-2-methylacrylamidine (Va) was formed, while reaction of IVb led to N-(N'-methylcarbamoyl)-3-hydroxy-2-methylacrylamidine (Vb). These compounds are ring-opened isomers of 5,6-dihydro-6-hydroxy-5-methylcytosine (Ia and IIa) and 5,6-dihydro-6-hydroxy-1,5-dimethylcytosine (Ib and IIb). Compounds Va and Vb each undergo thermal ring closure reactions to form two unstable compounds with chemical and UV spectral properties expected for Ia and IIa (or Ib and IIb). The latter compounds have been identified as minor products in UV-irradiated aqueous solutions of m5C and DMC. Evidence is also presented that the 2'-deoxycytidine photohydrates coexist with an opened-ring form, possibly similar in nature to Vb.
The environment of Trp57, introduced by the mutation of a tyrosine in the dynamic loop of porcine liver fructose-1,6-bisphosphatase (FBPase), was examined using time-resolved fluorescence and directed mutation. The Trp57 enzyme was studied previously by X-ray crystallography and steady-state fluorescence, the latter revealing an unexpected redshift in the wavelength of maximum fluorescence emission for the R-state conformer. The redshift was attributed to the negative charge of Asp127 in contact with the indole side chain of Trp57. Time-resolved fluorescence experiments here reveal an indole side chain less solvent exposed and more rigid in the R-state, than in the T-state of the enzyme, consistent with X-ray crystal structures. Replacement of Asp127 with an asparagine causes a 6 nm blueshift in the wavelength of maximum fluorescence emission for the R-state conformer, with little effect on the emission maximum of the T-state enzyme. The data here support the direct correspondence between X-ray crystal structures of FBPase and conformational states of the enzyme in solution, and provide a clear example of the influence of microenvironment on the fluorescence properties of tryptophan.
Hypericin in organic solvents displays two types of electronic spectra: one type which shows a distinct solvatochromic effect, the stable form, and the other, the unstable form, which lacks this property. The latter type is formed in dry nonprotic solvents (e.g. tetrahydrofuran, EtOAc) and can be converted to the stable form on addition of protic solvents. In order to establish the tautomeric structure of the unstable form we applied conventional nuclear magnetic resonance techniques as well as two-dimensional gradient-enhanced heteronuclear multiple-quantum correlation, gradient-enhanced ROESY and one-dimensional nuclear Overhauser effect difference experiments. All these techniques pointed to the fact that the unstable form has the 7,14-diketo tautomeric structure, like the stable form, and not the 1,6-diketo structure. Electronic spectroscopy indicated that the unstable form has acidic properties and therefore possesses two free OH groups at C3 and C4 at the bay region of the molecule.
The molecular structure of 1,4,6,8-tetramethylfuro[2,3-h]quinolin-2(1H)-one (FQ), a recent furocoumarin-like photosensitizer, has been modified with the aim of reducing its strong genotoxicity, by replacing the methyl group at 4 position with a hydroxymethyl one, and so obtaining 4-hydroxymethyl-1,6,8-trimethylfuro[2,3-h]quinolin-2(1H)-one (HOFQ). This modification gave rise to a strong reduction of lipophilicity and dark interaction with DNA. The formation of monoadducts (MA) was deeply affected, whereas the induction of bifunctional adducts between DNA and proteins (DPC(L>0)) was replaced by an efficient production of DNA-protein cross-links at zero length (DPC(L=0)), probably via guanine damage. Because of its angular molecular structure, HOFQ does not form interstrand cross-links (ISC): therefore, DPC(L=0) and MA represent the main lesions induced by HOFQ in DNA. In comparison with FQ (which induces MA and DPC(L>0)) and 8-methoxypsoralen (8-MOP) (MA, ISC, DPC(L>0)), HOFQ seems to be a more selective agent. In fact, contrary to FQ and 8-MOP, HOFQ, together with a noticeable antiproliferative activity, shows low levels of point mutations in bacteria and of clastogenic effects in mammalian cells. HOFQ is also an efficient apoptosis inducer, especially in comparison with 8-MOP, when tested at equitoxic experimental conditions; this property might be correlated with the complete HOFQ inability of inducing skin erythemas, a well-known side effect of classic furocoumarin photosensitization.
In order to provide a thorough characterization of a system with which to study the dielectric response of a protein, a well-defined system complex of a fluorescent probe and protein is required. We have argued that such a system is provided by coumarin 153 and apomyoglobin (Photochem. Photobiol. 79, 440-446 [2004]). In order to demonstrate further that coumarin 153 exhibits negligible nonspecific binding to the surface of apomyoglobin, we study its interactions with both the apo and holo proteins. We further make a similar comparison with 8-anilino-l-naphthalenesulfonic acid, for which an NMR structure with apomyoglobin has been obtained. Our results confirm the appropriateness of the system of coumarin 153 and apomyoglobin for the investigation of solvation by the protein matrix.
Top-cited authors
David Kessel
  • Wayne State University
Kristian Berg
  • Oslo University Hospital
Robert W. Redmond
  • Harvard University
Hasan Mukhtar
  • University of Wisconsin–Madison
Brian C Wilson
  • University of Toronto