ABSORPTION SPECTRAL SHIFTS OF CAROTENOIDS RELATED TO MEDIUM POLARIZABILITY

Abstract–Solvent induced absorption spectral shifts of the electronic transition from ground 1 A_g state to the excited 1B_u state in carotenoids have been studied. It is shown that the shift depends only on dispersion interactions in non-polar solvents. In polar media there is just a small extra contribution to the red-shift, due to other forms of interactions. The spectral shifts are well described by the theory, which expresses the shift relative to the gas phase value, as a function of solvent polarizability. The main conclusion is that the dominating mechanism behind the large red-shifted absorbance of carotenoids in the proteinacous environment, in vivo, is the mutual polarizability interactions between the carotenoids and the surrounding medium. The solution-phase values of the dipole moments of the lA_g to 1B_u transitions and the differences of isotropic polarizability between 1B_u and lA_g states of carotenoids in non-polar solvents are calculated and found to be around 13 D and 360 ų respectively. From the great overlap of absorption spectra between carotenoids in quinoline and carotenoids in vivo in purple bacterial antenna complexes, it can be expected that the carotenoids are surrounded by several aromatic amino acids in vivo. Comparisons have been done between the exicted states in carotenoids and in linear conjugated polyenes.     

PICOSECOND ABSORPTION STUDIES OF Zn-OCTAETHYLPORPHIN π-MONOANIONS

Abstract— Using the example of Zn-octaethylporphin π-monoanion solution in tetrahydrofuran, we studied the dynamics of ultrafast photoprocesses in porphyrin π-anions using a tunable picosecond absorption spectrometer. The deactivation kinetics of the lowest excited electronic (doublet) state of Zn-octaethylporphin iT-monoanion has been determined and was found to be followed by a single exponential function with the characteristic time 135 ps. It is shown that upon picosecond excitation to the long wavelength absorption band (λ_max= 830 nm) the process of two-quantum photoionization of the involved monoanion is easily realized, with the results that the observable kinetics of spectral changes exhibit a strong dependence on the excitation intensity.     

LOW TEMPERATURE POLARIZED ABSORPTION MICROSPECTROSCOPY OF SINGLE CRYSTALS OF THE REACTION CENTER FROM Rhodobacter sphaeroides WILD TYPE STRAIN 2.4.1

The absorbance and polarized absorbance spectra of single crystals of the reaction center complex isolated from Rhodobacter sphaeroides wild type strain 2.4.1 have been measured at 85 K. The crystals of the complex were obtained by the vapor diffusion technique. The spectroscopic experiments on the crystals were performed using an optical microspectrometer featuring a custom-built, liquid N2-flowing cold stage, the details of which are presented herein. These data demonstrate the feasibility of conducting optical spectroscopic experiments at cryogenic temperatures on single crystals of photosynthetic pigment-protein complexes.     

SPECTROSCOPY OF POLYENES—IV. ABSORPTION AND EMISSION SPECTRAL PROPERTIES OF POLYENE ALCOHOLS RELATED TO RETINOL AS HOMOLOGUES

A number of polyene alcohols related to retinol as homologues have been examined for their spectral and photophysical properties. The absorption spectra of the polyene alcohols with short polyene chainlength show an intense band system with its maximum at 3000–4000 cm^-1 above that of the main band system. The intensity of this higher-energy band system decreases sharply as the polyene chainlength is increased. All the polyene alcohols fluoresce strongly or moderately strongly at 77 K, the intrinsic fluorescence lifetimes being significantly longer than those expected from the oscillator strengths of the main, low-energy absorption band system. Fluorescence (relatively weak) is also observed at room temperature in the cases of polyene alcohols of long chainlength. A discussion is presented regarding the possible assignments of the various observed absorption band systems, state order and nature of the lowest excited singlet state, and aggregate formation (in 3-methylpentane at 77 K) and its effect on spectral and photodynamical behavior.     

ENERGY TRANSFER-ENHANCED CHEMILUMINESCENCE OF ADAMANTANONE (n,π*) AND ESTER (π,π*) SINGLET AND TRIPLET EXCITED STATES IN THE THERMOLYSIS OF SILYLOXYARYL-SUBSTITUTED SPIROADAMANTYL DIOXETANES†

Abstract— The thermal generation of singlet and triplet excited states from silyloxyaryl-substituted spiroadamantyl dioxetanes lab and the adamantylidineadamantane dioxetane (1c) was investigated by direct and enhanced chemiluminescence (CL). 9,10-Diphenylanthracene (DPA) and 9-fluorenone were used as energy acceptors in the singlet-singlet (S-S), naphthalene and europium chelate Eu(TTA)₃Phen (TTA = thenoyltrifluoroacetone, Phen = 1,10-phenanthroline) in the triplet-triplet (T-T) and 9,10-di-bromoanthracene (DBA) in triplet-singlet (T-S) energy transfer experiments. The direct chemiluminescence observed in the thermolysis of dioxetanes lab consisted of fluorescence derived from the singlet-excited adamantanones 2a,b. In the presence of naphthalene, selective T-S energy transfer with DBA (napthalene as quencher) displayed the adamantanone triplets 2a,b and with Eu(TTA)₃Phen (naphthalene as mediator) also the silyloxyaryl ester 3 triplets. From the Stern-Volmer constants (k_TNT_T⁰) the triplet lifetimes t⁰_t of these triplet state products were assessed. By using the Hastings-Weber standard, the total triplet excitation yield (φ^t) was estimated to be ca 20%. The energies of the first excited singlet and triplet states of the adamantanones 2a,b and the silyloxyaryl ester 3, the products of the thermally induced decomposition of dioxetanes la-c , were determined by semiempirical calculations (AMI-based configuration interaction), which included explicitly solvent effects on the excitation energies in terms of a self-consistent reaction field approach. The calculations revealed that the first excited singlet and triplet states of the adamantanones 2a,b are expectedly n,π*-type excitations while the silyloxyaryl ester 3 possesses π,π* character. The semiempirical computations suggest that excitation of the adamantanones 2a,b as well as the silyloxyaryl ester 3 is feasible in the thermolysis of the spiroadamantyl dioxetanes lab , which has been confirmed by the experimental energy transfer studies.     

SPECTROSCOPY OF POLYENES–III. ABSORPTION AND EMISSION SPECTRAL INVESTIGATION OF POLYENE SCHIFF BASES AND PROTONATED SCHIFF BASES RELATED TO VISUAL PIGMENTS

Abstract— A number of n -butylamine Schiff bases of polyenals related to retinals as homologues and analogues, and their protonated forms, have been studied for absorption and emission spectral properties. The polyene Schiff bases exhibit the same general features in their absorption spectra as those of the parallel polyenals except that the ^lB_u←¹A_g and π*← n singlet transitions are at substantially higher energy in the Schiff bases (the shift being larger for the π *← n transition). The Schiff bases with short polyene chainlength ( n = 2, 3 where n is the number of double bonds including C=N) do not fluoresce or phosphoresce in 3-methylpentane in the temperature range 298–77 K. The Schiff bases with intermediate chainlength ( n = 4, 5) show fluorescence at 77 K with intensity strongly dependent on the nature of solvent. The Schiff bases with relatively long chainlength ( n = 5–7) show strong or moderately strong fluorescence at 77 K and very weak fluorescence at 298 K ( n = 7) with intrinsic radiative lifetimes much longer than those estimated from the oscillator strength of the low-energy, strong absorption band (¹B_u←¹ A_g ). A discussion on the possible state order and nature of the fluorescing state of the various polyene Schiff base systems is presented.     

A SELF-CONSISTENT-FIELD STUDY OF THE EXCITED STATE π-ELECTRON CHARGES IN PHENOL

Abstract— –The semiempirical self-consistent-field (SCF) method was used to calculate the net π-electron charges for phenol in the first excited singlet and first excited triplet states. These calculations differ from the usual ground state calculations in that (i) recently available static excited state data were used wherever possible as the empirical basis for evaluating SCF parameters and (ii) the theory of density matrices was used to include the effects of all singly-excited configurations in the configuration interaction contributions to the excited state π-electron charge densities.     

ARYL AZIDES AS PROTEIN PHOTOLABELS: ABSORPTION SPECTRAL PROPERTIES AND QUANTUM YIELDS OF PHOTODISSOCIATION

Abstract A series of ring-substituted phenyl and naphthyl azides have been prepared. Electronic absorption spectral properties and quantum yields of disappearance have been determined in aqueous solution. For phenyl azide derivatives, the wavelengths of maximum absorption vary systematically, correlating with electronic effects of the ring substituents. Quantum yield values of 0.1–0.7 are obtained and are independent of a number of experimental conditions, including concentration. There is no simple correlation of the quantum yields of azide disappearance with electronic effects, except that the naphthyl azides have higher photochemical efficiencies than the phenyl azide derivatives. A series of potential protein photolabels has been identified based on electronic absorption characteristics and photochemical efficiencies.     

In vitro PHOTODYNAMIC INACTIVATION OF Herpes simplex VIRUS WITH SAPPHYRINS: 22 π-ELECTRON PORPHYRIN-LIKE MACROCYCLES

The photodynamic inactivation of HSV-1, a virus having a membranous envelope, with both a decaalkyl sapphyrin and its dicarboxy-substituted analog was studied. The decaalkyl sapphyrin was as efficient in the inactivation of HSV-1 on a per macrocycle basis as DHE, whereas the efficiency of the dicarboxy-substituted sapphyrin was approximately two orders of magnitude less. Fluorescence studies of sapphyrin's binding to liposomes and VSV suggested that the decaalkylsapphyrin bound monomerically to cholesterol-rich regions of the viral envelope, whereas its charged analog localized in a more polar environment.     

OPTIMIZATION OF THE PHOTO-ENZYMATIC REDUCTION OF THE CARBON-CARBON DOUBLE BOND OF α-β UNSATURATED CARBOXYLATES IN REVERSED MICELLES

Abstract A system is described for the photo-induced biocatalytic reduction of enoates in reversed micelles composed of detergent, buffer and mixtures of alcohol and octane as cosurfactant and dispersant respectively. The photosystem consisted of the sacrificial electron donor tributylamine, Zn(II) meso -mono(4-N-decyipyridyl)-tri(phenyl)porphyrin as photosensitizer and methylviologen as electron acceptor. The enzyme enoate reductase (EC 1.3.1.31) utilizes the photo-reduced methylviologen for the stereospecific reduction of enoates. The composition of the reversed micellar medium has been optimized with respect to both the photochemical and the enzymatic part of the system. The yield of the photosystem increased with increasing polarity of the reversed micellar solution. The activity and stability of enoate reductase depended strongly on the nature and concentration of the cosurfactant in the reversed micelles. Increasing the chain length of the cosurfactant stabilizes the enzyme in the presence of enoates, but it leads to a more rapid loss of activity of the reduced form of the enzyme. Reversed micelles were composed in which both operational and storage stability of enoate reductase are at least equal to that in water. The photochemical regeneration of methylviologen coupled to the enzymatic reduction of tiglic acid and cinnamic acid yielded a system that was active for over 100 h in the presence of a cofactor regenerating system in reversed micelles. The enzyme was as stable as in aqueous solution. Therefore this photo-enzymatic system can be used for the stereospecific reduction of apolar α-β unsaturated carboxylates.     

AN EPR STUDY OF π-CATION RADICALS IN DINUCLEOSIDE PHOSPHATES AND DNA PRODUCED BY PHOTOIONIZATION

In this work we have produced the π-cation radicals of a number of nucleotides, dinucleoside phosphates, and DNA in aqueous glasses (8M NaCIO₄) by photoionization and investigated these species by EPR spectroscopy. Results found for nucleotides and dinucleoside phosphates containing one type of DNA base, e.g. TpT, GpG, or dApdA, were used in the analysis of spectra found for mixed dinucleoside phosphates, e.g. TpdG. For TpdG and TpdA in neutral glasses photoionization takes place from the purine base and no transfer of charge to the pyrimidine base is found. In basic conditions both the adenine and thymine π-cations are observed in TpdA. In both neutral and basic conditions the results found for mixed dinucleoside phosphates containing guanine show that the guanine cation is formed preferentially by photolysis. This result was found to extend to DNA. Photolysis of DNA in 8M NaC1O₄ produced principally the guanine cation. Computer simulations using parameters determined by other workers from a study of γ-irradiated oriented DNA closely match the spectrum found in this work attributed to the guanine cation in dinucleoside phosphates and DNA. This work thus confirms the presence of the guanine cation in γ-irradiated DNA.     

UPTAKE AND BIOCONVERSION OF α-TOCOPHERYL ACETATE TO α-TOCOPHEROL IN SKIN OF HAIRLESS MICE

Abstract— The photoprotective effect of topically applied α-tocopheryl acetate (vitamin E acetate), a stable derivative of α-tocopherol (vitamin E), and its possible bioconversion to the active antioxidant species (α-tocopherol) was examined in skin tissue of female hairless mice (HRS/J) exposed to UV-B irradiation. Our results indicate that topically applied α-tocopheryl acetate is absorbed into and retained by skin tissue. Furthermore, skin tissue from UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments contained significantly higher levels (P < 0.001) of α-tocopheryl acetate than non-UV-B-irradiated mice that received identical daily topical α-tocopheryl acetate treatments. Finally, free α-tocopherol levels in skin also were significantly increased (P < 0.00 1) by topical applications of α-tocopheryl acetate and skin levels of free α-tocopherol were significantly greater (P < 0.001) in UV-B-irradiated animals that received daily topical α-tocopheryl acetate treatments than in non-UV-Birradiated animals. These results suggest that UV-B irradiation enhances both the absorption of α-tocopheryl acetate and its bioconversion to free α-tocopherol.     

PHOTODYNAMICALLY GENERATED 3-β-HYDROXY-5α-CHOLEST-6-ENE-5-HYDROPEROXIDE: TOXIC REACTIVITY IN MEMBRANES and SUSCEPTIBILITY TO ENZYMATIC DETOXIFICATION

Singlet oxygen (¹O₂)-mediated photooxidation of cholesterol gives three hydroperoxide products: 3β-hydroxy-5α-cholest-6-ene-5-hydroperoxide (5α-OOH), 3β-hydroxycholest-4-ene-6α-hydrope-roxide (6α-OOH) and 3β-hydroxycholest-4-ene-6β-hydroperoxide (6β-OOH). These species have been compared with respect to photogeneration rate on the one hand and susceptibility to enzymatic reduction/ detoxification on the other, using the erythrocyte ghost as a cholesterol-containing test membrane and chloroaluminum phthalocyanine tetrasulfonate (AlPcS₄) as a ¹O₂ sensitizer. Peroxide analysis was accomplished by high-performance liquid chromatography with mercury cathode electrochemical detection (HPLC-EC[Hg]). The initial rate of 5α-OOH accumulation in AlPcS₄/light-treated ghosts was found to be about three times greater than that of 6α-OOH or 6β-OOH. Membranes irradiated in the presence of ascorbate and ferric-8-hydroxyquinoline (Fe[HQ]₂, a lipophilic iron complex) accumulated lesser amounts of 5α-OOH, 6α-OOH and 6β-OOH but relatively large amounts of another peroxide pair, 3β-hydroxycholest-5-ene-7α- and 7β-hydroperoxide (7α,7β-OOH), suggestive of iron-mediated free radical peroxidation. When photoperoxidized membranes containing 5α-OOH, 6α,6β-OOH and 7α,7β-OOH (arising from 5α-OOH rearrangement) were incubated with glutathione (GSH) and phospholipid hydroperoxide glutathione peroxidase (PHGPX), all hydroperoxide species underwent HPLC-EC(Hg)-detect-able reduction to alcohols, the relative first order rate constants being as follows: 1.0 (5α-OOH), 2.0 (7α,7β-OOH), 2.4 (6α-OOH) and 3.2 (6β-OOH). Relatively rapid photogeneration and slow detoxification might make 5α-OOH more cytotoxic than the other peroxide species. To begin investigating this possibility, we inserted 5α-OOH into ghosts by transferring it from 5α-OOH-containing liposomes. When exposed to Fe(HQ)₂/ascorbate, these ghosts underwent GSH/PHGPX-inhibitable chain peroxidation, as indicated by the appearance of 7α,7β-OOH, phospholipid hydroperoxides and thiobarbituric acid reactive substances. Liposomal 5α-OOH also exhibited a strong, Fe(HQ)₂-enhanced, toxicity toward LI210 leukemia cells, an effect presumably mediated by damaging chain peroxidation. This appears to be the first reported example of eukaryotic cytotoxicity attributed specifically to 5α-OOH.     

β-CARBOLINE ALKALOIDS: MECHANISMS OF PHOTOTOXICITY TO BACTERIA AND INSECTS

Abstract— Phototoxicity of five naturally occurring (3-carboline alkaloids was assayed against a series of Escherichia coli strains and against the insect Trichoplusia ni (Lepidoptera: Noctuidae). Rank order efficacy of the compounds was comparable in both organisms. Although the bacterial assay demonstrated oxygen dependence, the degree of phototoxicity did not correlate with the relative efficiency of in vitro singlet oxygen or hydrogen peroxide photoproduction by the alkaloids. A better correlation was observed between chromatographic migration distances (lipophilicity) and phototoxicity. Therefore, hydrophobic mechanisms in which the alkaloids diffuse with varying efficiencies into cells or into the vicinity of target molecules may be important modes of phototoxicity for these compounds.