DEUTERIUM-ISOTOPE EFFECT ON THE FLUORESCENCE YIELDS AND LIFETIMES OF INDOLE DERIVATIVES–INCLUDING TRYPTOPHAN AND TRYPTAMINE

Abstract— The fluorescence yields and lifetimes of indole, five of its alkyl detivatives, tryptophan, and tryptamine have been determined in degassed, heavy and light water at room temperature. All of the compounds have radiative lifetimes nearly identical to the parent compound indole, and a comparison of these results with recently reported data on tryptophyl derivatives disclosed a striking uniformity in radiative lifetimes between indole and many amino acids and peptides which contain the indole group as the fluorescence unit. The fluorescence rate k _f in H₂O, was found to be 4.5 × 10⁷ sec^-1. The nonradiative decay rates were found to vary between 5.1 and 46 × 10⁷ sec^-1 and from a study of the deuterium-solvent isotope effect and the deuterium-substituent effect a mechanism for nonradiative deactivation is proposed which includes an isotopically dependent proton transfer and a pathway involving energy loss via the ring carbon hydrogen vibrations. Tryptophan at pH 7 was found to have a unique nonradiative decay scheme not evidenced at a pH 1 or pH 10.     

EXCITED STATES OF ANTHOCYANINS: THE CHALCONE ISOMERS OF MALVIDIN 3,5-DIGLUCOSIDE

Abstract The excited state properties of the chalcone isomers of malvidin 3,5-diglucoside (malvin) in acidic aqueous solution (0 < pH < 4) were investigated using steady-state and time-resolved fluorescence spectroscopy. The two chalcone isomers of malvin were first isolated by high-performance liquid chromatography and then characterized by UV/visible absorption and fluorescence spectroscopy. The results were supported by molecular orbital calculations. The rate constants for photodeprotonation ( k ₁= 1 × 10⁹ s^−l) and protonation ( k ₋₁= 1.3 × 10¹⁰ L mol⁻¹ s^−l) were determined, both from the multiexponential fluorescence decays and the fluorescence intensities measured at the emission wavelengths of the neutral and ionized chalcone forms. The results here obtained are relevant for the understanding of the photoreactivity of anthocyanins in acidic medium.     

ANALYSIS OF FLUORESCENCE KINETICS AND ENERGY TRANSFER IN ISOLATED α SUBUNITS OF PHYCOERYTHRIN FROM Nostoc sp.

Abstract— The fluorescence decay profiles, relative quantum yield, and transmission of the phycoerythrin a subunit, isolated from the photosynthetic antenna system of Nostoc sp., were measured using single picosecond laser excitation. The fluorescence decay profiles were found to be intensity independent for the intensity range investigated (4 × 10¹³ and 4 × 10¹⁵ photons-cm-² per pulse). The decay profiles were fitted to a model assuming both chromophores absorb and fluoresce. The inferred total deactivation rates for the two chromophores, in the absence of energy transfer and when the effects of the response time of the streak camera and the finite pulse width are properly included, are 1.0 × 10¹⁰s' and 1.0 × 10⁹ s ¹ for the s and f chromophores. respectively, whereas the transfer rate between the two fluorophorcs is estimated to be 1.0 × 10¹⁰ s⁻¹ giving a s→ f transfer rate on the order of (100 ps)⁻¹. Steady-atate polarization measurements were found to be equal to those calculated using the rate parameters inferred from the kinetic model fit to the fluorescence decays. The apparent decrease in the relative fluorescence quantum yield and increase of the relative transmission with increasing excitation intensity is suggestive of ground state depletion and upper excited state absorption. Evidence suggests that exciton annihilation is absent within isolated α subunits for the intensity range investigated (4 × 10¹³ to 4 × 10¹⁵ photons-cm ² per pulse).     

REACTIONS OF PHOTOCHEMICALLY FORMED TRANSIENTS FROM 2-NITROTOLUENE

Abstract— Kinetic data are reported for the thermal decay of colored transients formed by U.V. irradiation of aqueous solutions of 2-nitrotoluene. The transients display an acid-base equilibrium with a pK value of 3.7. The decay is catalyzed by acids and the following rate constants in liter sec^-l mole^-1 were evaluated for the base form of the transient reacting with an acid at 30.0°C: 3.5 × 10^-3 (H₂O), 2.6×10³ (CH₃COOH), 4.7×10⁴ (⁺NH₃CH₂COOH) and 4.2×10⁵(H+).     

THE ABSOLUTE VALUE OF THE REACTION RATE CONSTANT OF BILIRUBIN WITH SINGLET OXYGEN IN D2O

Abstract— The chemical reaction rate constant of bilirubin with singlet oxygen in basic aqueous solution has been redetermined to be 3.5 × 10⁸ M^-1 s^-1 by a competitive technique using a 1,3-diphenylisobenzofuran in sodium dodecyl sulfate micelles. Bilirubin also physically quenches a singlet oxygen with a rate constant of 9 × 10⁸ M ^-1 s^-1. The lifetime of singlet oxygen in D₂O solution has been determined to be 35 μ s . The absorption cross-section for the molecular oxygen ³∑_g-→¹δ _g + 1 v electronic transition at 1.06μn in aqueous solution is unexpectedly larger than the gas paase cross-section.     

RATES OF REACTION OF SINGLET OXYGEN WITH SULFIDES

Abstract—Reaction rate constants for the reaction of singlet oxygen with a series of 24 sulfides in chloroform have been measured by inhibition of the self-sensitized photooxidation of rubrene. The reaction rate constant is sensitive to steric effects, decreasing as the carbons α- to sulfur become more highly substituted. Addition of a methyl group to each of the carbons α- to sulfur decreases the rate constant by about a factor of 10. From a series of p - and m -substituted thioanisoles, a ρ of -1.67 ± 0.09 was found. A much better correlation was found with σ than with σ⁺ indicating there is no resonance interaction with the reaction center. Typical rate constants are: di- n -butyl sulfide, 2.3 × 10⁷ M ^-1 s^-1; CBZ-L-methionine methyl ester, 1.4 × 10⁷; di-s-butyl sulfide, 1.8 × 10⁶; di- t -butyl sulfide, 1.3 × 10⁵; and thioanisole, 2.3 × 10⁶.     

STUDIES ON SOME INTERMEDIATES AND PRODUCTS OF THE PHOTOREDUCTION OF 9,10-ANTHRAQUINONE *,†

Abstract— The mechanism of the photoreduction of 9,10-anthraquinone (AQ) in alcohol and hexane has been studied by flash photolysis. The fluorescence spectrum of the photoproduct, 9,10-dihydroxy anthracene shows a large shift between hexane and ethanol. The quantum yields of photoreduction for AQ are solvent-dependent, the reaction between the solvent radical and AQ determining the quantum yield.
The absorption spectrum of the 9,10-anthrasemiquinone (AQH.) has a long-wavelength absorption band with peaks at 631 and 678 nm. The second-order decay constants for AQH. were estimated to be 1.3 × 10⁹, 6.7 × 10⁸ and 2.0 × 10⁸ M ^-1 sec^-1 in ethanol, 2-propanol and ethylene glycol, respectively.
A long-wavelength absorption band was observed for 9,10-anthrasemiquinone radical anion, having peaks at 776 and 860 nm; epsi;_max= 1900 at 776 nm. This spectrum is compared with the spectra of 9,10-dihydroxy anthracene mono- and di-anions. The 9,10-anthrasemiquinone radical anion was found to photoreduce quantitatively to 9,10-dihydroxy anthracene mono-anion with a quantum yield of 0.1.     

PHOTOBLEACHING OF A CYANINE DYE IN SOLUTION AND IN MEMBRANES

Abstract— N,N'-bis(2-ethyl-1,3-dioxolane)-kryptocyanine (EDKC), a lipophilic dye with a delocalized positive charge, photosensitizes cells to visible irradiation. In phosphate-buffered saline (PBS), EDKC absorbs maximally at 700 nm (ε= 1.2 × 10⁵ M⁻¹ cm⁻¹) and in methanol, the absorption maximum is at 706 nm (ε= 2.3 × 10⁵ M⁻¹ cm⁻¹). EDKC partitions from PBS into small unilamellar liposomes prepared from saturated phospholipids and into membranes prepared from red blood cells (RBC) and binds to human serum albumin (HSA). The EDKC fluorescence maximum red shifts from 713 nm in PBS to 720–725 nm in liposomes and RBC membranes and the fluorescence intensity is enhanced by factors of 14–35 compared to PBS (φ= 0.0046). EDKC is thermally unstable in PBS (T_1/2= 2 h at 1.3 × 10⁻⁵ M EDKC), but stable in methanol. In liposomes and RBC membranes, EDKC is 10 times more stable than in PBS, indicating that it is only partially exposed to the aqueous phase. Quenching of EDKC fluorescence in liposomes and RBC membranes by trinitrobenzene sulfonate also indicates that EDKC is not buried within the membranes. Photodecomposition of EDKC was oxygen-dependent and occurred with a low quantum yield (6.4 × 10⁻⁴ in PBS). Singlet oxygen was not detected upon irradiation of EDKC in membranes or with HSA since the self-sensitized oxidation of EDKC occurred at the same rate in D₂O as in H₂O and was not quenched by sodium azide or histidine.     

CHLORIDE ENHANCEMENT OF QUENCHING OF TRIPLET METHYLENE BLUE BY Fe(II)

Abstract— The influence of chloride ion on the rate of decay of triplet methylene blue in 0.01 M acid in the absence and presence of ferrous ions was investigated by means of laser flash-photolysis monitored by kinetic spectrophotometry. Chloride weakly accelerates decay of ³MBH^2± in aqueous solution in the absence of Fe(II). Quenching of ³MBH²⁺ by Fe(II) is more strongly catalyzed by Cl^- in both water and 50 v/v% aq. CH₃CN. The uncatalyzed quenching constant, k ₅, is of the order of 1 × 10⁶ M ^-1 s^-1 while in 4.8 M aqueous chloride ( μ – 7.2 M ) k ₅= (37.2 ± 1.8) × 10⁶ M ^-1 s^-1. A possible role of chloride is as a bridging species in quenching via electron transfer between ³MBH²⁺ and Fe(II).     

FLUORESCENCE CHARACTERISTICS OF INDOLES IN NON-POLAR SOLVENTS: LIFETIMES, QUANTUM YIELDS AND POLARIZATION SPECTRA

Abstract— Fluorescence lifetimes, quantum yields and polarization spectra were measured for indole, 3-methylindole and 2,3-dimethylindole in non-polar solvents. The results indicate simultaneous emission from thermally equilibrated ¹L_a and ¹L_b levels, with ¹L_a→¹ A dominating the 2,3-dimethylindole emission, and ¹L_b→¹ A dominating the indole emission. These results are consistent with previous assignments of the 0-0 transitions in absorption for these compounds. Radiative rates are: ¹L_a→¹ A , 2·0 × 10⁸ S^-1 and ¹L_b→¹ A . 0·62 → 10⁸ S^-1. In addition, the temperature dependence of the excitation and emission spectra are presented, which show that aggregation occurs with these indoles in hydrocarbons below approximately - 110°C. Possible applications to tryptophyl emission in the hydrophobic interiors of proteins are briefly discussed.     

PHOTOBLEACHING OF MONO-l-ASPARTYL CHLORIN e6 (NPe6): A CANDIDATE SENSITIZER FOR THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract— Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono- l -aspartyl chlorin e₆ (NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 × 10⁻⁴; this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 × 10 ⁵ and 2.8 × 10⁻⁵, respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 × 10 ⁻⁶), while the Zn derivative was more sensitive (yield = 1.9 × 10⁻²). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photoblcaching two- to four-fold, while the electron acceptor, met-ronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.     

REEVALUATION OF THE SPECTRAL AND KINETIC PROPERTIES OF HO2 AND O2- FREE RADICALS

Abstract— The spectra and molar absorbances of the HO₂ and O₂^- free radicals have been redetermined in aqueous formate solutions by pulse and stopped-flow radiolysis as well as by ⁶⁰Co gamma-ray studies. The extinction coefficients at the corresponding maxima and 23°C are ²²⁵= 1400 ± 80 M ^-1 cm^-1 and ²²⁵= 2350 ± 120 M ^-1 cm^-1 respectively. Reevaluation of earlier published rate data in terms of the new extinction coefficients yielded the following rate constants for the spontaneous decay of HO₂ and O₂^-: K _Ho2+HO2= (8.60 ± 0.62) × 10⁵ M ^-1 s^-1; K _Ho2+O2-= (1.02 ± 0.49) × 10⁸ M ^-1 s^-1; K _Ho2+O2- < 0.35 M ^-1 s^-1. For the equilibrium HO₂→ O₂^-+ H⁺ the dissociation constant is K _Ho2= (2.05 ± 0.39) × 10^-5 M or p K _HO2= 4.69 ± 0.08. G (O₂^-) has been evaluated as a function of formate concentration.     

PHOTOSENSITIZED CYCLODIMERIZATION OF 5-FLUOROURACIL

Abstract—The photosensitization by acetone or N-methyllutidone yields a C₄-cyclodimer from 5-fluorouracil in aqueous solution. with a quantum yield of 2 × 10^--³. The dimer isolated has been characterized by UV, IR. NMR, and mass spectra. The stereochemistry of the dimer has been determined to be anti head-to-tail configuration from the NMR spectral analysis.     

A FLASH PHOTOLYSIS STUDY OF 1-METHYLINDOLE

Abstract— The conventional flash photolysis of 1-methylindole in aqueous media was studied at Λ_excitation≥290 nm. The transients observed 20 μs after excitation consisted mainly of the radical cation (R⁺). the hydrated electron (e^-_aq) and the triplet state (T). Electron counting experiments indicate that photoionization is the only source of R⁺ with e^-_aq/R⁺= 1.07±0.09 in neutral media. Quenching of the R⁺ yield with H⁺ indicates that the fluorescent state is the precursor to 80% of the photoionization events with the remainder probably arising from a prefluorescent state. The triplet decays with a lifetime of 29 μs in deaerated neutral media. This decay is unchanged by N₂O saturation, but T reacts with acrylamide with k ≥2.8 × 10⁹ M ^-1. In 2 M Br^-, R⁺ and T yields are increased by factors of 2–3. Consideration of fluorescence quenching and T enhancement by Br-permits an estimate of φ_Isc between 0.33 and 0.49. The increased R⁺yield at high Br-concentrations cannot be accounted for by induced photoionization or triplet state reactions.     

FLASH PHOTOLYSIS STUDIES OF OROTIC ACID

Abstract— The triplet state of orotic acid has been studied by flash photolysis. The rate for dimerization has been observed to vary from 2 × 10⁹ M ^-1 sec^-1 at pH 1 where both the triplet and ground state molecules are neutral, to under 10⁸ M^-1 sec^-1 above pH 9 where both the triplet and ground state molecules are doubly ionized. The p K of the triplet state has been measured as 4.6. The rate of oxygen quenching for the triplet is 2–3 × 10⁹ M^-1 sec^-1 while the rate of radiationless decay in solution is 0.73 × 10⁴ sec^-1. The triplet absorption spectra have been measured for the two ionic forms of the triplet.     

PHOTOLESIONS IN DNA UPON 193 nm EXCITATION

Aqueous solutions of plasmid (pBR322 and pTZ18R) and calf thymus DNA were excited by 20 ns laser pulses at 193 nm. The quantum yields of single- and double-strand break formation, interstrand cross-links, locally denatured sites, (6–4)photoproducts and biological inactivation (Φ_ssb, Φ_dsb, Φ_icl, Φ_ids, Φ_6–4 and Φ_ina, respectively) were measured. The quantum yields are virtually independent of intensity, demonstrating a one-quantum process. The obtained values in aerated neutral solution in the absence of additives are Φ_ssb= 1.5 × 10^--³, Φ_dsb, = 0.06 × 10^--³ (dose: 10–200 J m^-2), Φ_icl=Φ_Ids= 0.1 × 10³ and Φ_6–4= 0.5 × 10^--³ Both Φ_ssb and Φ_dsb decrease strongly with increasing concentrations of TE buffer (0.01–10 m M ). Biological inactivation of the pTZ18R plasmid was determined from the transformation efficiency of Escherichia coli bacteria strains AB1157, AB1886 uvr and A82480 uvr rec; the Φ_ina values are 1.4 × 10³, 2.1 × 10³ and 3 × 10^--³, respectively. The monoexponential survival curves in all cases show that a single damage site leads to inactivation (one single hit). The biological consequences of different photoproducts are discussed.     

OXODIPYRROMETHENES AS REACTIVE SINGLET OXYGEN ACCEPTORS. MEASUREMENT OF THEIR CHEMICAL REACTION RATES BY A LASER FLASH PHOTOLYSIS TECHNIQUE

Abstract— Singlet oxygen has been generated directly in 1,1,2-trichloro, 1,2,2-trifluoroethane solution by irradiation of the oxygen dimol 2³∑-_g→2¹δ_g transition with a pulsed dye laser and the ³∑_g^-→¹δ_g+ lv transition with a continuous Nd-YAG laser. The rates of chemical reaction and physical quenching of singlet oxygen so generated has been measured for a series of substituted oxodipyrro-methenes. The results show that the oxodipyrromethenes react with singlet oxygen at rates comparable to that for 1,3-diphenylisobenzofuran. The rate of quenching of singlet oxygen by ground state oxygen has been measured to be 2.5±0.3 × 10³ M^-l s^-1.     

THE SINGLET OXYGEN REACTIVITY OF BILIVERDIN

Abstract— In 1, 1, 2-trichlorotrifluoroethane solution biliverdin physically quenches singlet oxygen at a rate of 8 × l0^sM-¹s^-1 and reacts chemically at 6 × 10 ⁵M-¹s^-1 to give a red product. In D, O solution the rate constants are PD dependent and range from 1.5–6 times 10¹⁰M-¹s^-1 for quenching and the chemical rate varies from 3–5 × 10⁸ M^-1 s^-1 to give colorless products.     

Dynamics of Flavin in Flavocytochrome b2: A Fluorescence Study

Abstract— The dynamics of the flavin bound to the flavocytochrome b₂ from Hansenula anomala were studied by fluorescence intensity quenching and quenching emission anisotropy with iodide. The fluorescence intensity of bound flavin is decreased 13-fold as compared to the free molecule. The remaining fluorescence decays with two lifetimes equal to 0.963 ± 0.040 and 4.635 ± 0.008 ns and fractional intensities of 0.036 ± 0.002 and 0.964 ± 0.002, respectively. The bimolecular diffusion constant was found to be 3.33 × 10⁹ M ^-1 s^-1 when the flavin is bound to the enzyme and 8.3 × 10⁹ Mv s^-1 when the flavin is free in solution. Thus, the flavin in flavocytochrome b₂ is accessible to the solvent, but the amino acid residues of the binding site inhibit the diffusion of iodide. The rotational correlation time of bound flavin was found to be 2.015 ± 0.365 ns, a value higher than that (155 ps) of free flavin in solution. Our results are discussed on the basis of local dynamics of the flavin.     

ISOLATION OF UNIQUE DIMER SPECTRA OF DYES FROM THE COMPOSITE SPECTRA OF AGGREGATED SOLUTIONS

Abstract— Aggregation of fluorescein, eosin and rose bengal in aqueous solutions has been studied. Mathematical expressions are derived to calculate the monomer mole fractions in moderately and highly concentrated solutions. The average values of the dimer dissociation constant, K_d , for fluorescein, eosin and rose bengal in aqueous solutions at pH 12 are 0.20, 9.0×10^-3 and 40 × 10^-3 moles/l respectively. A method is developed here to isolate the dimer spectrum from the composite spectrum. The uniqueness of this method is established by comparison with other known methods. The dimer spectra of all the three dyes are reported.