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.     

ELECTRON TRANSFER FROM THE EXCITED STATE OF TYROSINE TO COMPOUNDS CONTAINING DISULFIDE LINKAGES

Abstract— The flash photolysis of aqueous solutions of tyrosine has been studied in the presence of various concentrations of the cyclic disulfide sodium lipoate (thioctic acid, Na⁺ salt). In addition to the formation of phenoxyl radicals and hydrated electrons (and possibly H atoms) from the photoionization of tyrosine, the characteristic spectrum of the radical anion RSSR^- of lipoate was also observed in neutral as well as in alkaline solutions. From the dependence of these yields upon the concentration of lipoate, it was found that a long–lived triplet excited state of tyrosine, rather than the singlet excited state, is involved in these reactions. The negative radical ions RSSR^- are formed by two distinct pathways: (a) Na⁺–lipoate reacts with the solvated electrons which are ejected from the tyrosine triplets ³Tyr → RO.+ e ^-_aq+ H⁺ followed by e ^-_aq+ RSSR → RSSR^-, and (b) by direct interaction of lipoate with triplet excited tyrosine, resulting in the transfer of a negative charge from tyrosine to the disulfide linkage. At high lipoate concentrations, the singlet excited state of lipoate is quenched, k ₄= 1.6 × 10¹⁰ M ^-1 sec^-1, but this reaction does not lead to the formation of RSSR^- radical ions.     

A FLASH PHOTOLYSIS STUDY OF 5-METHOXYINDOLE

Abstract— The microsecond flash photolysis of 5-methoxyindole in aqueous solutions has been studied at γ_exc≥ 290 nm. Transients identified in this time realm in neutral solutions are: e_aq^-, the 5-methoxyindole radical cation (γ_max≅ 440 nm), the neutral transient with γ_max≅ 530 nm) and an unidentified oxygen sensitive transient with γ_max≅ 435 nm. Radical cations and e^-_aq are shown to be produced in equal amounts consistent with a photoionization process as the only source of both transients. H⁺ quenching of fluorescence and radical cation production gives equivalent Stern-Volmer constants indicating that photoionization occurs from the fluorescent state. The unidentified oxygen sensitive transient exhibits a pK _a of2–2.5 and is quenched at lower pH values indicating that it also has a fluorescent state precursor.     

THE TRIPLET STATE OF 8-METHOXYPSORALEN

Abstract— Transient absorption spectra produced by laser flash photolysis of an aqueous solution of 8-methoxypsoralen (8-MOP) have been studied. The biphotonic production of hydrated electrons and of the radical ions, 8-MOP ⁺ and 8-MOP^- is reported. The hydrated electron was found to react with ground state 8-MOP with k ˜ 3 × 10¹⁰ M ^-1 s^-1. In order to obtain a true triplet-triplet absorption spectrum. contributions from the radical ions were subtracted from the overall transient absorption. In addition, contributions from e^-_aq to the transient spectrum were removed by using N₂O, low laser intensity to minimize photoionization or by measuring the transient O.D. after the electron has decayed. These three methods each produced the same triplet-triplet spectrum which differs in the red region from previously reported spectra.     

LASER FLASH PHOTOLYSIS AND PHOTOINACTIVATION OF SUBTILISIN BPN'

Abstract— Laser flash photolysis of subtilisin BPN'at 265 nm has shown that photoionization of tryptophanyl (Trp) and tyrosinyl (Tyr) residues are the principal initial photochemical reactions. The initial products are the corresponding oxidized radicals. Trp and Tyr, and hydrated electrons (e_aq) which react with the enzyme at: k (e_aq+ subt. BPN') = 2.1 × 10¹⁰ M⁻¹ s⁻¹. The photoionization quantum yield was 0.032 ± 0.005 at 265 nm, which was enhanced 3.5-fold by simultaneous excitation at 265 and 530 nm. The photoionization yields were unchanged by 3 M bromide ion and 8 M urea. which did affect the enzyme fluorescence excited at 265 and 295 nm. A similar lack of correlation between the effects of perturbants on the photionization yields and fluorescence yields was found for subtilisin Carlsherg. The results indicate that the monophotonic and biphotonic ionization of the Trp residues does not involve the thermally-equilibrated. lowest excited singlet state and that singlet energy transfer from Tyr to Trp does not contribute to Trp photoionization. The photoinactivation quantum yield was 0.014 for 265 nm laser excitation. which was not changed by simultaneous 530 nm excitation. The corresponding quantum yield was 0.009 for low intensity 254 nm radiation, indicative of a biphotonic contribution to photoinactivation. The results are explained by postulating that photolysis of Trp-113 leads to disruption of hydrogen bonding to Asn-117 and a shift in the primary chain sequence associated with the aromatic substrate binding sites. The photoionization quantum yields in subtilisin BPN'and subtilisin Carlsberg agree with a model based on the assumption that exposed Trp and Tyr residues contribute independently at intrinsic photoionization efficiencies characteristic of the chromophores.     

ONE-ELECTRON OXIDATION AND REDUCTION OF AZAPROPAZONE AND PHENYLBUTAZONE DERIVATIVES IN AQUEOUS SOLUTION: A PULSE RADIOLYSIS STUDY

Abstract— Using pulse radiolysis techniques, 3 azapropazone and 3 phenylbutazone derivatives all structurally related to the potentially photosensitive anti-inflammatory drug, azapropazone, have been reacted with the free radical oxidants N₃, Br₂^- and (SCN)₂^- as well as with e^-_aq a strong reductant. It is demonstrated that for 5 derivatives, azapropazone (Az), 2-[a-Carboxy-valeryll-3-dimethylamino-7-meth1-1,2-dihydro-1,2,4-benzotriazine (Mi307), phenylbutazone (PB), oxyphenylbutazone (OPB), and ketophenylbutazone (KPB), N₃^- and Br₂^- appear to react via a one-electron removal process. For the other derivative, 8-hydroxy azapropazone (8-OH-Az), Nj and (SCN); oxidise via a one-electron process, while Br₂^- probably fqrms a free radical adduct.
The absolute spectra of the one-electron oxidised and reduced transient species for all six derivatives are thus given in this work and are a basis to the understanding of the action of light on these drugs.     

ON THE PHOTOIONIZATION ENERGY THRESHOLD OF TRYPTOPHAN IN AQUEOUS SOLUTIONS

Abstract— To investigate the existence and energy position of a photoionization threshold. tryptophan (Trp) has been photoionized in desecrated neutral aqueous or alcoholic solution under monochromatic light of variable frequency, in presence of N₂O to scavenge the photoelectron.
Present findings and some literature data converge to show the existence of a threshold for the one photon ionization process. This threshold is located at 4.5 ± 0.1 eV and 4.85 ± 0.1 cV for Trp in aqueous and ethanol solutions. respectively, which corresponds to a lowering with respect to the gas phase ionization potential of 3.4 and 3.0 eV.
The photoionization quantum yields for Trp is found about 4 times greater at 250 nm than at Λ_cx= 265 nm, where φe^-_4M=0.080±0.025. In such spectral range. at most one photoelectron out of 4–5 escaping geminate recombination would lead to Trp photodegradation in acrated solutions.
These results also point out that the neutral radical Trp. which has been previously observed for Λ_cx > 275 nm, i.e. below the ionization threshold energy—would not necessarily derive from Trp ⁺ deprotonation or cation-electron dissociative recombination. Similarly, the opening of the indole ring with formylkynurenine (FK) formation which is observed under aerobic conditions and Λ_cx >, 280 nm would not imply an electron attachment on O₂ but reactions such as Trp +³O₂ or Trp*+³O₂ or else
     

WAVELENGTH EFFECTS ON THE PHOTOPROCESSES OF INDOLE AND DERIVATIVES IN SOLUTION

Abstract— The two main primary photoprocesses (electron ejection and H-atom release) for indole, 5-methoxyindole and N-methylindole in various polar and nonpolar solvents were studied as a function of the excitation energy and were correlated with the corresponding fluorescence quantum yields. In hydrocarbon solvents, N–H bond cleavage is the main primary photoprocess from the ¹B_b band of the substrates with the exception of N-methylindole. In alcohols, both processes are of negligible importance. Hydrated electrons (e⁻_aq) are ejected from the relaxed singlet states of all three compounds in aqueous solutions with a similar yield for excitation at 280 and 254 nm (¹L_a and ¹L_b states). The yield increases when the excitation is into the ¹B_b band. The quantum yields of the two primary processes from the higher excited states are generally lower than the fraction of molecules not converting to the fluorescent state. This is explained by an efficient back reaction in competition with a thermally activated radical release from an intermediate state or radical pair formed from the S₂ (¹B_b) state. The non-occurrence of a photoionization energy threshold is discussed.     

Photophysical Properties of the Cationic Form of Neutral Red

Abstract— Photophysical properties of the cationic form of neutral red (NRH⁺), a phenazine-based dye of biological importance, have been investigated in several protic and aprotic solvents using optical absorption, steady-state and time-resolved fluorescence and picosecond laser flash photolysis techniques. Absorption and fluorescence characteristics of the dye in protic solvents indicate the existence of intermolecular hydrogen bonding between the NRH⁺ and solvent molecules in the ground state as well as in the excited state. Measurements of the fluorescence lifetime in normal and heavy water also support the formation of intermolecular hydrogen bonding. Time-resolved transient absorption spectra obtained in the picosecond laser flash photolysis experiments show only the absorption band due to the S_n← S₁ absorption. The picosecond transient absorption results do not indicate any spectral shifts attributable to the hydrogen bond formation dynamics between the excited NRH⁺ and the protic solvent molecules. It is inferred that the hydrogen bonding dynamics are much faster than the time resolution of our picosecond setup (∼35 ps).     

CHEMILUMINESCENT REACTIONS OF SO

Abstract— The rapid bimolecular reaction SO + O₃= SO₂+O₂+ 106 kcal/mole
yields electronically excited SO₂ in the ³B ₁ and ¹B₂ states with some vibrational excitation, as well as SO₂ in its electronic ground state. It is shown that k₁ = 1.5 x 10¹² exp (-2100/ RT ) cm² mole^-1 s-¹ and that the formation of electronically excited SO₂ involves higher activation energies.     

PRIMARY PRODUCTS OF LIQUID WATER PHOTOLYSIS AT 1236, 1470 AND 1849 Å

Abstract— The yield of the primary products of the liquid water photolysis at 1236 and 1470 Å is reported. It was found that besides the dissociation of the excited water molecules into H and OH radicals probably e⁻_aq is also formed. The H and OH radicals were scavenged by means of formate, and the e⁻_aq together with a part of H₂O* by adding carbon dioxide. The quantum yields determined at 1236 Å, are: Φ(H, OH) = 1.03 & 0.02, 0.06 <Φ( e⁻_aq , H₂O*) < 0.12 and at 1470 Å,: Φ(H, OH)=0.72±0.02, 0.037 < Φ( e⁻_aq , H₂O*) <0.075. The quantum yield of high purity water at 1849 Å in the absence of any scavengers is Φ(H, OH)=0.022. Previously published data by us for 1849 Å are also given: Φ(H, OH)=0.33 ± 0.01 and 0.02 < Φ ( e⁻_aq H₂O*) < 0.04. Reaction mechanisms are proposed.     

ACID-BASE EQUILIBRIA OF THE EXCITED SINGLET AND TRIPLET STATES AND THE SEMI-REDUCED FORM OF ACRIDINE ORANGE

Abstract— The acid-base equilibria of the excited singlet and triplet states of acridine orange (AO) were studied by flash-photolysis and fluorometric methods. The dye is a stronger base in the first excited singlet state (pK_s= 13.3) than in the triplet and ground states (pK_r= 10.3: pK_c, = 10.2); acridine orange follows the trend observed with some other heterocyclic compounds, viz. pK_s > pK_r= pK,_c. At room temperature, an anomalous fluorescence occurs from the dye in basic media: the assignment of this emission is discussed.
The semi-reduced dye was studied as a function of pH. In a large pH range (3–14), only the protolytic equilibrium between the cationic (AOH₂⁺) and the neutral (AOH) radicals was observed; the pK value corresponding to this equilibrium was found to be in the range of pH 5–6.     

PHOTOCHEMISTRY OF BENZOTHIAZOLE MODELS OF PHEOMELANIN

Abstract— The photochemistry of several 4-hydroxy- and 4-methoxybenzothiazoles has been investigated by laser flash photolysis. In aqueous solutions of pH3–12, the 4-hydroxybenzothiazole chromophore undergoes monophotonic photoionization to afford e^-_aq with quantum yields on the order of 0.06; no evidence for triplet species was obtained. The spectra and stability of the resultant free radicals were determined using pulse radiolysis. In contrast, triplet transients with life-times on the order of 8 mUs are readily observable upon irradiation of the 4-methoxybenzothiazole analog. Triplet sensitization experiments with the water-soluble carotenoid crocetin were employed to obtain the triplet extinction coefficients and subsequently the triplet quantum yields. The significance of these differences in photochemical behavior is discussed in relationship to the photochemistry and photobiology of the epidermal melanin pigment pheomelanin.     

THE EFFECTS OF HIGH-INTENSITY UV-RADIATION ON NUCLEIC ACIDS AND THEIR COMPONENTS—I. THYMINE

Abstract— The set of final products of thymine conversion induced by high-intensity UV irradiation (λ= 266nm, intensity 10²⁴-5 × 10²⁹ photons·s⁻¹·m⁻², pulse duration 10ns) of the dilute aqueous solution to the first approximation is similar to that formed with ionizing irradiation (γ-irradiation of aqueous solution or autoradiolysis of a solid 2-[¹⁴C]-thymine). The data obtained suggest that high-intensity UV-induced photochemical conversion of thymine involves photoionization and/or photodissociation. These processes pass through the higher excited state(s) populated as a result of the second photon absorption by excited (most probably in the T₁ triplet state) thymine molecules.     

Fast Redox Perturbation of Aqueous Solution by Photoexcitation of Pyranine

Abstract— Intense illumination (60-120 MW/cm²) of an oxygen-free aqueous solution of pyranine (8-hydroxypyrene-l,3,6-tri-sulfonate) by the third harmonic frequency of an Nd-Yag laser (355 nm) drives a two successive-photon oxidative process of the dye. The first photon excites the dye to its first electronic singlet state. The second photon interacts with the excited molecule, ejects an electron to the solution and deactivates the molecule to a ground state of the oxidized dye (φ⁺). The oxidized product, φ⁺, is an intensely colored compound (Λ_max= 445 nm, ε= 43 000 ± 1000 M ⁻¹ cm⁻¹) that reacts with a variety of electron donors like quinols, ascorbate and ferrous compounds. In the absence of added reductant, φ⁺ is stable, having a lifetime of -10 min. In acidic solutions the solvated electrons generated by the photochemical reaction react preferentially with H⁺. In alkaline solution the favored electron acceptor is the ground-state pyranine anion and a radical, φ, of the reduced dye is formed. The reduced product is well distinguished from the oxidized one, having its maximal absorption at 510 nm with e = 25 000 ± 2000 M^-l cm⁻¹. The oxidized radical can be reduced either by φ^- or by other electron donors. The apparent second-order rate constants of these reactions, which vary from 10⁶ up to 10⁹M⁻¹ s⁻¹, are slower than the rates of diffusion-controlled reactions. Thus the redox reactions are limited by an energy barrier for electron transfer within the encounter complex between the reactants.     

PHOTOCHEMICAL HYDROXYLATION OF 7,8-DIMETHYLALLOXAZINE (LUMICHROME)-A FLASH PHOTOLYSIS STUDY

Abstract— Flash photolysis experiments on the hydroxylation of lumichrome (L) in aqueous 0.5 M H₂SO₄ solution in the presence of O₂ or Ni²⁺ as triplet quenchers and quantum yield measurements confirm the assignment of the photoreactive species to the protonated form of the excited singlet state. A mechamism concerning the photochemical step is proposed, accounting for the formation of protonated 9-hydroxy-5,10-dihydrolumichrome (LOH₃⁺). This primary stable photoproduct was characterized by spectral and kinetic data. The dark reactions originating from LOH₃⁺ were investigated, and data regarding the successive steps are presented. The reaction LOH₃⁺ L→ LO + LH₃⁺ is demonstrated to be a two-electron reduction. The rate constant for the reaction of LH₂⁺ with O₂ is much larger than that for the oxidation of LH₃⁺ by oxygen.     

LASER FLASH PHOTOLYSIS STUDIES OF DNA-COMPLEXED ETHIDIUM BROMIDE

Abstract— Laser flash photolysis studies of DNA-complexed ethidium bromide were undertaken. We have observed a singlet-singlet (S₁-S_n) absorption process for DNA-complexed ethidium bromide. The observed lowest singlet excited state lifetime was 21 ± 2 ns. The molar difference extinction coefficient was measured to be 2.4 ± 0.4 × 10³M^-1 cm^-1 at 370 nm. The assignment of this transition was confirmed by time resolved fluorescence measurements.     

TRANSIENT PHOTOCHEMISTRY OF SAFRANIN-O

Abstract— We have characterized the spectra, acidity constants and decay kinetics of the triplet and semireduced radical species of Safranin-O. Between pH 3.0 and 10.6, there are three triplet species denoted ³DH^{2 +}₂, ³DH⁺ and ³D, the p K _as being 7.5 and 9.2. All three triplet species exhibit first order decay, the rate constant for ³DH⁺ being ca. 5-fold lower than the rate constants of ³DH⁺ and ³D. Ascorbic acid and ethylenediaminetetraacetic acid (EDTA) quench the triplet state under appropriate pH conditions and the pH dependencies of the yield of semireduced dye indicate that ³DH⁺ is more reactive than ³DH⁺ or ³D. With EDTA as the reducing agent, there is the additional requirement that at least one of the amino nitrogens be deprotonated to obtain a significant yield of semireduced dye. In these reactions, ascorbic acid is oxidized reversibly, but EDTA is oxidized irreversibly, so that with the latter reducing agent photolysis causes buildup of the leucodye, which on subsequent photolysis can reduce triplet state dye. With ascorbic acid as the reducing agent, the regeneration of the ground state dye is reversible, the decay of the semireduced radical being second order. In general, the transient photochemistry of Safranin-O resembles that of Thionine, the major difference being that the lifetimes of ³DH^{2 +}₂ and ³DH⁺ are much longer for Safranin-O than for Thionine.     

REACTIVITY OF ACRIDINE DYE TRIPLET STATES IN ELECTRON TRANSFER REACTIONS

Abstract—Rate constants, k _q , for the reaction of cationic and neutral acridine orange and 10-methylacridine orange triplet states (³AOH ⁺, ³AO, ³MAO⁺) with a series of electron donors have been measured. Two different protolytic forms of the semireduced dye radical are produced by acridine orange triplet quenching at various pH^M values in methanolic solution.
It is found that k ₄ decreases with increasing oxidation potential of the reducing agent for all triplet states in a manner which is expected for electron transfer reactions. The different reactivities of the cationic and neutral triplet forms can, therefore, be attributed to the difference in reduction potentials of these species. The difference in reduction potentials is related to the p K ^M values of triplet state, p K _T^M , and semireduced dye radical, p K ^M_S , by thermodynamic consideration. p K _T^M (³AOH⁺/³AO) is determined to be 11.2. From thisp K _S^M (AOH./AO;) is estimated to be 17–18. This is in striking contrast to the protolytic equilibrium of the semireduced dye radicals found to be pK^F= 4.1. We conclude that the last value represents the second protonation equilibrium (AOH⁺₂./AOH). This conclusion is confirmed by spectroscopic data.     

DETERMINATION OF SINGLET OXYGEN RATE CONSTANTS IN AQUEOUS SOLUTIONS

Abstract— A very efficient quenching of singlet oxygen (¹O₂) by N₃^- ions has been applied to the determination of rate constants of reactions of ¹O₂ with various substrates (A). This determination has been made possible by choosing experimental conditions which give simple competition between N₃^- and A for ¹O₂ formed in the steady state irradiation of convenient sensitizing dye (S). The consumption of oxygen by the substrate, as followed with an oxygen analyzer, decreases in the presence of low concentrations of N₃^-. Using neutral air saturated aqueous solutions containing the dye phenosafranine + A and varying concentrations of N₃^-, the ¹O₂ rate constants for reactions with biological substrates and some radiation protective agents have been determined.