TRIPLET EXCITED STATES OF PYRIMIDINE NUCLEOSIDES AND NUCLEOTIDES

Abstract— The triplet absorption spectra, lifetimes, extinction coefficients, e_TT, and intersystem crossing quantum yields to the lowest triplet T₁, ø_T1, of thymidine, thymidine monophosphate, uridine and uridine monophosphate, have been determined in various solvents at 300 K.
The effect of H-bonding on ø_T1, of these nucleosides and nucleotides and also of uracil has been determined and discussed. This effect allows, an ordering of ^l,3 n, π* and ^1,3 π, π* states in protic and aprotic solvents.     

LUMINESCENCE STUDIES OF QUINIZARIN AND DAUNORUBICIN

Abstract— The absorption and emission spectra of quinizarin (1,4-dihydroxy-anthraquinone) have been investigated in hydrocarbon and alcoholic solvents. Fluorescence spectra in 3 different Shpolskii matrices were recorded at 14 K. Vibrational analyses of these spectra revealed the presence of 3, 8, and 9 sites in octane, heptane, and hexane matrices, respectively. The fluorescence lifetime was found to be 6.5 ns in hexane and EPA. Fluorescence photoselection measurements in EPA (77 K) showed that the first 4 electronic transitions of quinizarin are polarized parallel, parallel, perpendicular, and parallel to the long molecular axis and can be assigned, in order of increasing energy, to ¹B₂, ¹B₂, ¹A₁ and ¹B₂ (ππ*)→¹A₁(C_2v,) transitions, respectively. The fluorescent transition is assigned as ¹B₁ (ππ*)→¹A¹. The absence of phosphorescence is attributed to the intramolecular hydrogen bonding present which displaces the parent anthraquinone n →* states above the ππ* states, thereby rendering the intersystem crossing (S₁-T₁) radiationless pathway inefficient.
Photoselection measurements on daunorubicin, a substituted quinizarin and known anticancer drug, revealed an absorption band polarization pattern identical to that of quinizarin. These results are in part at variance with assumptions used in previous work on the intercalation specificity of daunorubicin with DNA.     

A THEORETICAL TREATMENT OF THE ELECTRONIC STATES OF THIONINE AND RELATED MOLECULES

Abstract— –Problems associated with the protolytic equilibria of thionine and related molecules in their lowest excited electronic states were investigated. The theoretical arguments are based on semi-empirical SCF MO (CI) calculations for the π-electronic system of these molecules; all singly excited configurations were included in the CI. The results indicate that the basic form of thionine in its ground, first excited singlet and lowest triplet state is protonated at the heterocyclic N atom. The difference of the p K values of these three states can be explained in terms of the calculated charge densities. The photochemical reactivity of the lowest triplet of the acidic form of thionine (³TH₂²⁺) differs greatly from that of the lowest triplet of the basic form (³TH⁺). Some arguments for the assignment of nπ* character to ³TH₂²⁺ and ππ* character to ³TH⁺ are advanced.     

FLUORESCENCE OF THYMINE IN AQUEOUS SOLUTION AT 300° K

Abstract— –Fluorescence of thymine in neutral aqueous solution at room temperature has been detected using the multiscaling operation of a multichannel analyzer. The emission maximum (2.96 μm^-1) and 0-0 transition energy (3.37-3.45 μm^-1) are close to those determined at liquid nitrogen temperature in mixed solvents. The quantum efficiency of fluorescence excited at 3.77 μm^-1 is calculated to be 1.04 × 10^-4.
The corrected relative excitation spectrum shows significant differences from the absorption spectrum when both are determined under identical conditions of concentration and spectral bandwidth on the same instrument. The quantum yield of fluorescence decreases about 2-fold as the energy of excitation is increased beyond the 0-0' transition and follows the relation 1/φ°α E _excit..
This behavior is discussed in terms of (a) n π* and ππ* states, (b) emission from a minor tautomer and (c) kinetics of competing deactivation processes.     

PHOTOPHYSICAL PROPERTIES OF NUCLEIC ACID COMPONENTS—1. THE PYRIMIDINES: THYMINE, URACIL, N, N-DIMETHYL DERIVATIVES AND THYMIDINE

Abstract— Low-temperature (and some room temperature) absorption and emission, fluorescence and phosphorescence, data including quantum yields and lifetimes have been obtained from the title pyrimidine bases as a function of the nature of the solvent environment. Modest vibrational resolution has been observed for the first time in the absorption spectra, particularly for thymine and uracil. The excitation spectra also show structure. The quantum yields of fluorescence (φ_F) and phosphorescence are independent of the excitation wavelength. Thymine, thymidine and uracil have profoundly different photophysical properties in polar-aprotic vs polar-protic solvents. The N, N-dimethyl substitution of thymine and uracil produces photophysical changes comparable to the solvent change for the unsubsti-tuted bases. The species involved in the emission processes is the keto (lactam) form. It is probable that ^1,3(n,π*) state(s) has(have) changed order relative to a lowest ¹(π,π*) state as a consequence of both the solvent change and N, N-dimethyl substitution. The lowest triplet state is assigned as ³(n π*). We propose that an important factor contributing to the previously reported excitation wavelength dependence of φ_F and φ_T1 (φ_isc) for nucleic-acid components is the equilibrium coexistence of H-bonded and non-H-bonded forms each having different photophysical properties. Consideration is given of the impact of the significantly different photophysical properties of nucleic-acid bases as a function of the nature of the solvent upon the photochemical properties.     

THE INFLUENCE OF MOLECULAR CONFORMATION ON THE STABILITY OF ULTRAVIOLET STABILIZERS TOWARD DIRECT AND DYE-SENSITIZED PHOTOIRRADIATION: THE CASE OF 2-(2'-HYDROXY-5'-METHYPHENYL)BENZOTRIAZOLE (TIN P)

Abstract— The mechanism for photodegradation of the ultraviolet photostabilizer 2-(2'-hydroxy-5'-methylphenyl)benzotriazole (TIN P) upon direct and dye-sensitized (singlet molecular oxygen [O₂(1Δ_g)]-mediated) irradiation was studied. From the experimental TIN P photodegradation rate data, and low temperature (77 K) fluorescence and phosphorescence quantum yields, one can conclude that the photodegradative process involves phosphorescent states of TIN P. The open conformer of TIN P quenches O₂(¹Δ_g) by physical scavenging with a rate constant (k_q) in dimethylsulfoxide of 2.8 times 10⁶ M ^-1 s^-1. The intramolecular hydrogen-bonded conformer does not appreciably interact with O₂(¹Δ_g). In the presence of a relatively high concentration of OH- (either 5 times 10^-2 M KOH in ethanol or water at pH 13), the ionic form of TIN P (with an ionized phenol group) physically and chemically quenches O₂(¹Δ_g). The reaction rate constant ( k _r) is 1 times 10 ⁸ M ^-1 s^-1, and the ratio k _q/ k _r is approximately three in alkaline aqueous media.     

DEPROTONATION DYNAMICS OF LOCAL ANESTHETICS IN HYDROPHOBIC MEDIA: DIBUCAINEHCI IN WATER/ALCOHOL OR SURFACTANT MIXTURES

Abstract— The emitting chromophores, emitting states, state orderings and phosphorescence lifetimes of neutral, hydrogen-bonded, and protonated dibucaines in water/alcohol mixtures were determined experimentally and were confirmed theoretically using a HAM/3 method. These photophysical properties are used to probe the deprotonation dynamics of dibucaine-HCl in the water/hydrophobic media. It is found that the protonated dibucaine in an aqueous solution is deprotonated and/or forms hydrogen-bonded dibucaine when it is brought into contact with the alcohols and surfactants. The observed deprotonation processes could lend some insight on the molecular basis of pharmacological action of dibucaine.     

CALCULATED RATES AND EQUILIBRIA OF THERMAL INTERCONVERSION OF TRIPLET (3g-) AND SINGLET (1Δg and g+) MOLECULAR OXYGEN

Abstract— From spectroscopic data and rate constants in the literature, equilibrium constants and rates of thermal formation of singlet oxygen (¹Δ_g and ¹Σ_g⁺) were calculated for a number of conditions. For the gas phase we estimate K _eq(¹Δ_g³Σ_g^-) = 1.67 exp(-94.31 KJ/RT) and K _eq(¹Σ_g⁺/³Σ_g^-) = 0.33 exp(-157.0 KJ/RT). The calculated rate constants for the ³Σ_g⁺→¹Δ_g transition of O₂ at 25°C varied from 2.5 × 10^-11 s^-1 in water to 4.8 × 10^-16 s^-1 in air, assuming equal solvent interactions with the ground and excited states. Physical quenchers for singlet oxygen are expected to be catalysts for its thermal formation. Equations are presented which allow one to estimate whether such catalysis by quenchers will result in a pro-oxidant effect.     

CHEMILUMINESCENCE AND ENERGY TRANSFER FROM SINGLET OXYGEN PAIRS

Abstract— Chemiluminescence and photochemiluminescence of systems generating excited (singlet) molecular oxygen has been studied at low temperature in fluid media. A possible interpretation is proposed: that dimers of singlet oxygen are directly or indirectly responsible for the observed emission, the species [¹δg +¹σg⁺] predominating. Energy transfer to a fluorescent molecule is possible only when conditions of spectral overlap, required for transfer by inductive resonance, are fulfilled. Evidence is also presented for the formation of a transient complex between oxygen and a sensitiser or acceptor molecule [S…O₂], which is the precursor of singlet oxygen in these systems. However, other possible mechanisms cannot be excluded.     

STUDIES ON THE TRIPLET EXCITED STATE OF 4-THIOURIDINE

Abstract— The absorption spectra, lifetimes, extinction coefficients and intersystem crossing quantum yields of the lowest triplet T ₁ of 4-thiouridine have been determined both in acetonitrile and in water. An ordering of ^1,3(n,π)* and ^1,3(π,π)* states is suggested. Triplet quenching rate constants with various pyrimidine bases or amino acids are reported.     

THE PRODUCTION OF SINGLET MOLECULAR OXYGEN BY ZINC(II) PHTHALOCYANINE IN ETHANOL AND IN UNILAMELLAR VESICLES. CHEMICAL QUENCHING AND PHOSPHORESCENCE STUDIES

Abstract— Zn(II)phthalocyanine (ZnPc) generates O₂(¹Δ_g) with a quantum yield of ca. 0.4 upon photocxcitation at 354 or 600 nm in ethanolic solution as determined by time-resolved phosphorescence studies at 1270 nm and photooxidation experiments using 1,3-diphenylisobenzofuran (DPBF) as substrate. The quantum yield of photooxidation slightly increases upon incorporation of ZnPc into unilamellar liposomes of dipalmitoylphosphatidylcholine. Under our irradiation conditions (600 nm, 18^°C, and short light exposure times), DPBF(5–50 μM) undergoes photooxidation by a pure Type II mechanism; the rate constant for the O₂(¹Δ_g) + DPBF reaction is (1.1 ±0.1) x 10⁹ M^-1 s^_1 in ethanol solution and determined to be about two orders of magnitude smaller when both ZnPc and DPBF are embedded into liposomes.     

Effect of Magnesium and Calcium Complexation on the Photochemical Properties of Norfloxacin

Abstract— The fluoroquinolone antibiotics can induce skin photosensitivity in some patients and this has been ascribed to the generation of reactive oxygen species, such as singlet oxygen (O₂[¹Δ_g]). We have studied the photochemical properties of the different ionized forms of the fluoroquinolone norfloxacin upon complexation with Mg²+ and Ca²+ ions, as it is proposed that the antibiotic exists mainly as a complex in the blood plasma. We found that the norfloxacin cation (pH < 6) shows no photodegradation after UVA irradiation and has a low quantum yield of O₂(¹Δ_g) generation. The norfloxacin cation does not complex. Ca²+ or Mg²+ ions; when these ions are added to the solution, we observed no changes in the fluorescence quantum yields (φ_flu) and singlet oxygen yields (φ_Δ). In contrast, the neutral (6 ± pH > 8.5) and anionic (pH > 9) forms of norfloxacin are able to complex calcium and magnesium, and their generation of O₂ (¹Δ_g) is decreased by complexation. The neutral zwitterionic form and the anionic form also quench singlet oxygen by both chemical and physical pathways regardless of complex formation, while physical quenching is observed for the cation. At pH > 7.4, norfloxacin photobleaches and complexation to Ca²+ and Mg²+ increases the rate at which photobleaching occurs. Thus, both the pH of the medium and complexation with metal cations may affect the phototoxic potential of this antibiotic.     

CHEMILUMINESCENCE AND THE FORMATION OF SINGLET OXYGEN IN THE OXIDATION OF CERTAIN POLYPHENOLS AND QUINONES

Abstract— The possibility of ¹O₂ (¹Δ_g) participation in the oxidation of polyphenols and quinones has been investigated in two systems: (1) the system involving autooxidation leading to oxidative polymerization and destruction, and (2) the modified Trautz-Schorigin reaction, i.e. oxidation of polyphenols and HCHO with H₂O₂ in concentrated alkaline solutions. The red band with maximum at 635 nm observed in chemiluminescence of pyrocatechol, adrenaline, pyrogallol, gallic acid, adrenochrome and p -benzoquinone corresponds to the transition 2O₂(¹Δ_g) → 2O₂(³Σ^-_g). Emission bands in the range 475–540 nm arise from the superposition of the 2O₂(¹Δ_g) → 2O₂(³Σ^-_g) transition and radiative deactivation of excited oxidation products. In system (2) chemiluminescence has a broad band from 580 nm beyond 800 nm and much higher intensity than in system (1). Formaldehyde was found to enhance light emission in system (1) by a factor of about 30. The influence of solvents, including D₂O in which ¹O₂ has varying lifetimes, on kinetics of chemiluminescence as well as quenching effect of β-carotene, hydroquinone, cysteine, bilirubin and biliverdin strongly support the involvement of ¹O₂ in the chemiluminescence of both systems.     

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.     

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.     

Photophysical properties of the lowest excited singlet and triplet states of thio- and seleno-psoralens

The decay processes of the lowest excited singlet and triplet states of five heteropsoralens (HPS) were investigated by steady-state and shift-phase fluorometry and by laser-flash photolysis in different solvents. The emission spectra of HPS are detectable only in trifluoroethanol (TFE), where fluorescence lifetimes (τ_F) and quantum yields (φ_F) were measured. The triplet lifetimes (τ_T), triplet (φ_T) and singlet-oxygen production (φ_Δ) quantum yields were determined in benzene, ethanol and TFE by laser-flash photolysis. Semiempirical (INDO/1-CI) calculations allowed the nature of the lowest excited singlet and triplet states and transition probabilities to be obtained. Theoretical and experimental results indicate that the two lowest excited singlet states S₁ and S₂ of HPS are close-lying and different in nature (π,π* and n,π*). The "proximity effect" between these two states controls the photophysical properties of HPS as it does for the other furocoumarins. However, HPS have a peculiar behavior with respect to the related compounds because they are fluorescent and have, in three cases, detectable intersystem crossing only in TFE. This behavior can be tentatively explained by a different energy gap and/or order between the S₁ and S₂ states.     

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.     

PHOTOPHYSICAL PROPERTIES OF NUCLEIC ACID COMPONENTS—II. EFFECT OF N, N'DIMETHYL SUBSTITUTION ON INTERSYSTEM CROSSING IN PYRIMIDINE BASES

Abstract— The intersystem crossing quantum yields to the lowest triplet states of N, N'dimethyluracil and N, N'dimethylthymine are 0.02 and ≥0.004, respectively. The very low yields are interpreted in terms of a ¹( n , π*) to ¹(π.π*) state order change compared with the unsubstituted parents.     

POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC TUMOR THERAPY—I. PHOTOPHYSICAL PROPERTIES OF TWO CHLORIN DERIVATIVES

Abstract— Photophysical properties of two chlorin type molecules (CHLI) and (CHLII) were investigated in different solvents. Quantum yields of fluorescence φ_F of S, → T, intersystem crossing φ_T, and of singlet oxygen (¹Δ_g) formation φ_Δ, as well as the Stern-Volmer constants for the quenching of the S, states by oxygen and the bimolecular rate constants of quenching of ¹Δ_g by the chlorins were measured. The values of φ_T and φ_Λ can be given as 0.57 and 0.58 for CHLI and 0.69 and 0.58 for CHLII. The values of the fluorescence quantum yields, the strong absorption of the chlorins in the red (Λ > 630 nm) and the high values of the quantum yields for ¹Δ_g formation recommend the chlorin derivatives as potential markers and photosensitizers for tumor therapy.     

THE QUENCHING OF SINGLET OXYGEN BY AMINO ACIDS AND PROTEINS

Abstract— The physical quenching of singlet molecular oxygen (¹Δ_g) by amino acids and proteins in D₂O solution has been measured by their inhibition of the rate of singlet oxygen oxidation of the bilirubin anion. Steady-state singlet oxygen concentrations are produced by irradiating the oxygenated solution with the 1–06 μm output of a Nd-YAG laser, which absorbs directly in the electronic transition ¹Δ_g+ 1 v →³Σ_g^-. The rate of quenching by most of the proteins studied is approximated by the sum of the quenching rates of their amino acids histidine, tryptophan and methionine, which implies that these amino acids in the protein structure are all about equally accessible to the singlet oxygen. The quenching constants differ from those obtained by the ruby-laser methylene-blue-photosensitized method of generating singlet oxygen, or from the results of steady-state methylene-blue-photosensitized oxidation, where singlet oxygen is assumed to be the main reactive species. The singlet oxygen quenching rates in D₂O, pD 8, are (10⁷ℒ mol^-1 s^-1): alanine 0–2, methionine 3, tryptophan 9, histidine 17, carbonic anhydrase 85, lysozyme 150, superoxide dismutase 260, aposuperoxide dismutase 250.