AN EPR STUDY ON THE TRIPLET STATE OF PURINE FREE BASE IN AQUEOUS GLASSES AT 77 K

Abstract— Since purine free base in aqueous solution exists in different ionic forms at different pHs and most of its photoreactions are likely to involve the triplet excited states of these different ionic forms, electron paramagnetic resonance studies (EPR) have been performed in order to determine intersystem crossing quantum yields and other characteristic parameters of the excited triplet state of these forms. Intersystem crossing yields decrease with a decrease in pH, being 0.62, 0.37 and 0.10 in 8 M NaOH, 8 M NaCIO₄ and 6M H₃P0₄ glasses, respectively. Differences in triplet decay lifetimes 3.4, 2.5 and 3.1 s, as well as in root mean-square zero-field splitting (ZFS) parameter, D_*, (0,1304, 0.1512 and 0.1353 cm_-1) are also observed for the anionic, neutral and cationic species of purine free base. The EPR signals of the triplet state of the neutral and anionic forms have been observed simultaneously in the pH range of7–10.     

A LASER FLASH PHOTOLYSIS STUDY OF PYRENE-1-ALDEHYDE. INTERSYSTEM CROSSING EFFICIENCY, PHOTOREACTIVITY AND TRIPLET STATE PROPERTIES IN VARIOUS SOLVENTS

Abstract— Triplet-and singlet-related photoprocesses of pyrene-1-aldehyde (PA) in various solvents have been investigated in detail using 337.1 and 355 nm laser flash photolysis in conjunction with time-correlated determination of fluorescence lifetimes (τ_F) and steady-state photochemical and absorption-emission spectral measurements. In benzene, the lowest triplet of PA (43 < E_T < 46 kcal/mol) has a lifetime of about 50 µs (τ_T) and displays the absorption maximum at 443 nm with a maximum extinction coefficient (ε_max) of 21000 M ^-1cm^-1; the corresponding ketyl radical has a sharp absorption maximum at 428 nm (ε_max≥ 25000 M ^-1cm^-1). The quantum yields (φ_T) of lowest triplet occupation are high in nonprotic solvents (0.6–0.8), decrease in protic solvents (alcohols) as the polarity of the latter is increased, and maintain a complementary relationship with the quantum yields (φ_F) of fluorescence. Quantum yields (φ_PC) of loss of PA due to photoreactions in some solvents have also been determined under conditions of steady irradiation at 366 nm; φ_PC is in the range 0.1–0.2 in electron-rich olefinic solvents such as cyclohexene and tetramethylethylene. These results concerning τ_F, τ_T, φ_F. φ_T and φ_PC as well as the effects of 1,2,4-trimethoxybenzene and 2,5-dimethyl-2,4-hexadiene as quenchers for fluorescence, triplet yield, and photochemistry are discussed in the light of possible state orders for PA in polar and nonpolar environments.     

THE PHOTOCHEMISTRY OF ADENOSINE: INTERMEDIATES CONTRIBUTING TO ITS PHOTODEGRADATION MECHANISM IN AQUEOUS SOLUTION AT 298 K AND CHARACTERIZATION OF THE MAJOR PRODUCT

Abstract— The steady-state (254 nm) photolysis of 9–(β-d-erythropentofuranosyl)adenine (adenosine) in aqueous solution was studied. Photodestruction yields on the order of 1.3 × 10⁻³⁾ were determined at room temperature by measuring the initial decrease in the absorption maximum as a function of irradiation time. The use of high performance liquid chromatography (HPLC) permitted a more exact determination of the yield (2.5 × 10³). The formation of photoproducts was also studied using HPLC. In the photolysis of 50 μ M aqueous solutions of adenosine under anaerobic conditions at least 11 stable photoproducts are formed that absorb at 260 nm, the wavelength of maximum absorption of adenosine. The major photoproduct was also isolated and characterized as adenine; its formation yield was determined to be 4.5 × 10⁴. This yield is affected by the presence of oxygen and by the initial concentration of adenosine employed. Fluorescence emission and excitation spectra were used to monitor the formation of highly fluorescent photoproducts that emit with maxima at 365, 398 , and 430 nm and absorb in the wavelength region of 240–380 nm.
The reactive species in the photodestruction mechanism were established using substrates that react selectively with the respective short-lived species. Photoionization is a primary photoprocess implied by these studies. The triplet state of adenosine also contributes to the photodestruction mechanism.     

The Photobiological Differences of Gilvocarcins V and M Are not Related to their Transient Intermediates and Triplet Yields

Abstract— The transient absorption spectra of the intermediates produced by the 355 nm laser excitation of gilvocarcin derivatives have been investigated in various solvents. The spectra consist of a triplet-triplet absorption in the visible region and a residual absorption observed between 340 and 700 nm due to a long-lived species, assigned to the radical cation. A broad-fast decaying band with a maximum at around 700 nm attributed to the solvated electron is also seen in solutions containing a low DMSO/water volume ratio and at 266 nm irradiation of a 50% methanol/water solvent mixture. The molar absorption coefficient of the triplet state of gilvocarcin V (GV) and gilvocarcin M (GM), determined by the energy transfer method, is independent of the solvent properties and has a value of 3.0 × 10⁴/ M cm. The triplet decay rate constants for both drugs are between 1 and 5 × 10⁴/s. A similar initial yield and triplet decay rate constant of GV were observed in the presence of 3.4 m M thymine. Thus, a quenching rate constant of the GV's triplet state by thymine is estimated to be lower than 10⁶/Ms. The triplet quantum yields of both antibiotics determined by using the comparative method are higher in dimethylsulfoxide (DMSO) (0.18) than are those corresponding to 25% DMSO/water (0.06). The decrease in φ_T in the presence of water could be attributed to an enhanced internal conversion rate constant from the S₁ state or to an increase in the photoionization yield. The similarity of the transient intermediates and their yields for GV and GM suggest that their photobiological differences are due to other factors such as DNA binding constants, preferential localization of the drugs in the cell or the enhanced reactivity of the vinyl group toward cellular components.     

MECHANISM OF THE PHOTOCHEMICAL REACTION OF DIPHENYLAMINES WITH CARBON TETRACHLORIDE

Abstract. The quantum yields of HCI (φ_HC1) formation have been measured for the photolysis of N -methyldiphenylamine (MeDPA), triphenylamine (TPA) and diphenylamine (DPA) in the presence of CCl₄ in polar solvents. The quantum yields of N-methylcarbazole formation (φ_mφca) have also been determined for the system MeDPA-CCl₄. With increasing CCl₄ concentration, φ_HCl increases as φ_MeCA decreases, and φ_HCl reaches maximum values 2.7 at 1 M CCl₄. Using laser photolysis, transient spectra have been recorded for MeDPA in the absence and presence of CCl₄ in polar and non-polar solvents, and for TPA. Transient absorption due to the triplet states and photocyclization products (without CCU), exciplexes, the (C₆H₅)₂ NCHi radical, the MeDPA⁺ cation radical, the (TPA⁺., CCl₄) ion pair, and the TPA⁺ cation radical have been identified. The mechanistic implications of these results are discussed.     

A Photophysical Study of Purines and Theophylline by Using Laser-Induced Optoacoustic Spectroscopy

Abstract— The photophysics of purinic compounds (purine, 6-meth-ylpurine, 6-aminopurine [adenine], 6-chloropurine, 6-methoxypurine) and theophylline in acetonitrile solution were studied by pulsed laser-induced optoacoustic spectroscopy (LIOAS) exciting at 266 nm. The effect of O₂, Xe and MnCl₂ on the photophysical behavior of these compounds was studied; as well, the formation quantum yield of purine and 6-methylpurine triplet states were determined, with φ_T= 0.88 ± 0.03 for both compounds. Multiphotonic and depletion processes were observed at high laser fluences. In order to explain this behavior, theoretical UV-visible absorption electronic spectra from both the S₀ and S₁ state have been calculated for purines and theophylline by using the semiempirical PM3 and ZINDO/S methods.     

AN EPR STUDY OF THE SPECIES PRODUCED DURING THE UV PHOTOLYSIS OF HETEROCYCLIC COMPOUNDS IN METHYLTETRAHYDROFURAN AT 77 K

Abstract —The ultraviolet irradiation (290 nm ≤Λ≤ 390 nm) of indole, purine, indazole, acridine and quinoline in 2-methyltetrahydrofuran glass at 77 K produces trapped radicals. Two electron-paramagnetic-resonance (EPR) signals are found at 77 K during illumination, one at high magnetic field (3–25 times 10^-1 T) assigned to the matrix radical and the other at low field (1.3 times 10^-1 to 1–5 times 10^-1 T) attributed to the lowest triplet state of the heterocyclic molecule. Quantum yields for triplet production at 77 K are 0–34 for indole, 0.51 for purine, 0.55 for indazole, 0.15 for acridine, and 0.94 for quinoline. The rate of formation of matrix radicals varies as the n _R^th power of the incident light intensity, I ₀^nR, where 1.6 ≤ n _R=≤ 2. Solvent radical yields, which depend on the light intensity, have been determined. Under the experimental conditions, no signals attributable to trapped electrons or cations have been observed. The dependence of the reciprocal value of the rise lifetime of the low field EPR signal as a function of the intensity of exposure is in accordance with a biphotonic mechanism.     

LASER FLASH PHOTOLYSIS OF INDOLE IN THE PRESENCE OF AMINO ACIDS

The laser flash photolysis of indole at 265 nm in the presence of glycine, proline and hydroxy proline was studied. The relative yields of c _aq, triplet state, and indole cation radical were determined in the absence and in the presence of the amino acids. The yields were determined as a function of laser intensity and the values at very low intensity were compared with the fluorescence quenching results. It was concluded that in these conditions the photoionization of indole occurs via the fluorescent state. From the curves of triplet yield vs laser intensity, the triplet quantum yield extrapolated at low laser intensity was obtained, φr = 0.55 φ 0.05, relative to the literature value of 0.15 for φ_eag. This gives φ_F+φ_eaq= 1.0 ± 0.1 at room temperature. When proline and hydroxy proline were used as singlet quenchers, the yield of In was greater than the yield of c_aq. This was considered as evidence that a fraction of the quenching processes leads to complete electron transfer from indole to the amino acids.     

LUMIFLAVIN-SENSITIZED PHOTOOXYGENATION OF INDOLE

Abstract— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 10⁹ M ^-1 s^-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, ¹O₂. But the reaction between ¹O₂ and indole (7 times 10⁷ M^_l s^_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via ¹O₂ processes, though appreciable ¹O₂ was formed.     

Photophysical Properties of Tin Ethyl Etiopurpurin I (SnET2) and Tin Octaethylbenzochlorin (SnOEBC) in Solution and Bound to Albumin

Abstract— The photophysical characteristics of two second-generation PDT photosensitizers, tin ethyl etiopurpurin I (SnET₂) and tin octaethylbenzochlorin (SnOEBC), have been measured in homogeneous solution and when bound to bovine serum albumin (BSA). The ground state and triplet state absorption spectra have been characterized, as have triplet lifetimes and quantum yields for intersys-tem crossing, singlet oxygen formation and photobleaching. In total, these parameters provide a complete set of data that can be used to quantitatively compare the photosensitizing efficiencies of these molecules. The photo-bleaching quantum yield of SnET₂ is increased dramatically when it is bound to BSA, thus limiting the production of singlet oxygen at incident fluences above 1 J/cm². In contrast, the quantum yield of photobleaching of SnOEBC is at least an order of magnitude lower than that of SnET₂ under these conditions and does not significantly limit the photosensitization process for typical in vivo or in vitro fluences. This difference is expected to play a significant role in determining the relative photosensitizing ability of these compounds in vivo.     

Generation and Characterization of Psoralen Radical Cations

Abstract— Radical cations of psoralen, 8-methoxypsoralen(8-MOP) and 5-methoxypsoralen have been generated by photosensitized electron transfer in acetonitrile and aqueous buffer/acetonitrile (1:1) and have absorption maxima at 600, 650 and 550 nm, respectively. The radical cations have lifetimes of 5 p.s under these conditions, are unreactive toward oxygen and show behavior typical of ar-ylalkene radical cations in their reactivity toward nucle-ophiles and the precursor psoralens. Direct 355 nm excitation of 8-MOP in aqueous buffer at physiological pH results in monophotonic photoionization to give 8-MOP_*+ with a quantum yield of 0.015.The 8-MOP_*+ reacts with both guanosine and adenosine mononucleotides ( k = 2.5 times 10₉ and 3.4 times 10₇ M_-1 s₁, respectively) via electron transfer to give the purine radical cations, but does not react with pyrimidine mononucleotides. These results suggest that reactions of psoralen radical cations generated by electron transfer or photoionization may be involved in psoralen/UVA therapy.     

PHOTOKINETIC ASPECTS OF SPECIFIC HYDROGEN BONDING IN ALL-TRANS RETINAL AT ROOM TEMPERATURE

Room-temperature hydrogen-bonding of all- trans retinal (ATR) with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in cyclohexane results in fluorescence enhancement and triplet yield quenching; these effects, as well as the associated absorption spectral changes in both ground and triplet states and the kinetics of H-bonding in the triplet state, have been studied by steady-state absorption-emission and laser flash-photolytic transient measurements. The fluorescence enhancement is predominantly controlled by the H-bonding in the ground state (static interaction) and gives a value of 440 M ^-1 for the corresponding equilibrium constant which is very similar to the value (420 M ^-1) obtained from the analysis of absorption spectral data as a function of [HFIP˜. The quantum yield of triplet occupation (ø_T) of the H-bonded complex, ATR-HFIP, in cyclohexane is non-negligible and is about one-third of ø_T of free ATR. The kinetic data of H-bonding equilibration in the triplet state, observable on a nanosecond time scale, indicate that the triplet ATR is a stronger base than the ground state as far as H-bonding with HFIP is concerned.     

AN EFFICIENT OXYGEN INDEPENDENT TWO-PHOTON PHOTOSENSITIZATION MECHANISM

A novel oxygen-independent photosensitization mechanism from the upper triplet state (T_n) of rose bengal has been demonstrated by selectively populating T_n by sequential two-color laser excitation. Products formed from T_n inhibit red blood cell acetylcholinesterase and decrease viability of P388D₁ mouse macrophage monocyte cells as measured by trypan blue exclusion assay. Laser flash photolysis studies indicate that T_n reacts efficiently, as evidenced by permanent photobleaching of T₁ absorption, with chemical yields approaching unit efficiency. This mechanism may have application for oxygen deficient photosensitization under high intensity, pulsed laser irradiation.     

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.     

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.     

RESONANCE RAMAN and ABSORPTION SPECTRA OF ISOMERIC RETINALS IN THEIR LOWEST EXCITED TRIPLET STATES

Abstract— The triplet-triplet absorption spectra of 9-cis-, 13-cis- and all-fraw-retinal as well as the time-resolved resonance Raman spectra of the lowest electronically excited triplet states of 9-cis-, 11-cis, 13-ciy and all-trans-retinal in aromatic solvents at room temperature have been obtained under conditions ensuring the isomeric purity of the starting materials. The triplet states were produced by triplet energy transfer from a sensitizer in pulse-radiolysis experiments. The overall results suggest that the isomeric retinals form either different relaxed triplet species or different mixtures of relaxed triplet species. The possible implications about the size of the energy barriers separating the various triplet species are discussed. The resonance Raman spectra obtained by using either anthracene (E_T= 177.7 kJ mol^-1) or naphthalene (E_T= 254.8 kJ mol^-1) as sensitizers were virtually identical for the corresponding triplet states from each of the isomers 11-cis-, 13-a's- and all-tams-retinal, suggesting that the relaxed triplet species or the mixture of relaxed triplet species formed from each isomer is independent of the energy of the sensitizer.     

LASER PHOTOLYSIS OF RETINAL AND ITS PROTONATED AND UNPROTONATED n-BUTYLAMINE SCHIFF BASE

Abstract —The pulsed ruby laser (347 nm) flash photolysis technique has been used to measure the triplet-triplet absorption spectra and triplet lifetimes of trans -retinal, N-frans -retinylidene- n -butylamine (NRBA), and protonated NRBA (NRBAH⁺) at room temperature. In methylcyclohexane solution, the triplet lifetimes are in the range 10–20 μs and decrease in the order NRBAH⁺ > NRBA > trans -retinal. Intersy stem-crossing efficiencies (φ_ISC) were determined by a comparison technique using anthracene and 1,2-benzanthracene as reference compounds. For trans -retinal, φ_ISC is 0–50 pM 0–05 in methylcyclohexane and 0–08 in methanol, which confirms that earlier values of 0–11 and 0–017 in these solvents are in error. For NRBA and NRBAH⁺ in methylcyclohexane, Φ_ISC values are 0008 and < 0–001, respectively. Evidence is presented for a significant solvent effect in the isomerization of retinal via the triplet state, and that cis φ trans isomerization occurs from the triplet state of NRBAH⁺. The relation between the intersystem-crossing properties of model compounds and the photochemistry of rhodopsin is discussed.     

ACTINOMETRY IN MONOCHROMATIC FLASH PHOTOLYSIS: THE EXTINCTION COEFFICIENT OF TRIPLET BENZOPHENONE AND QUANTUM YIELD OF TRIPLET ZINC TETRAPHENYL PORPHYRIN

Abstract— The extinction coefficient ε_T, of triplet benzophenone in benzene has been directly determined by absolute measurements of absorbed energy and triplet absorbance, Δ D ⁰_T, under demonstrably linear conditions where incident excitation energy, E ₀, and ground state absorbance, A ₀, are both extrapolated to zero. The result, 7220 ± 320 M ^-1 cm^-1 at 530 nm, validates and slightly corrects many measurements relative to benzophenone of triplet extinction coefficients made by the energy transfer technique, and of triplet yields obtained by the comparative method.
As E ₀ and A ₀ both decrease, Δ D ⁰_T becomes proportional to their product. In this situation, the ratio R = (1/ A ₀)(dΔ D ⁰_T/d E ₀) = (ε_T - ε_G)φ_T. Measurements of R , referred to benzophenone, give (ε_T - ε_G)φ_T for any substance, without necessity for absolute energy calibration.
Both absolute and relative laser flash measurements on zinc tetraphenyl porphyrin (ε_T - ε_G at 470 nm = 7.3 × 10⁴ M ^-1 cm^-1) give φ_T= 0.83 ± 0.04.     

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.     

LASER FLASH PHOTOKINETIC STUDIES OF ROSE BENGAL SENSITIZED PHOTODYNAMIC INTERACTIONS OF NUCLEOTIDES AND DNA

Abstract— Laser flash photolysis studies of the production of the triplet state of the xanthene dye, rose bengal (RB), have been carried out. The reactions of this state with oxygen to form singlet oxygen and the superoxide anion radical have been observed and yields measured. Quenching of RB(T₁) by oxygen leads to approximately 75% singlet oxygen and 20% superoxide. The reactivity of these species-RB(T₁), O₂(¹Δ_g) and O₂^-—with four nucleotides and DNA have been determined. Only guanine residues showed any noticeable reaction at neutral pH. At higher pH guanine rate constants increased. The consequences to biological photodynamic processes are discussed.