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.     

DECAY KINETICS OF THE TRIPLET EXCITED STATE OF LUMIFLAVIN

Abstract— The rate of decay of the triplet state of lumiflavin in deaerated phosphate buffer at pH 6.9, has been studied using the flash photolysis technique, at various concentrations and flash energies. The decay of the transient appears as a mixture of first and second order processes, the relative importance of which depends on the experimental conditions.
The following competitive reactions have been found to explain the course of the reaction of the triplet excited state of lumiflavin.     

THE PHOTOCHEMISTRY OF RIBOFLAVIN—VI. THE PHOTOPHYSICAL PROPERTIES OF ISOALLOXAZINES

Abstract— Many of the photophysical properties of riboflavin and several other N-10 substituted isoal-loxazines have been measured and these include: quantum yields of fluorescence at 77 K and 298 K, φ, quantum yields of phosphorescence at 77 K, φ_P, lifetimes of the triplet state by electron spin resonance and phosphorescence at 77 K, φp,. and the quantum yield of intersystem crossing. φ_isc. For riboflavin in an alcoholic matrix at 77 K the limiting values were: φ _J = 0.32, φ_P= 0.007, φp = 0.20s and φ_isc= 0.7. At 298 K, φ _f for riboflavin in water and alcohol were 0.25 and 0.32, respectively. The results for the photophysical processes are compared with several photochemical processes known to involve the triplet state of riboflavin in aqueous solution. It is concluded that the φ _isc decreases from 0.7 in alcohol to 0.6 in water at 298 K.     

DETERMINATION OF THE pk VALUES OF THE LUMIFLAVIN TRIPLET STATE BY FLASH PHOTOLYSIS

Abstract— The triplet-triplet absorption spectra in aqueous solution of the acid (³LfH₂⁺), the neutral (³LfH) and the basic (³Lf^-) forms of lumifiavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The p K _a values of the corresponding protolytic equilibria of the lumifiavin triplet were found to be 4.4₅±0.1 and 9.8±0.15.     

EXCITED-STATE REACTIONS OF OXIDIZED FLAVIN DERIVATIVES

Abstract— The reactivity of flavin mononucleotide and of lumiflavin triplets was studied by flash and laser photolysis. The rate constants of the triplets with oxygen, with flavin ground-state molecules, and with Br^- ions were determined. Although in solution at room temperature, the protonated flavin triplet, ³F1H⁺, is not formed directly from its very short lived singlet state, a transient, which we think is this triplet, results from protonation of the neutral triplet. This conclusion is based on a comparison between the neutral and the protonated triplet spectra in a low-temperature glass. It is proposed that the protonated triplet can also be formed by sensitization via the phenanthrene triplet.     

INTERMEDIATES IN THE ROOM TEMPERATURE FLASH PHOTOLYSIS OF ADENINE AND SOME OF ITS DERIVATIVES

The transient absorption spectra of aqueous solutions of adenine, 2′-deoxyadenosine, 2′-deoxyadenosine-5′-phosphate and 2′-deoxyadenylyl-(3′-5′)-2′-deoxyadenosine have been determinated at different pH values using conventional flash photolysis. Reactives intermediates produced in the flash photolysis of these adenine derivatives present similar absorption regions: two higher intensity bands in the UV and 560–720 nm wavelength region and a third weaker band at 420–560 nm. On the basis of the effects produced by triplet quenchers and/or electron scavengers the bands have been assigned to hydrated electrons, radical cations, radical anions and/or neutral radicals resulting from neutralization reactions of the charged radicals. The results indicate that the bases photoionize via a triplet state under these conditions.     

THE EFFECT OF 8α-SUBSTITUTION ON FLAVIN TRIPLET STATE AND SEMIQUINONE PROPERTIES AS INVESTIGATED BY FLASH PHOTOLYSIS*

Abstract— Flash photolysis techniques have been used to study the effect of 8α-substitution on flavin triplet state formation and decay and on the properties of neutral and anionic serniquinones. Compared with riboflavin, the N(1) and N(3) isomers of 8α-histidylriboflavin show a lower triplet yield (?10%) and a faster rate of decay (? 4-Cfold). Acetylation of the histidyl a-amino groups and of the flavin ribityl side chain results in a 2-fold increase in triplet yield and a 2-fold slower rate of decay. The yield of neutral 8α-substituted flavin semiquinones upon flash photolysis in the presence of EDTA was approximately 50% that given by riboflavin. These substituted flavin neutral semiquinones dismutated at a rate 2–3 times slower than the corresponding unsubstituted form, although the anionic semiquinones dismutated at approximately the same rate. In the presence of oxygen, the kinetics of semiquinone decay changed from second order to pseudo-first order upon raising the pH, thus showing anionic semiquinone oxidation as seen previously with unsnbstituted flavins. The pK values for the ionization of the neutral 8α-substituted Aavin semiquinones are 1–1.5 units lower than the unsubstituted form. The anionic 8α-substituted flavin semiquinones react with oxygen at a rate 2–10 times more slowly than does the riboflavin form. Such alterations in properties probably reflect the electron-withdrawing effect of the 8α-substituents on the flavin ring system.     

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.     

THE EFFECT OF COORDINATING LIGANDS ON THE TRIPLET STATE OF CHLOROPHYLL

Under laser excitation at 457.9 and 514.5 nm, a frozen solution of chlorophyll a in n -octane displays fluorescence peak maxima at 2K that may be assigned to two distinct monomeric chlorophyll species. Using zero-field fluorescence-detected magnetic resonance the triplet state properties of the two chlorophyll species have been assigned to the monoligated and biligated chlorophyll monomer in which water serves as the ligand coordinated to the magnesium metal center. These triplet state properties for chlorophyll in solution are then utilized in interpreting triplet state results for in vivo chlorophylls associated with the light harvesting chlorophyll protein complex. It is shown that the triplet state data are consistent with attachment of the chlorophyll molecule to the protein site with a single ligand coordinated to the chlorophyll metal center.     

ENERGY CONVERSION IN THE DECAY OF TRIPLET LUMIFLAVIN IN THE PRESENCE OF FERRI- AND FERROCYANIDE

Abstract— Flash photolysis at 450 nm has been used to study the quenching of the excited triplet state of lumiflavin and the transient species formed in subsequent reactions in deaerated phosphate buffer (pH 6.9).
The effect of the presence of ferricyanide on the life time of triplet lumiflavin has been studied. The results suggest an energy transfer reaction without concurrent electron transfer reactions. The rate constant for the process was 2.8 times 10⁹ M ^-1 s^-1. The analogous reaction with ferrocyanide could not be observed because of the efficient electron transfer reaction (δG = -20.6 kcal mol^-1) leading to the formation of the semireduced lumiflavin and ferricyanide. The rate constant for this reaction was 3.3 times 10⁹ M ^-1 s^-1. The semireduced lumiflavin radical was found to disappear in a second order reaction with a rate constant of 1.7 times 10⁹ M ^-1 s^-1. It was found to react with ferricyanide with a rate constant of 0.7 times 10⁹ M ^-1 s^-1.
A model for the various photochemical and photophysical processes involved in the decay and quenching of the lumiflavin triplet state is suggested and discussed.     

PHOTOCHEMISTRY OF THE PHOTOTOXIC POLYACETYLENE PHENYLHEPTATRIYNE*

Abstract— Investigation of the photochemistry of the phototoxic polyacetylene phenylheptatriyne, PHT, was undertaken to obtain further information on competing photo-oxidative type II and non-oxidative processes observed in vivo. Laser excitation (308 or 337 nm) led to the formation of a strong triplet signal with a lifetime of 28 μs in MeOH. The triplet was efficiently quenched by the triplet quencher 1,3-octadiene (k_q - 1.6 × 10⁹M^-1s^-1). Quenching by O₂ occurred with a rate constant (1.7 × 10⁹M^-1s^-1) comparable to the rate of electron transfer to methylviologen (1.4 × 10⁹M^-1 s^-1). The formation of singlet oxygen established earlier (type II reaction) in the former case and the semioxidized PHT radical in the latter case are consistent with the competing phototoxic processes observed in vivo.     

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.     

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.     

THE EFFECTS OF AGGREGATION ON THE FLUORESCENCE and THE TRIPLET STATE YIELD OF HEMATOPORPHYRIN

Abstract— The fluorescence and triplet state yields of hematoporphyrin have been measured in wuter/methanol mixtures. There is a closely linked response of these yields to aggregation of the hematoporphyrin molecule. The monomer, which is the principal species present at high methanol content, has a triplet state yield of 0.91 and a fluorescence yield of 0.09. By contrast, hematoporphyrin solutions with a high water content containing aggregates have lower fluorescence and triplet state yields, e.g. 0.018 and 0.56, respectively, in water. Static, singlet state quenching in some of the aggregates is responsible for the reduced fluorescence yield. The results also show that in addition to these aggregates there are other types of aggregates where there is an increased singlet to ground state radiative transition probability, resulting from the interaction between transition dipoles in adjacent molecules.     

THE FLAVIN-SENSITIZED PHOTOOXIDATION OF NUCLEOTIDES—II. THE PARTICIPATION OF THE FLAVIN TRIPLET STATE

Abstract— –A study has been made of the effects of a series of nucleotides upon the electronic excited states of lumiflavin in order to determine the mechanism of their flavin-sensitized oxidation. A hydrogen-abstraction mechanism is ruled out, because if the nucleotide acts as a reducing agent for the excited dye molecules, it should increase the rate of reduction of the dye when the irradiation is carried out in the absence of oxygen. However, each of the nucleotides studied was found to reduce the rate of anaerobic photoreduction. While oxidation by an intermediate species such as the dye 'moloxide' or singlet oxygen is not entirely ruled out, our evidence suggests that the initial reaction is between the nucleotide and the flavin triplet. This results in a loss of the triplet excitation energy and is a very efficient reaction, guanosine monophosphate shewing 36 per cent of the triplet quenching efficiency of potassium iodide. The relative rates of reaction of the nucleotides with the flavin triplet exactly parallels their quantum yields of sensitized photo-oxidation. The formation of ground-state complexes between flavin and nucleotide and the participation of the singlet excited state of the flavin are not considered to be important.     

REACTIVITY OF EXCITED STATES OF FLAVIN AND 5-DEAZAFLAVIN IN ELECTRON TRANSFER REACTIONS

Abstract— Quenching of the excited states of lumiflavin and 3-methyl-5-deazalumiflavin by methyl-and methoxy-substituted benzenes and naphthalenes in methanol was investigated. The observed difference in the reactivity of acid and neutral lumiflavin triplets is explained thermodynamically by applying the Michaelis cycle, as being due to the higher reduction potential of the acid triplet. In this connection the p K values of lumiflavin triplet (p K ^M= 6.5) and semiquinone (p K ^M= 11.3) have also been determined in methanol. The difference in the reactivity between the singlet and triplet states of lumiflavin is found to be greater as predicted by the difference in excitation energy. The reactivities of the excited states of flavin and 5-deazaflavin differ only slightly in contrast to the marked difference in the ground state reactivities of electron transfer reactions. This is explained in terms of the model of Rehm and Weller. The pH dependence of the electron transfer quenching of 5-deazaflavin triplet was investigated in water, yielding a triplet p K of 2.5. In contrast to the flavin, this triplet p K does not significantly differ from the p K of the 5-deazaflavin ground state. From this, different sites of protonation are deduced for the photoexcited triplet states of flavin and 5-deazaflavin.     

TRIPLET EXCITED STATE OF THE 4'5' PHOTOADDUCT OF PSORALEN AND THYMINE

Abstract— The triplet-triplet absorption spectrum of the 4'5' psoralen-thymine mono-adduct has been determined in water and methanol using the technique of laser flash photolysis. The extinction coefficient of the triplet was measured by the energy-transfer method with retinol triplet as standard, and used to determine the singlet → triplet intersystem crossing quantum yield for 353 nm excitation. Reaction rate constants for mono-adduct triplet with thymine and tryptophan were measured in water. Long-lived transient absorptions detected after quenching the mono-adduct triplet with thymine and tryptophan are assigned mainly to the corresponding mono-adduct radical anion, whose spectrum was established in separate pulse radiolysis studies of the mono-adduct in aqueous formate.
The significant singlet → triplet quantum yields found for the mono-adduct might be consistent with the involvement of triplet excited mono-adduct in DNA cross-link formation, as also may be the high reactivity obtained for the triplet with thymine. The initial quenching products observed resulted from a charge-transfer reaction.     

PHOTOCHEMISTRY AND PHOTOPHYSICS OF GUANINE-CONTAINING DINUCLEOSIDES

Abstract— Yields and action spectra are reported for photochemistry. fluorescence. and total lumincscence at 405 nm due to UV excitation (240–300 nm) or dilutc (-0.1 mM ) solutions or guanosine 5–monophosphate (GMP) and the dinucleosides linking guanine with adenine (ApG and GpA). cytosine (CpG) and uracil (GpU) in neutral ethylene glycol-water (7:3) glasses at 140–165 K. Phosphorescence lifetimes were determined at 140 K. Less complete data are presented for GpC, UpG and dpGpT. Quantum yields for all three processes were usually found to increase as the excitation wavelength increases. Although intramolecular exciplex formation was not dominant under these conditions interactions were strong enough to frustrate attempts at interpretation of results within the Forster very weak coupling framework. There is evidence that the GMP photochemistry proceeds from the triplet state. Surprisingly, this photochemistry is not quenched in ApG, GpA, and dpGpT at 163 K although the adenine (A) and thymine (T) moieties are known to have lower triplet states. At 140 K the phosphorescence from ApG and GpA was entirely characteristic of A but both G and T components were observed from dpGpT.     

ANTHRALIN-DERIVED TRANSIENTS—II. FORMATION OF THE RADICAL BY SPONTANEOUS FRAGMENTATION OF BOTH SINGLET AND TRIPLET STATES OF THE 10,10''-DEHYDRODIMER: RADICAL PAIR MULTIPLICITY EFFECTS

The singlet and triplet states of the anthralin (1,8-dihydroxy-9-anthrone) dehydrodimer have been produced selectively in benzene via pulsed laser excitation and pulse radiolysis respectively. The lifetime of S1 is less than or equal to 30 ps, that of T1 short but unspecified. Both states fragment spontaneously to yield a pair of anthralin radicals. The singlet radical pair predominantly undergoes geminate recombination within the solvent cage. In contrast, the corresponding triplet radical pair undergoes essentially exclusive cage escape to give the anthralin free radical (lambda max 370, 490 and 720 nm) which recombines under normal diffusive conditions. Both recombination processes lead, at least in part, to one or more species which have been assigned as tautomeric forms of the original dimer. The anthralin free radical in benzene is insensitive to the vitamin E model 6-hydroxy-2,2,5,7,8-pentamethylchroman and reacts only slowly with oxygen.     

螺吡喃激发态的反应活性

研究了6′-硝基-1,3,3-三甲基吲(口乃木)苯骈螺吡喃,Ⅰ,和6′-硝基-1-苯基-3,3-二甲基吲(口乃木)苯骈螺吡喃,Ⅱ,的光化学,Ⅰ的直接光解和敏化光解均得到份菁结构的光解产物Ⅲ,量子产率分别是0.42和0.32.Ⅱ直接光解时生成相应的有色光解产物Ⅳ,量子产率是0.40,氮杂蒽酮为敏化剂的敏化光解量子产率是0.58,无疑地,单线态和三线态均参予螺吡喃的发色反应,用这些结果和折算关系式得到,在Ⅰ的直接光解中,Ⅲ主要来自单线态,而在Ⅱ的直接光解中,Ⅳ主要来自三线态.从Ⅰ或Ⅱ的敏化联乙酰燐光实验中求得三线态参数.它们是:φ_ISC^Ⅰ＝0.11,φ_ISC^Ⅱ＝0.48;³K_dt^Ⅰ＝3×10⁴秒^-1,³K_dt^Ⅱ＝9×10⁴秒^-1,类似地,³K_r^Ⅰ＝0.12×10⁴秒^-1,³K_r^Ⅱ＝2.52×10⁴秒^-1,这样,³φ_r^Ⅰ值较小是由于³φ_ISC^Ⅰ值较小,而三线态寿命随结构变动不大.最后,取苯乙烯为模型,用它的电子能量与乙烯双键扭曲角度关系图推断出,来自单线态的双离子具有螺环构型,而来自三线态者具有平面状构型.