POTENTIAL PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY—II. PHOTOPHYSICAL PROPERTIES OF [26] PORPHYRIN

Abstract— –Photophysical properties of [26] porphyrin (26 P) were investigated in chloroform. The quantum yields of fluorescence, of S₁→ T₁ intersystem crossing and singlet oxygen formation were measured. The purity, stability, the strong absorption in the red (δ_max= 783 nm; ε_max= 28 000 M ¹ cm^-1) and the ability of singlet oxygen formation recommend 26 P as potential photosensitizer for tumor therapy.     

PREPARATION AND PHOTOPHYSICAL STUDIES OF PORPHYRIN-C60 DYADS

Abstract Porphyrin-C₆₀ dyads in which the two chromophores are linked by a bicyclic bridge have been synthesized using the Diels-Alder reaction. The porphyin singlet lifetimes of both the zinc (Pz_n-C₆₀) and free base (P-C₆₀) dyads, determined by time-resolved fluorescence measurements, are ≦17 ps in toluene. This substantial quenching is due to singlet-singlet energy transfer to C₆₀ The lifetime of Pzn-¹C₆₀ is -5 ps in toluene, whereas the singlet lifetime of an appropriate C₆₀ model compound is 1.2 ns. This quenching is attributed to electron transfer to yield P_zn^bull;+-C₆₀^bull;-. In toluene, P-¹C₆₀ is unquenched; the lack of electron transfer is due to unfavorable thermodynamics. In this solvent, a transient state with an absorption maximum at 700 ran and a lifetime of-10 μs was detected using transient absorption methods. This state was quenched by oxygen, and is assigned to the C₆₀ triplet. In the more polar benzonitrile, P-¹C₆₀ underoes photoinduced electron transfer to give P^•+-C₆₀^bull;-. The electron transfer rate constant is −2 × 10¹¹ s⁻¹.     

A Water-Soluble Luminescence Oxygen Sensor

We developed a water-soluble luminescent probe for dissolved oxygen. This probe is based on (Ru[dpp(SO₃Na)₂]₃) Cl₂, which is a sulfonated analogue of the well-known oxygen probe (Ru[dpp]₃)Cl₂. The compound dpp is 4,7-diphenyl-1,10-phenanthroline and dpp(SO₃Na)₂ is a disulfonated derivative of the same ligand. In aqueous solution in the absence of oxygen (Ru[dpp(SO₃Na)₂]₃)Cl₂ displays a lifetime of 3.7 μs that decreases to 930 ns on equilibrium with air and 227 ns on equilibrium with 100% oxygen. The Stern–Vohner quenching constant is 11330 M⁻¹. This high oxygen-quenching constant means that the photoluminescence of Ru(dpp[SO₃Na]₂)₃Cl₂ is 10% quenched at an oxygen concentration of 8.8 x 10⁻⁶ M , or equilibration with 5.4 torr of oxygen. The oxygen probe dissolved in water displays minimal interactions with lipid vesicles composed of dipalmityl-L-α-phosphatidyl glycerol but does appear to interact with human serum albumin. The absorption maximum near 480 nm, long lifetime and large Stokes'shift allow this probe to be used with simple instrumentation based on a light-emitting diode light source, allowing low-cost oxygen sensing in aqueous solutions. To the best of our knowledge this is the first practical water-soluble oxygen sensor.     

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.     

FLUORESCENCE LIFETIMES OF CHLOROPHYLL a: SOLVENT, CONCENTRATION AND OXYGEN DEPENDENCE

Fluorescence lifetimes (τ_f) of chlorophyll a (Chi a ) have been measured by the single-photon-counting technique over a wide range of concentrations (˜10^-7˜10^-4 M ) in deoxygenated pyridine, diethyl ether, toluene and methanol. At pigment concentrations ˜1 μ M , reabsorption of fluorescence induces significant artifacts on measured values of τ_f which are dependent on detection wavelength and the specific geometry of the experiment. There is a clear dependence of τ_f on the nature and degree of solvation, including both coordination of the central magnesium and hydrogen-bonding of the solvent (viz. alcohols) to the macrocycle. Quenching of the excited singlet state by molecular oxygen was measured quantitatively in ether, and a bimolecular rate constant markedly slower than the diffusion-controlled limit was obtained.     

极性敏感的BDP分子溶剂化效应的光谱性质

合成了具有分子内电荷转移(ICT)性质的三重态光敏剂分子BDP,研究了其稳态吸收光谱、荧光光谱、荧光寿命、飞秒/纳秒瞬态吸收光谱及诱导产生单线态氧的能力等性质,发现强极性溶剂对BDP分子的溶剂化效应降低了其ICT态和第一激发三重态(T1态)的能量,从而降低了BDP分子单线态氧的产量.     

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.     

AN INVESTIGATION OF RETINAL AS A SOURCE OF SINGLET OXYGEN

Abstract— All -trans retinal is dissolved in alcohols and illuminated at 365 nm in the presence of a singlet oxygen acceptor, 2,5-dimethylfuran. Illumination produces the photosensitized oxidation of the acceptor which is measured by the disappearance of its 215 nm absorption band. A kinetic study is carried out and β_DMF is 1.6 × 10^--⁴ M . The quantum yield of ¹O₂ production from the light-excited retinal is estimated to 0.096. The retinal sensitized photooxidation of dimethylfuran is inhibited by a ¹O₂ quencher, 1,4-diazabicyclo(2,2,2)-octane, and enhanced by deuteration of the solvent. Deuterated solvents are known to increase ¹O₂ lifetime.
The production of ¹O₂ from retinal is briefly discussed in relation to the damage which may be induced by light in the visual cells.     

Energetics of Photoinduced Electron Transfer in the Indole-O2 Cluster in Gas Phase: Possible Consequences for Photoexcited Tryptophan in Solution

Abstract— A direct process for an activationless electron transfer from photoexcited tryptophan to molecular oxygen is proposed. By photodetachment of mass-selected indole-O_2- clusters in gas phase a neutral indole₊ O_2- charge-separated exciplex state is found at 2.25 0.2 eV above the neutral ground state. By theory also, the existence of an excited charge separated state at 3.05 0.2 eV is postulated. In gas phase both charge-separated cluster states are energetically below the first singlet states ₁L_b and ₁L_a and the lower even below the first triplet state T₁ of indole. In gas-phase clusters these energetics imply a very efficient quenching of photoexcited indole by fast electron transfer to oxygen. We discuss a similar mechanism for tryptophan-O₂ in aqueous environment and find it without activation barrier and presumably extremely fast. In the collisional tryptophan_*-O₂ complex the efficiency and the time scale of the charge transfer process should be mostly solvent independent. In polar solvent a complete charge separation and free superoxide formation are expected. We correlate this model with previous fluorescence and phosphorescence quenching data of excited tryptophan by O₂ and propose electron transfer to be the relevant process.     

CHEMILUMINESCENCE FROM AUTOXIDIZING 6-HYDROXYDOPAMINE: THE INVOLVEMENT OF ACTIVATED FORMS OF OXYGEN

Abstract— The autoxidation of the catecholamine neurotoxin 6-hydroxydopamine (20 μ M ) gave rise to a chemiluminescence which was greatly stimulated by FeSO₄ (20 μ M ) or by hydrogen peroxide addition (20 μ M to 2 m M ). The luminescence of both 6-hydroxydopamine alone or 6-hydroxydopamine plus hydrogen peroxide was strongly inhibited by catalase and by superoxide dismutase (both at 10 μg/m/); bovine serum albumin at 10 μg/m/ had no inhibitory effect. The luminescence was also strongly inhibited by several potent hydroxyl radical trapping agents and also by low concentrations of the ¹O₂ quencher DABCO (l,4-diazabicyclo-2.2.2.-octane). Chemiluminescence was greatly enhanced in D₂O, a solvent in which ¹O₂ has a prolonged lifetime. These data demonstrate the involvement of hydrogen peroxide, the superoxide radical and the hydroxyl radical in the chemiluminescence. The data are also consistent with some role for ¹O₂.     

Near-infrared Light Emission from Nanomolar-level Singlet Molecular Oxygen Generated with an Ultra low Concentration Mixture of Hypochlorite and Hydrogen Peroxide

Abstract— We report the detection of a weak near-infrared light emission originating from 8 nM singlet molecular oxygen (¹O₂) produced in a mixture of 1 m M hypochlorite (OC1^-) and 8 n M hydrogen peroxide (H₂O₂). The measurements were made with a highly sensitive detection system for ultraweak light emission in the 1.0-1.5 μm wavelength region. The emission intensity exhibited linear dependence for H₂O₂ concentrations in the range of 8-670 n M . The mixture containing a lower concentration (33 μ M ) of OCl^- pseudocontinuously emitted near-infrared light for 5 s. The rate constant for ¹O₂ production obtained from the kinetic analysis agrees with that previously reported. Our results demonstrate the possibility of measuring very low concentrations of ¹O₂ in a OCi-/H₂O² mixture as well as ¹O₂ production in intact living systems.     

AFFINITY FOR OXYGEN IN PHOTOREDUCTION OF MOLECULAR OXYGEN AND SCAVENGING OF HYDROGEN PEROXIDE IN SPINACH CHLOROPLASTS

Abstract— The apparent K _m for O₂ in the photoreduction of molecular oxygen by spinach class II chloroplasts and photosystem I subchloroplast fragments was determined. In both cases, a value of 2 ∼ 3 μ M O₂ was obtained. The reaction rate constant between O₂ and P-430, the primary electron acceptor of PS I, is estimated to be ∼ 1.5 × 10⁷ M ^-1 s^-1 and the factors affecting the production of superoxide by the photoreduction of O₂ in chloroplasts are discussed. Preliminary evidence is presented indicating the occurrence of an azide-insensitive scavenging system for H₂O₂ in chloroplast stroma.     

THE Py SCALE OF SOLVENT POLARITIES. SOLVENT EFFECTS ON THE VIBRONIC FINE STRUCTURE OF PYRENE FLUORESCENCE and EMPIRICAL CORRELATIONS WITH ET and Y VALUES

Abstract— Pyrene fluorescence spectra have been run in 62 solvents of widely differing solvent polarity. As has been noted previously, the intensity ratio of the first (the 0–0 band) and third bands in vibronic fine structure of these spectra are very sensitive to solvent polarity. These I ₁/ I ₃ values, however, are not sensitive to hydrogen bonding aspects of solvent-solute interactions. Correlations are reported with Winstein's Y values and with Dimrotb's E _T values. On this basis the I ₁/ I ₃ values for pyrene fluorescence are suggested as the basis for a new empirical scale of solvent polarity, called the Py scale, which offers certain conveniences over other scales of solvent polarity.     

ESTIMATION OF ENERGY DISTRIBUTION AND REDISTRIBUTION AMONG TWO PHOTOSYSTEMS USING PARALLEL MEASUREMENTS OF FLUORESCENCE LIFETIMES AND TRANSIENTS AT 77 K

Abstract— A single-sample method for estimating energy distribution and redistribution among the two photosystems using fluorescence lifetimes and transients at 77 K is presented. In this method,α(the fraction of photons absorbed by photosystem I, PSI) is F_1(α)/(F_1(α)+ (τ_{F 1(M)}/τ_{F 2(M)}).F_2(M)) where, F_1(α) is the fluorescence intensity from PSI excited by photons initially absorbed by the latter, τ_{F 1(M)} and τ_{F 2(M)} are the maximum lifetimes of fluorescence from chlorophyll- a in PSI (1) and II (2), and, F_2(M) is the maximum fluorescence intensity from PSII (P level). Analysis of the intensities and lifetimes of wavelength resolved fluorescence of thylakoids (pH 7.0), with and without cations, leads to the following conclusions: The addition of 10 m M Na⁺ to cation-depleted thylakoids (pH 7.0) increases α by ˜ 10%, while the subsequent addition of 10 m M Mg²⁺ leads to three principal concomitant changes (in the order of importance): a 50% decrease in PSII to PSI energy transfer, a 20% increase in other radiation-less losses, and a 10% decrease in α.     

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.     

THE PHOTOPHYSICS OF p-AMINOBENZOIC ACID

Abstract— The lowest-lying allowed UV transition in p -aminobenzoic acid (PABA) is assigned Γ→¹L_a based on quantitative absorption and fluorescence studies, as well as semiempirical PM3 multielec-tron configuration interaction calculations. The oscillator strengths, fluorescence quantum efficiencies and lifetimes are reported for PABA in several polar, nonpolar, protic and aprotic solvents (aerated) at 296 K. Reasonable agreement is found between the observed radiative rate constant and that calculated from the absorption and fluorescence spectra. Shifts in the absorption and fluorescence spectra in aprotic solvents are analyzed in terms of the Onsager reaction field model; results are consistent with an increase in dipole moment of ca 4 D between the relaxed S₀ and S₁, states. No evidence is found for the emission from the amino-twisted form of PABA in all solvents studied although calculations show that the amino-twisted S, state is highly polar, but higher in energy by ca 35 kJ/mol ( in vacuo ). The fluorescence efficiency is excitation wavelength independent in both methylcyclohexane and water. The temperature dependence of the nonradiative rate constant (from S₁) was studied in several solvents. Nonradiative decay may be due to intersystem crossing, which would be fast enough to compete with thermally activated intramolecular NH₂ twisting. The phosphorescence spectrum and lifetime obtained in an EPA glass at 77 K are reported, and the triplet energy of PABA is estimated.     

MECHANISM OF CONCENTRATION QUENCHING OF A XANTHENE DYE ENCAPSULATED IN PHOSPHOLIPID VESICLES

Encapsulating a xanthene dye in phospholipid vesicles produces vesicle solutions that contain dye at very high microscopic concentrations, but have a low overall optical density, thereby eliminating reabsorption. Using this system, we have studied the effects of concentration on the fluorescence lifetime of one such dye, sulforhodamine 101. We have observed that the lifetime decreases as a function of encapsulated dye concentration, which is indicative of collisional quenching. The lifetime decreases from 4.5 nsec for sulforhodamine in dilute aqueous solution to 0.69 ns at an encapsulated concentration of 33 m M . The bimolecular rate constant for this event is 2.6 10¹⁰ M ^-1 s^-1, consistent with a diffusion controlled event. However, the quenching constant calculated from changes in intensity is 2.2 10¹¹ M ^-1 s^-1. Thus, collisional quenching is not the predominant mechanism of quenching. The absorption spectra of dye in vesicles indicate an important contribution from static complex formation. Förster distance calculations indicate that energy transfer can also occur to a significant extent, with a predicted efficiency of transfer of 34% at a dye concentration of only 1 m M     

FLUORESCENCE AND PHOTOOXIDATION OF 1—ANILINONAPHTHALENE—8—SULFONIC ACID IN REVERSED MICELLAR SOLUTIONS AND THE EFFECT OF CCl4

Abstract— The 1-anilinonaphthalene-8-sulfonic acid solubilized in dodecylammonium propionate reversed micellar cyclohexanic solutions, emitted a strong fluorescence, and was photooxidized under aerobic conditions. Carbon tetrachloride (CCl₄) highly quenched the fluorescence and remarkably enhanced the oxidation reaction. The fluorescence quenching obeyed the Stern-Volmer relation, and the photooxidation was caused by the singlet oxygen generated by the photosensitization of the dye. From the kinetic analysis, it was known that the intersystem crossing rate from the dye excited singlet to triplet was enhanced by CCl₄. Carbon tetrachloride did not quench the triplet state. The ratio of quantum yields for the oxidation in the presence and absence of CCl₄ was independent of the oxygen concentration in the reaction mixture. The fluorescence quenching constant and the intersystem crossing rate were obtained at various solubilized water contents.     

CHEMICAL REACTION RATES OF AMINO ACIDS WITH SINGLET OXYGEN

Abstract— The chemical reactions of amino acids with singlet oxygen have been measured in D₂O solution where the singlet oxygen was generated directly by irradiation of the oxygen ³∑_g^-→¹δ_g+ lv electronic transaction with the 1.06 μm output of an Nd-Yag laser. Chemical reaction was measured as amino acid loss by an amino acid analyzer or by fluorescence in the cases of tryptophan and tyrosine.
The chemical rate constants, in units of 10⁷ M ^-1s^-1, are histidine 10, tryptophan 3, methionine 1.7, tyrosine 0.8 and alanine 0.2, In the cases of histidine, methionine and alanine the interaction appears to be entirely chemical, i.e. there is no evidence for physical quenching in addition to the chemical reaction. The histidine chemical reaction rate constant shows an increase with pD with a p K of 6.9.