THE ROLE OF GROUND STATE COMPLEXATION IN THE ELECTRON TRANSFER QUENCHING OF METHYLENE BLUE FLUORESCENCE BY PURINE NUCLEOTIDES

The effect of three purine nucleotides on the fluorescence of methylene blue in aqueous buffer has been investigated. Guanosine-5'-monophosphate (GMP) and xanthosine-5'-monophosphate cause fluorescence quenching while adenosine-5'-monophosphate causes a red shift in the fluorescence maximum. All three nucleotides form ground state complexes with the nucleotides as indicated by absorption spectroscopy. The fluorescence changes at nucleotide concentrations less than 30 mM are best described by a static mechanism involving the formation of non-fluorescent binary and ternary complexes in competition with dimerization of the dye. Quenching of the fluorescence decay (tau = 368 ps) at high GMP concentrations (10-100 mM) occurs at the rate of diffusion. The mechanism of fluorescence quenching may involve electron transfer within the singlet excited dye-nucleotide complex although published values of the oxidation potentials of various purine derivatives would suggest that all three nucleotides should cause quenching. Evidence for electron transfer was obtained from flash photolysis experiments in which 100 mM GMP was found to cause the appearance of a long lived transient species absorbing in the region expected for semimethylene blue.     

Graphene as a quencher of electronic excited states of photochemical probes

Graphene sheets quench the singlet and triplet excited states of a series of six photochemical probes including pyrene, acridine orange, tris(2,2?-bipyridyl)ruthenium(II) dichloride, methylene blue, meso-tetrakis(phenylsulphonate)porphyrin, and 5,10,15,20-tetraphenyl-21H,28H-porphine zinc. It was found that Stern-Volmer fluorescence quenching can fit to one or two different quenching regimes depending on the probe. In addition, the quenching can be either static or dynamic depending on the fluorophore. The occurrence of several quenching regimes has been interpreted considering that quenching arises from the crowding of the fluorophore on both graphene faces, or site isolation on the graphene sheets. Laser flash photolysis has shown that the triplet lifetime of the probes generally decreases due to graphene quenching and that no new transients appear except in the case of methylene blue, where a new absorption spectrum characterized by a continuous absorption band is observed and attributed to graphene radical ion. This spectroscopic evidence suggests that the most general quenching mechanism is energy transfer from the singlet or triplet excited state of the dye to graphene. This raises the issue of determining the energy of the electronic excited states of graphene.     

The excited states interaction of safranine-O with PAMAM and DAB dendrimers in methanol

The quenching of the excited singlet and triplet states of the synthetic dye safranine-O by low generation PAMAM and DAB dendrimers was investigated in methanol. The rate constants for the quenching of the excited singlet state depend on the number of primary amino groups in the dendrimer. The first-order rate constant for the decay of the triplet state presents a downward curvature as a function of the quencher concentration. This behavior was interpreted in terms of the reversible formation of an intermediate complex in the excited state. From a kinetic analysis of the quenching mechanism the equilibrium constant K_exc could be extracted. The values of K_exc may be related to the proton affinity of the quencher. The results were interpreted in terms of a reversible proton transfer quenching. This was further confirmed by the transient absorption spectra obtained by laser flash photolysis. The transient absorption immediately after the triplet state quenching could be assigned to the unprotonated form of the dye. At later times the spectrum matches the semireduced form of the dye. The overall process corresponds to a one-electron reduction of the dye mediated by the deprotonated triplet state.     

Proton and electron transfer in the excited state quenching of phenosafranine by aliphatic amines

The quenching of the excited singlet and triplet states of phenosafranine by aliphatic amines was investigated in acetonitrile and methanol. The rate constants for the quenching of the excited singlet state depend on the one-electron redox potential of the amine suggesting a charge transfer process. However, for the triplet state, quenching dependence on the redox potential either is opposite to the expectation or there is not dependence at all. Moreover, in MeOH the first-order rate constant for the decay of the triplet state, k(obs) presents a downward curvature as a function of the amine concentration. This behavior was interpreted in terms of the reversible formation of an intermediate excited complex, and from a kinetic analysis the equilibrium constant K(exc) could be extracted. The log K(exc) shows a linear relationship with the pKb of the amine. On the other hand, for the triplet state quenching in acetonitrile k(obs) varies linearly with the amine concentration. Nevertheless, the quenching rate constants correlate satisfactorily with pKb and not with the redox potential. The results were interpreted in terms of a proton transfer quenching, reversible in the case of MeOH and irreversible in MeCN. This was further confirmed by the transient absorption spectra obtained by laser flash photolysis. The transient absorption immediately after the triplet state quenching could be assigned to the unprotonated form of the dye. At later times the spectrum matches the semireduced form of the dye. The overall process corresponds to a one-electron reduction of the dye mediated by the deprotonated triplet state.     

Photophysical and Photochemical Studies of Pyridoxamine

The absorption and fluorescence emission of pyridoxamine were studied as function of pH and solvent properties. In the ground state, pyridoxamine exhibits different protonated forms in the range of pH 1.5–12. Fluorescence studies showed that the same species exist at the lowest singlet excited state but at different pH ranges. The phenol group is by ca. 8 units more acidic in the excited state than in the ground state. On the other hand, the pyridine N‐atom is slightly more basic in the lowest excited state than in the ground state. Excitation spectra and emission decays in the pH range of 8–10 indicate the protonation of the pyridine N‐atom by proton transfer from the amine group, in the ground and singlet excited states. Spectroscopic studies in different solvents showed that pyridoxamine in the ground or excited states exhibits intramolecular proton transfer from the pyridine N‐atom to the phenol group, which is more favorable in solvents of low hydrogen‐bonding capacity. The cationic form with the protonated phenolic group, which emits at shorter wavelength, is the dominant species in nonprotic solvents, but, in strong proton‐donor solvents, both forms exist. The fluorescence spectra of these species exhibit blue shift in protic solvents. These shifts are well‐correlated with the polarity and the H‐donor ability of the solvent.     

A novel sensitized chemiluminescence: Infrared emissions from thiazine dyes sensitized by singlet oxygen

An unusual infrared chemiluminescence emission (8130Å) of methylene blue, and other thiazine dyes, sensitized by singlet molecular oxygen is reported. This chemiluminescence does not correspond to the ordinary fluorescence of the dye and cannot be explained by previously proposed mechanisms for singlet oxygen sensitized emissions of dyes. From energetic considerations singlet molecular oxygen in its ¹Σ_g⁺ state is postulated as the sensitizing agent for the thiazine dye chemiluminescences. Schemes in which ¹Σ_g⁺ oxygen transfers electronic excitation energy (a) to the lowest triplet state of the dye, (b) to a combined multiplicity state of the lowest triplet state of the dye, and triplet molecular oxygen, or (c) to a charge-transfer state between the dye and oxygen, are compared. The chemiluminescence of methylene blue in aqueous solution may be used as a luminescent probe for ¹Σ_g⁺ oxygen.     

ON THE ACRIDINE AND THIAZINE DYE SENSITIZED PHOTODYNAMIC INACTIVATION OF LYSOZYME—SINGLET OXYGEN SELF-QUENCHING BY THE SENSITIZERS

Abstract— The quantum yield of the photodynamic inactivation of lysozyme increases in the sequence acridine orange, methylene blue, proflavine and acriflavine (1:5:6:12). At least up to protein concentrations of 0.1 m M , singlet oxygen is exclusively responsible for the inactivation of the enzyme. For methylene blue, acriflavine and proflavine the quantum yields decrease considerably with increasing dye concentrations. From measurements in H₂O and D₂O buffer solutions it was concluded that in the case of methylene blue the effect is mainly caused by the quenching of singlet oxygen [rate constant (3–4) × 10⁸ M ⁻¹ s⁻¹]. For the acridine sensitizers both singlet oxygen and dye triplet quenching processes have to be taken into consideration. It has been found that all sensitizers act as competitive inhibitors of the enzymatic reaction of lysozyme. However, the dye-protein interaction near the active center cannot be responsible for the observed dye self-quenching effect.     

KINETIC PROPERTIES OF THE TRIPLET STATES OF METHYLENE BLUE AND OTHER PHOTOSENSITIZING DYES

Abstract— The long-lived (> 1 μsec) transients formed in the flash excitation of the representative photosensitizers methylene blue, eosin Y and pyrene have been investigated and various criteria have been used to distinguish between triplet state intermediates and chemical intermediates. Previous assignments of the triplet transients of methylene blue appeared less secure in view of the photochemical reactivity of this dye and its lack of phosphorescence. Earlier assignments of monomeric and dimeric triplet transients of methylene blue are substantiated, however, by the observations that the rate constant for quenching by oxygen is approximately 1/9^th diffusion controlled and the formation rates are commensurate with singlet decay rates and by the observation of triplet-triplet annihilation. Additional evidence in support of monomer triplet assignments for methylene blue and eosin Y is provided by the effect of heavy atom quenchers Cs⁺, Hg²⁺ and T1⁺ on decay rates. Due to chemical reactivity, quenching by I⁻appears less suitable as a diagnostic test for triplet state intermediates. The effect of N₃⁻, which is known to quench singlet oxygen molecules and to alter the course of photosensitized oxidations, on the triplet decay of methylene blue, eosin Y and pyrene is also investigated.     

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.     

Photoluminescence of 2-aminofluorene: a relook

The absorption and fluorescence spectra of 2-aminofluorene (2-AF) have been studied as a function of solvent polarity and acid concentration. Using the multiparametric approach of Taft et al., it is clear that 2-AF is a better proton acceptor in the S₀ state and a proton donor in the S₁ state. Excitation of 2-AF to three electronically excited states has shown that fluorescence is always observed from the lowest excited singlet state, but fluorescence quantum yield increases with the increase of λ_exc. The decrease in fluorescence quantum yield with increase in solvent polarity and hydrogen bonding is due to solvent-induced quenching. A correspondence is observed between the decrease and increase in fluorescence intensities of the neutral and monocation, respectively, in the pH range from 6 to 3. Proton-induced fluorescence quenching of neutral 2-AF is noticed in the pH range 3 to 1. pK_a and values were determined for different prototropic equilibria and are discussed.     

Electron vs energy transfer in arrays featuring two Bodipy chromophores axially bound to a Sn(IV) porphyrin via a phenolate or benzoate bridge

In this report we describe the synthesis of multichromophore arrays consisting of two Bodipy units axially bound to a Sn(IV) porphyrin center either via a phenolate (3) or via a carboxylate (6) functionality. Absorption spectra and electrochemical studies show that the Bodipy and porphyrin chromophores interact weakly in the ground state. However, steady-state emission and excitation spectra at room temperature reveal that fluorescence from both the Bodipy and the porphyrin of 3 are strongly quenched suggesting that, in the excited state, energy and/or electron transfer might occur. Indeed, as transient absorption experiments show, selective excitation of Bodipy in 3 results in a rapid decay (τ ≈ 2 ps) of the Bodipy-based singlet excited state and a concomitant rise of a charge-separated state evolving from the porphyrin-based singlet excited state. In contrast, room-temperature emission studies on 6 show strong quenching of the Bodipy-based fluorescence leading to sensitized emission from the porphyrin moiety due to a transduction of the singlet excited state energy from Bodipy to the porphyrin. Emission experiments at 77 K in frozen toluene reveal that the room-temperature electron transfer pathway observed in 3 is suppressed. Instead, Bodipy excitation in 3 and 6 results in population of the first singlet excited state of the porphyrin chromophore. Subsequently, intersystem crossing leads to the porphyrin-based triplet excited state.     

Probing the active site of trypsin with rose bengal: insights into the photodynamic inactivation of the enzyme

In this work the active site of trypsin has been probed with the dye rose bengal. The dye binds competitively to the enzyme, and it can be used as a probe of the active site of the enzyme. On the basis of the emission wavelength, the binding site of trypsin is relatively polar and is similar to that of acetone in its polarity. The triplet state of rose bengal is quenched by trypsin. This quenching may be caused by the tryptophan and tyrosine residues that are in the near vicinity of the trypsin active site. This quenching can compete with the formation of singlet oxygen from the excited triplet state of rose bengal. We demonstrate that the singlet oxygen involved in the photoinactivation of trypsin is produced by the free rose bengal in solution and the bound dye is incapable of producing singlet oxygen. This explains the lack of correlation between photoinactivation efficiency and sensitizer binding capability previously reported by Wade and Spikes.     

Photophysics and Photochemical Studies of the Vitamin B6 Group and Related Derivatives

The photophysics and photochemical properties of vitamin B6 constituents and analogs were studied as function of pH and solvent. The p K of the phenolic oxygen and the pyridine ring nitrogen depends on the electron donor-acceptor ability of the 4-substituent, and agrees with the calculated proton affinity. For all studied compounds, the fluorescence properties showed that the phenolic oxygen is 8 units more acidic in the lowest singlet excited state than in the ground state. The pyridine N-atom is slightly more basic in the excited state. At pH of biological significance, pH 6–8, pyridoxamine and 4-pyridoxic acid are the more efficient chromophores with higher fluorescence yield and longer lifetime. Spectroscopic studies showed that the tautomeric equilibrium depends on the nature of the 4-substituent. The quenching of the singlet excited state of pyridoxamine and 4-pyridoxic acid by amino acids, free or in a peptide, and DNA bases at pH 7 was studied by time-resolved fluorescence techniques. The quenching rate constants are well correlated with the redox properties of the pyridoxinic compound and amino acids, and are related to the free energy change in the electron transfer process. Guanosine and pyrimidine bases also are efficient quenchers, involving an electron transfer reaction.     

LASER FLASH SPECTROSCOPY OF METHYLENE BLUE WITH NUCLEIC ACIDS. EFFECTS OF IONIC STRENGTH AND pH

Laser flash spectroscopic measurements were made on methylene blue complexed to the synthetic polynucleotides poly[d(G-C)] and poly[d(A-T)] in solutions of varying ionic strength and pH. Triplet decay rates and rates of triplet quenching by oxygen have been measured for the polymer/dye solutions. The triplet decay and oxygen quenching rates of methylene blue in complex with poly[d(A-T)] are much less sensitive than those with poly[d(G-C)] with respect to variations in ionic strength. It is also shown that protonation of the triplet state of MB+ with poly[d(A-T)] is slower than that of free dye. These results indicate strong binding of the dye to poly[d(A-T)]. Excitation of the MB+/poly[d(A-T)] complex at 665 nm yielded single exponential decay kinetics, in contrast with excitation at 600 nm where double exponential kinetics were measured. This is tentatively assigned to excitation of dye dimer bound to this polymer.     

A physiochemical study of azo dyes: DFT based ESIPT process

Azo linked dye derivatives were synthesized and characterized by NMR, mass and elemental analysis. An excited state intramolecular proton transfer (ESIPT) in hydroxy Schiff base has been analyzed, and found that two distinct ground state isomers of I and II are responsible for the observed dual emission. DFT calculation on energy, dipole moment, charge distribution of the rotamers in the ground and excited states support the ESIPT process. PES calculation indicates that the energy barrier for the interconversion of two rotamers is too high in the excited state than the ground state. By varying the addition of base concentration to hydroxy Schiff base, two isobestic points were found which confirm the equilibrium among the trans enol form, anion and the cis enol form. Fluorescence quenching with metal ions reveal that hydroxy Schiff base can be used as a new fluorescence sensor to detect the Cu(2+) ion.     

Proton activity in nafion films: probing exchangeable protons with methylene blue

A simple approach to monitor the H(+) activity of a proton-exchange membrane (Nafion) is introduced by incorporating methylene blue as an indicator dye. The dye exhibits characteristics absorption maxima at 665 and 745 nm corresponding to its singly and doubly protonated forms, respectively. The apparent proton activity of Nafion as monitored from the appearance of doubly protonated methylene blue absorption is equivalent to 1.2 M H2SO4. By monitoring the spectral changes associated with the protonation equilibrium of the dye, it is possible to probe the rate and the exchangeable proton sites within the Nafion film. For the Nafion 117 film, we estimate the total exchangeable proton sites to be 2.5 x 10(19) sites/cm(2) or 4.2 x 10(-5) mols/cm(2). The equilibrium constant for the H(+)/Na(+) exchange for the bound sites is determined to be 2.2. The feasibility of methylene blue as a probe to monitor proton activity during the operation of a direct methanol fuel cell has been explored.     

THE INTERACTION OF THE EXCITED STATES OF SAFRANINE T WITH ALIPHATIC AMINES IN ORGANIC SOLVENTS

Abstract—
The reactions of the excited states of safranine T with aliphatic amines have been studied in methanol and acetonitrile. Quenching of the singlet and triplet states occurs by different mechanisms. Whereas the former excited state is quenched by a charge-transfer mechanism, the triplet state is quenched through proton transfer from the excited dye to the amine. This process leads to the unprotonated form of the dye in the triplet state, which is later quenched by amines to form the corresponding semireduced species. The monoprotonated triplet also undergoes self-quenching in both solvents (k = 1.2 × 10⁸ M ^-1 s^-1).     

CHARACTERIZATION OF THE RED LIGHT INDUCED REDUCTION OF A PARTICLE ASSOCIATED b-TYPE CYTOCHROME FROM CORN IN THE PRESENCE OF METHYLENE BLUE*

Methylene blue transfers electrons to a membrane-associated b-type cytochrome in particulate fractions from corn coleoptiles. The K_m for methylene blue is less than 1 µM under optimal conditions. This reaction is destroyed by boiling, but not by 7 M urea. Kinetic analyses of the influence of light intensity on cytochrome reduction suggest that a first order photochemical reaction is limiting. Free EDTA may serve as an electron donor in this system at least at high methylene blue and protein concentrations. The photoactivity does not coincide either with mitochondrial or endoplasmic reticulum markers, and may be localized in plasma membrane. There is an estimated 5 times 10^-10 mol photoreducible cytochrome per g coleoptile tissue. Studies on the effect of pH on the reaction in the presence of methylene blue or thionine indicate that dye photoreduction itself is not rate-limiting. Wavelength dependence studies suggest that it is methylene blue monomer and not dimer which mediates the reaction. Although oxygen is apparently required for the reaction, neither superoxide nor excited singlet oxygen appear to be involved. The reaction mechanism is still unknown.     

REDUCTION OF THE EXCITED SINGLET STATE OF CHLOROPHYLL AND PHEOPHYTIN AT THE ORGANIC CRYSTAL/AQUEOUS SOLUTION INTERFACE

Abstract— Reduction of the excited singlet state of chlorophyll a , chlorophyll b , and pheophytin a at the surface of perylene, anthracene, and chrysene single crystals has been measured as hole injection current. The dependence of the quantum yield on the standard free-energy difference of the reaction was in accordance with theoretical expectations without correcting for an interaction energy. The maximum quantum yield of only about 10⁻² holes per absorbed photon is ascribed to a very small effective lifetime of the excited singlet state due to concentration quenching of the excited singlet state in the adsorbed dye layer.     

LASER FLASH SPECTROSCOPY OF METHYLENE BLUE WITH NUCLEIC ACIDS

Absorption spectra, fluorescence spectra and quantum yields, triplet state absorption spectra, yields and rates of decay have been obtained for methylene blue (MB+), MB+/CT-DNA complexes and complexes of MB+ with the synthetic polynucleotides poly[d(G-C)] and poly[d(A-T)]. Strong fluorescence quenching is observed in the complexes of the dye with CT-DNA and poly[d(G-C)] concomitant with a decrease in the triplet yield. The fluorescence and triplet yield of MB+ with poly[d(A-T)] are similar to those of the free dye. The triplet decay times are increased in all three polymer/dye complexes and show a decreased sensitivity to oxygen quenching. These results are interpreted in terms of the binding of the dye to these polymers and the implications of polymer binding on the photosensitizing properties of the dye are discussed.