STEADY-STATE FLUORESCENCE METHOD FOR EVALUATING EXCITED STATE PROTON REACTIONS: APPLICATION TO FLUORESCEIN

Abstract Fluorescein is a complex fluorophore in the sense that it displays four prototropic forms (cation, neutral, monoanion and dianion) in the pH range 1–9. In experiments with fluorescein-labeled proteins we have sometimes observed complex nanosecond emission kinetics, which could be due to conversion of the excited monoanion into the excited dianion through an excited state proton exchange with a proton acceptor in the labeled protein. However, the literature is ambiguous on whether this possible excited state proton reaction of fluorescein does occur in practice. In this article we describe a general steady-state fluorescence method for evaluating excited state proton reactions of simple as well as complex pH-sensitive fluorophores and apply it to evaluate excited state proton reactions of fluorescein. The method depends on finding a buffer that can serve as an excited state proton donor-acceptor but does not significantly perturb ground state proton equilibrium and especially does not form ground (or excited state complexes) with the fluorophore. Our results show that the excited monoanion-dianion proton reaction of fluorescein does occur in the presence of phosphate buffer, which serves as a proton donor-acceptor that does not significantly perturb ground state proton equilibria. The reaction becomes detectable at phosphate buffer concentrations greater than 20 mM and the reaction efficiency increases with increase in phosphate buffer concentrations. The reaction is most clearly demonstrated by adding phosphate buffer to a solution of fluorescein at constant pH 5.9 with preferential excitation of the monoanion. Under these conditions, the excited monoanion converts to the dianion during its lifetime. The conversion is detected experimentally as an increase in dianion and decrease in monoanion fluorescence intensities with increase in phosphate buffer concentration. The absorption spectrum is not significantly perturbed by the increase in phosphate buffer concentration. To quantitate the reaction, we have recorded titration graphs of fluorescence intensity versus pH for fluorescein solutions at low (5 mM) and high buffer (1 M) concentrations with preferential excitation of the monoanion and preferential detection of the dianion emission. We have also developed theoretical expressions that relate fluorescence intensity to pH in terms of the concentration of the four prototrophic forms of fluorescein, extinction coefficients, fluorescence efficiencies and ground and excited state pK_a. The theoretical expressions give very good fits to the experimental data and allow evaluation of fundamental parameters such as pK_a and fluorescence efficiencies. The analysis of the experimental data shows that the excited monoanion-dianion reaction does not significantly occur at 5 mM phosphate buffer concentration. However, at 1 M buffer concentration the reaction is sufficiently fast that it practically achieves equilibrium during the lifetimes of the excited fluorescein monoanion and dianion. The pK_a* of the excited monoanion-dianion proton reaction is around 6.3. The results and methods presented here should be useful in the development and testing of pH-sensitive labeling fluorophores and fluorescent indicators.     

Theoretical Study of Fluorescence Spectra Utilizing the pKa Values of Acids in Their Excited States

Assignment of the fluorescence spectrum of firefly luciferin in aqueous solutions was achieved by utilizing not only emission energies but also theoretical absorption spectra and relative concentrations as estimated by pK_a values. Calculated Gibbs free energies were utilized to estimate pK_a values. These pK_a values were then corrected by employing the experimental results. It was previously thought that the main peak near 550 nm observed in the experimental fluorescence spectra at all pH values corresponds to emission from the first excited state of the luciferin dianion [Ando et al. (2010) Jpn. J. Appl. Phys. 49, 117002–117008]. However, we found that the peak near 550 nm at low pH corresponds to emission from the first excited state of the phenolate monoanion of luciferin. Furthermore, we found that the causes of the red fluorescence at pH 1–2 are not only the emission from phenol monoanion but also the emission from the protonated species at nitrogen atom in the thiazoline ring of dianion.     

Raman and FTIR spectroscopies of fluorescein in solutions

Raman and Fourier transform-infra red (FT-IR) spectroscopies of fluorescein in aqueous solutions have been investigated in the pH range from 9.1 to 5.4. At pH 9.1 fluorescein is in the dianion form. At pH 5.4, fluorescein is a mixture of monoanion (approximately 85%), dianion and neutral forms (together approximately 15%). The fluorescence quantum yield drops from 0.93 for the dianion form to 0.37 for the monoanion form. The Raman and FT-IR studies focused on the frequency range from 1000 to 1800 cm(-1) which contains the skeletal vibrational modes of the xanthene moiety of fluorescein. At pH 9.1, the spectroscopic feature of fluorescein dianion are consistent with a picture of an electron delocalized among the xanthene moiety and two identical oxygens attached to opposite ends of the xanthene moiety, forming a very symmetric structure. The characteristic of fluorescein dianion is the presence of the phenoxide-like stretch at 1310 cm(-1). At pH 5.4, fluorescein monoanion has lost the symmetric structure characteristic of the dianion. The spectra of the monoanion have distinctive contributions from the phenolic bend at 1184 cm(-1). The assignments of the vibrational bands shown in Raman and FT-IR spectra are given based on both literature and the ab initio calculations at the Hartree-Fock level with HF/6-31 + +G* basis set. Excellent correlation is found between the experimental and calculated spectra.     

AN UNUSUAL EXCITED STATE SPECIES OF ortho- HYDROXY-CINNAMIC ACID*

The fluorescence of ortho-hydroxycinnamic acid was studied in aqueous solution over the pH 0-14 range and in methanol. At pH values above 9.7, intense anion fluorescence is observed. In the pH 4-9 range emissions are from both the monoanion (albeit weakly) and from the dianion due to photodissociation. In the pH 0-4 range a blue and a green fluorescence was found, which is attributed to emissions from the unchanged molecule and from an unusual excited state tautomer respectively. The latter is assumed to be the phenolate anion of cinnamic acid and represents a tautomer with no equivalent in the electronic ground state. It is assumed to be formed by adiabatic photodissociation during the lifetime of the first excited singlet state.     

Effect of solvent-water mixtures on the prototropic equilibria of fluorescein and on the spectral properties of the monoanion

A spectral resolution procedure was used to resolve the absorption, excitation and emission spectra of the fluorescein monoanion in a number of solvent-water mixtures. This permitted an analysis of the effect of the solvent environment on the spectral properties of the monoanion and on the lactone/monoanion/dianion transitions of fluorescein. The monoanion excitation and emission spectra show relatively small changes with changing environment, a behavior that is related to the hydrogen-bonding environment of the solvent-water mixtures. There is also a general increase in the quantum yield of the monoanion from 0.36 in water to values up to 0.49 in the solvent-water mixtures. The presence of solvent also results in a general increase in the lactone content and in the monoanion:dianion and lactone:monoanion ratios. General polarity effects alone cannot account for the observed effects on the prototropic transitions indicating that specific solute-solvent effects involving hydrogen bonding perturb the prototropic equilibria of fluorescein.     

Acid-base equilibriums of lumichrome and its 1-methyl, 3-methyl, and 1,3-dimethyl derivatives

Lumichrome photophysical properties at different pH were characterized by UV-vis spectroscopy and steady-state and time-resolved fluorescence techniques, in four forms of protonation/deprotonation: neutral form, two monoanions, and dianion. The excited-state lifetimes of these forms of lumichrome were measured and discussed. The results were compared to those obtained for similar forms of alloxazine and/or isoalloxazine, and also to those of 1-methyl- and 3-methyllumichrome and 1,3-dimethyllumichrome. The absorption, emission, and synchronous spectra of lumichrome, 1-methyl- and 3-methyllumichrome, and 1,3-dimethyllumichrome at different pH were measured and used in discussion of fluorescence of neutral and deprotonated forms of lumichrome. The analysis of steady-state and time-resolved spectra and the DFT calculations both predict that the N(1) monoanion and the N(1,3) dianion of lumichrome have predominantly isoalloxazinic structures. Additionally, we confirmed that neutral lumichrome exists in its alloxazinic form only, in both the ground and the excited state. We also confirmed the existence and the alloxazinic structure of a second N(3) monoanion. The estimated values of pK(a) = 8.2 are for the equilibrium between neutral lumichrome and alloxazinic and isoalloxazinic monoanions, with proton dissociation from N(1)-H and N(3)-H groups proceeding at the almost the same pH, while the second value pK(a) = 11.4 refers to the formation of the isoalloxazinic dianion in the ground state.     

11B NMR study of p-carboxybenzeneboronic acid ions for complex formation with some monosaccharides.

The 11B NMR spectra of p-carboxybenzeneboronic acid (PCBA) ions demonstrated that their chemical shifts depend on the pH. At a lower pH, PCBA exists as a mixture of neutral PCBA and the monoanion with a trigonal structure, and at higher pH as the dianion with a tetrahedral structure. In the intermediate pH region, both the mono- and dianion coexist. The pKa of the monoanion of PCBA has been estimated to be 8.53. The complex formation constants of PCBA with several monosaccharides suggest that PCBA-fructose complex is most predominant.     

Voltammetry of maleic acid in aqueous and mixed environments

The electrochemical behavior of maleic acid is studied on rotating disk electrodes and stationary electrodes of individual Cu, Cd, Pt, and Ta metals, Cu and Cd amalgams, and Cd alloys with Sn, Cu, In, Pb, Ni, and Ag in aqueous solutions and water mixtures with acetone, acetonitrile, ethanol, dimethylformamide, and pyridine. Basic kinetic parameters for the maleic acid reduction to succinic acid are determined. Depending on the solution composition, all three forms of maleic acid (nondissociated acid, monoanion, dianion) may undergo reduction. The difference between diffusion coefficients and kinetic reduction parameters of the acid and monoanion is insignificant and that of the monoanion and dianion is noticeable. This is due to an intramolecular hydrogen bond in the monoanion, which decreases the negative charge on its anion center. The effect the electrode nature and the mixed-media composition have on the maleic acid reduction kinetics is discussed     

Selective reduction of fullerene C60 by metals in neutral and alkaline media. Interaction of C60 with KOH

The reduction of fullerene C₆₀ by Zn and Mg in DMF was studied both in the presence and absence of KOH. Fullerene C₆₀ was reduced in these systems to form the C₆₀ ^n– (n = 1, 2, and 3) anions. The anions were detected by optical and ESR spectroscopies. It was found that Mg reduced C₆₀ to the monoanion, Mg/KOH and Zn reduced C₆₀ to the dianion, and Zn/KOH reduced C₆₀ to the trianion. Like KCN, potassium hydroxide adds to fullerene upon interaction with C₆₀ in DMF. The reaction of C₆₀ with KOH in benzonitrile was accompanied by the generation of the fullerene monoanion. A possible mechanism of the formation of fullerene monoanions in the presence of KOH is discussed. The degradation of the C₆₀ ^n– anions in air was studied.     

Molecular size effect in NCO and NCS dianion resonances

Cross sections for electron-impact detachment and electron-impact dissociation of NCO- and NCS- were measured from about 3 to about 40 eV. The former are found to follow a classical prediction with a threshold energy of 9.1 +/- 0.1 eV for NCO- and 8.9 +/- 0.2 eV for NCS-. When the incoming electron binds to the monoanion, a short-lived dianion complex is formed, which is revealed as a resonance in the cross section. For NCO- a resonance is evident at 9.3 +/- 0.2 eV, which implies that the dianion lies above the monoanion by this amount of energy. In the case of NCS- two resonances are evident at 8.4 +/- 0.2 and 19.0 +/- 0.5 eV, respectively. The low-energy NCS dianion is less unstable than the dianion of NCO, which in turn is less unstable than the CN dianion (10-eV resonance). Thus the resonance shifts down in energy with the increasing size of the anion, a fact which is attributed to a decrease in Coulomb energy between the spatially separated electrons.     

The seventeen- and eighteen-electron metallacarbaboranes [1,1,1-(CO)3-2-Ph-closo-1,2-MnCB9H9]n- (n = 1, 2): a structurally characterized,redox-related pair

An eleven-vertex manganese-monocarbaborane dianion, upon one-electron oxidation, gives a stable radical monoanion in which the unpaired electron is delocalized over the cluster.     

Simulation analysis of formycin 5′-monophosphate analog substrates in the ricin A-chain active site

Summary Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2-positions, and from hydroxyl substitution at the 2-position.     

Generation and reactions of a selenoamide dianion

The selective generation of selenoamide monoanion and dianion was achieved by reacting N-benzyl selenobenzamide with BuLi. Alkylation of the dianion with 1 equiv of electrophile took place at the carbon atom adjacent to the nitrogen atom, and subsequent hydrolysis produced functionalized selenoamides in good to high yields. Ring opening of oxiranes using the dianion proceeded with high regio- and stereoselectivity to form N-3-hydroxy-1-phenylalkyl selenobenzamides. The stereochemistry of the major isomer derived from cyclohexene oxide was determined by X-ray molecular structure analysis. [reaction: see text]     

Mechanism for basic hydrolysis of N-nitrosoguanidines in aqueous solution

A kinetic study was carried out on the hydrolysis of two N-nitrosoguanidines, 1-nitroso-1-methyl-3-tolylsulfonylguanidine (TSGNO) and 1-nitroso-1-methyl-3-benzoylguanidine (BCGNO). We observed an absence of buffer catalysis using H(2)PO(4)(-)/HPO(4)(2)(-), H(3)BO(3)/H(2)BO(3)(-), and HCO(3)(-)/CO(3)(2)(-) regulators and a complex dependency of the rate constant on the pH. We discovered the existence of three simultaneous reaction paths: spontaneous decomposition of the neutral form of the N-nitrosoguanidine, decomposition of the monoanion, and decomposition through the form of the dianion. The analysis of the kinetic data has allowed us to obtain the acidity constant for the formation of the monoanion of the N-nitrosoguanidine, with values of p = 11.5. The reaction rate for the process through the monoanion, k(2), decreases as the acidity increases. The application of the principle of nonperfect synchronization shows that the basicity and reactivity do not correlate when there exists a possibility of stabilization of the negative charge by resonance. This behavior is consistent with the mechanism E1cB whereby the stabler the negative charge, the slower the elimination reaction. When dealing with the case of the elimination through the neutral form we observe that the reaction rate increases together with the capacity of stabilization of the positive charge on the nitrogen atom adjacent to the imino group. For the reaction through the dianion we used a maximum value of k(3) = 10(10) s(-)(1) to estimate the value of p for the formation of the dianion of the N-nitrosoguanidine, obtaining values of p < 24.     

A new donor–acceptor double‐cable carbazole polymer with perylene bisimide pendant group: Synthesis,electrochemical, and photovoltaic properties

We report here electrochemical synthesis of novel soluble donor–acceptor (D–A) polymer with suitably functionalized perylenetetracarboxylic diimide dye derivative covalently linked to carbazole moiety (Cbz‐PDI). The band gap, E_g was measured using UV–Vis spectroscopy and compared with that obtained by cyclic voltammetry (CV). Efficient intramolecular electron transfer from carbazole‐donor to perynediimide‐acceptor leads to remarkable fluorescence quenching of the perylene core. Furthermore, spectroelectrochemical property and surface morphology of the polymer film were investigated. Characteristic monoanion and dianion radical bands on the UV–Vis absorption spectra attributed to the electrochemical reduction of the neutral polymer were observed. During the reduction process, red color of the film turned into blue and violet, respectively. Finally, the photovoltaic performance of the D–A double‐cable polymer was checked and nearly 0.1% electrical conversion efficiency is obtained under simulated AM 1.5 solar light with 100 mW/cm² radiation power. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6280–6291, 2009     

“Dumbbell”‐ and “Clackers”‐Shaped Dimeric Derivatives of Monocarba‐closo‐dodecaborate

We designed, synthesized, and characterized two types of dimeric forms of monocarba‐closo‐dodecaborate, namely, a “dumbbell”‐shaped dianion having a C?C bond and a “clackers”‐shaped monoanion having an iodonium linker. The unique architectures of these anionic molecules were established by X‐ray analysis. Spectroscopic analysis, DFT calculations, and reactivity experiments revealed high anionic and chemical stability of both anions, which are crucial properties for weakly coordinating anions.     

Phosphoryl transfer from phenyl and 4-nitrophenyl phosphates in aprotic and protic solvents : Amine catalysis and formation of oxyphosphorane and metaphosphate intermediates

The behavior of 4-nitrophenyl dihydrogen phosphate, ArOPO₃H₂, and of its tetra-n-butylammonium and tetramethylammonium salts, ArOPO₃H^?R₄N⁺, ArOPO₃^2?2(R₄N⁺), was studied in aprotic solvents, in the absence and in the presence of increasing amounts of alcohols or water. The reactions were investigated in the absence of amines, and in the presence of hindered and unhindered amines, diisopropylethylamine and quinuclidine. The course of the reactions was followed at 35° or at 70° by ³¹P and ¹H NMR spectrometry. Values for the approximate half-times of the reactions were estimated (± 25 %) from the times at which reactant signal intensity becomes equal to product signal intensity. The mononitrophenyl ester transfers its phosphoryl group to alcohols and water from the diprotonated acid by the addition-elimination mechanism via oxyphosphorane intermediates, and from the monoanion and dianion by the elimination-addition mechanism via the monomeric metaphosphate intermediate, PO₃^?. Formation of PO₃^? is faster from dianion than from monoanion in acetonitrile and in alcohol solutions. Conversely, PO₃^? is generated at a faster rate from monoanion than from dianion in aqueous solution. This effect results from a decrease in the rate of formation of PO₃^? in the solvent series: acetonitrile > alcohols > water. The rate depression as a function of the medium is greater for the dianion than for the monoanion, and is attributed to greater solvation of the more polar phosphate ground state than of the less polar transition state in the more polar protic solvents. Unhindered amines add to 4-nitrophenyl phosphate monoanion, but not to the dianion. The oxyphosphorane intermediate thus formed collapses to aroxide ion and a protonated dipolar phosphoramide which is rapidly deprotonated by the relatively basic 4-nitrophenoxide: ArOPO₃H^? + CH(CH₂CH₂)₃N(acetonitrile ? CH(CH₂CH₂)₃N⁺P(O)(OH)O^? + ArO^?? CH(CH₂ CH₂)₃N⁺PO₃^2?+ ArOH → CH(CH₂CH₂)₃N + PO₃^?. The postulated formation of PO₃^? by this route explains why the addition of quinuclidine to an acetonitrile solution containing the monoanion salt, ArOPO₃H^?R₄N⁺, and t?BuOH produces t-butyl phosphate at a faster rate than the addition of diisopropylethylamine to the same solution. 2,4-Dinitrophenyl phosphate, which was previously studied by the same techniques, reacts via oxyphosphorane intermediates from the diprotonated and the monoanion forms, and via monomeric metaphosphate, from the dianion form.     

Spectral-luminescence and quantum-chemical study of the anionic forms of 5-fluorouracil

A spectral-luminescence study of neutral (pH 7) and alkaline (pH 11 and 14) aqueous solutions of the anticancer drugs 5-fluorouracil (FU) and tegafur has been performed. The fluorescence spectra of the N3- and N1-centered anions of 5-fluorouracil, its dianion, and the tegafur monoanion with emission maxima at wavelengths (λ_em) of 358, 372, 366, and 358 nm and photoluminescence quantum yields (φ) of 11.2 × 10^–4, 35.1 × 10^–4, 26.5 × 10^–4, and 8.6 × 10^–4, respectively, have been recorded for the first time. The fluorescence characteristics of the FU anionic forms have been related to the magnetic shielding constant as one of the criteria of aromaticity.     

An NMR and quantum-mechanical investigation of tetrahydrofuran solvent effects on the conformational equilibria of 1,4-butanedioic acid and its salts

Vicinal proton-proton NMR couplings have been used to compare the influences of water and tetrahydrofuran (THF) as solvents on the conformational equilibria of 1,4-butanedioic (succinic) acid and its mono- and dianionic salts. An earlier NMR investigation (Lit, E. S.; Mallon, F. K.; Tsai, H. Y.; Roberts, J. D. J. Am Chem. Soc. 1993, 115, 9563-9567) showed that, in water, the conformational preferences for the gauche conformations for butanedioic acid and its monoanion and dianion were, respectively, approximately 84%, 66%, and 43%, essentially independent of the nature of the cation or concentration. We now report the corresponding gauche percentages calculated in the same way for 0.05 M solutions in THF to be 66%, 90-100%, and 46-64%. Substantial evidence was adduced for the rotational angle between the substituents in the monoanion being approximately 70 degrees. The positions of conformational equilibria of the salts in THF, particularly of the dianion, were found to be rather insensitive to concentration and temperature, but more sensitive to the amount of water present. Ab initio quantum-mechanical calculations for 1,4-butanedioate dianion indicate that, as expected for the gas phase, the trans conformation of the dianion should be heavily favored over the gauche, but, in both THF and water, the gauche conformation is calculated to predominate with rotational angles substantially less than 60 degrees. This conclusion is, in fact, generally consistent with the experimental vicinal proton couplings, which are wholly inconsistent with the trans conformation.