Dina Pines

Real-Time Observation of Carbonic Acid Formation in Aqueous Solution

A Glimpse of Wet Carbonic Acid Both carbon dioxide and bicarbonate play extraordinarily widespread roles in biochemical and geochemical reactions. It is surprising therefore that carbonic acid, the intermediate in the water-coupled interconversion of these two compounds, has never been directly characterized in aqueous solution. Adamczyk et al. (p. 1690, published online 12 November) have succeeded in glimpsing the elusive acid by inducing an aqueous photoacid (a compound rendered transiently more acidic upon light absorption) to react with dissolved bicarbonate. Using infrared spectroscopy, they show that the carbonic acid product persists for nanoseconds. Analysis of its formation kinetics affords a direct pKa value of 3.5, substantially lower than the effective value derived from observations of the net bicarbonate/carbon dioxide equilibrium. The use of a photoacid enables the long-sought characterization of the conjugate acid of bicarbonate. Despite the widespread importance of aqueous bicarbonate chemistry, its conjugate acid, carbonic acid, has remained uncharacterized in solution. Here we report the generation of deuterated carbonic acid in deuterium oxide solution by ultrafast protonation of bicarbonate and its persistence for nanoseconds. We follow the reaction dynamics upon photoexcitation of a photoacid by monitoring infrared-active marker modes with femtosecond time resolution. By fitting a kinetic model to the experimental data, we directly obtain the on-contact proton-transfer rate to bicarbonate, previously inaccessible with the use of indirect methods. A Marcus free-energy correlation supports an associated pKa (Ka is the acid dissociation constant) of 3.45 ± 0.15, which is substantially lower than the value of 6.35 that is commonly assumed on the basis of the overall carbon dioxide–to–bicarbonate equilibrium. This result should spur further exploration of acid-base reactivity in carbon dioxide–rich aqueous environments such as those anticipated under sequestration schemes.

Photophysical characterization of trans-4,4′-disubstituted stilbenes

Abstract Absorption and emission spectra of twenty trans -4,4′-disubstituted stilbenes have been measured in four solvents: cyclohexane (CH), chlorobenzene (CB), 2-butanone (methylethyl ketone, MEK) and dimethylsulfoxide (DMSO) at room temperature. Fluorescence quantum yields ( Φ f ) and fluorescence lifetimes ( τ f ) have been measured for these stilbenes. The lifetimes and quantum yields of fluorescence were found to be dependent on the donor-acceptor properties of the substituents and correlate with the Hammett ν-constants. In addition, we experimentally observed the appearance of a second emitting state which is close energetically to the lowest exited singlet state 1 t ∗ in cases of strong donor-acceptor substituents in polar solvents.

Coexistence of Domains with Distinct Order and Polarity in Fluid Bacterial Membranes

In this study we sought the detection and characterization of bacterial membrane domains. Fluorescence generalized polarization (GP) spectra of laurdan‐labeled Escherichia coli and temperature dependencies of both laurdans GP and fluorescence anisotropy of 1,3‐diphenyl‐1,3,5‐hexatriene (DPH) (rDPH) affirmed that at physiological temperatures, the E. coli membrane is in a liquid‐crystalline phase. However, the strong excitation wavelength dependence of rlaurdan at 37°C reflects membrane heterogeneity. Time‐resolved fluorescence emission spectra, which display distinct biphasic redshift kinetics, verified the coexistence of two subpopulations of laurdan. In the initial phase, <50 ps, the redshift in the spectral mass center is much faster for laurdan excited at the blue edge (350 nm), whereas at longer time intervals, similar kinetics is observed upon excitation at either blue or red edge (400 nm). Excitation in the blue region selects laurdan molecules presumably located in a lipid domain in which fast intramolecular relaxation and low anisotropy characterize laurdans emission. In the proteo‐lipid domain, laurdan motion and conformation are restricted as exhibited by a slower relaxation rate, higher anisotropy and a lower GP value. Triple‐Gaussian decomposition of laurdan emission spectra showed a sharp phase transition in the temperature dependence of individual components when excited in the blue but not in the red region. At least two kinds of domains of distinct polarity and order are suggested to coexist in the liquid‐crystalline bacterial membrane: a lipid‐enriched and a proteo‐lipid domain. In bacteria with chloramphenicol (Cam)–inhibited protein synthesis, laurdan showed reduced polarity and restoration of an isoemissive point in the temperature‐dependent spectra. These results suggest a decrease in membrane heterogeneity caused by Cam‐induced domain dissipation.

Ultrafast Excited-State Proton-Transfer Reaction of 1-Naphthol-3,6-Disulfonate and Several 5-Substituted 1-Naphthol Derivatives

The 1-naphthol molecule has been the subject of intense research activity for the past 60 years due to its complex behavior as a photoacid upon optical excitation. We have utilized femtosecond mid-infrared spectroscopy and time-resolved fluorescence spectroscopy to investigate the excited-state proton-transfer reaction of 1-naphthol-3,6-disulfonate (1N-3,6diS) and several 5-substituted 1-naphthol derivatives. The proton dissociation rate constant of 1N-3,6-diS was found to be about 3 times faster and the pKa* about 2 pKa units more acidic than the values previously reported in the literature. A Marcus (free-energy) plot of excited-state proton dissociation rate constants vs the excited-state equilibrium constant of the photoacids, Ka*, was constructed using the C-5 series of 1-naphthol derivatives. The newly measured values for the ESPT rate constant and pKa* of 1N-3,6diS was found to fit well with the Marcus correlation. We discuss our findings in the context of the photoacidity phenomenon in general, and the photoacidity of 1-naphthol and its derivatives in particular.

Apparent Stoichiometry of Water in Proton Hydration and Proton Dehydration Reactions in CH3CN/H2O Solutions

Gradual solvation of protons by water is observed in liquids by mixing strong mineral acids with various amounts of water in acetonitrile solutions, a process which promotes rapid dissociation of the acids in these solutions. The stoichiometry of the reaction XH(+) + n(H(2)O) = X + (H(2)O)(n)H(+) was studied for strong mineral acids (negatively charged X, X = ClO(4)¯, Cl¯, Br¯, I¯, CF(3)SO(3)¯) and for strong cationic acids (uncharged X, X = R*NH(2), H(2)O). We have found by direct quantitative analysis preference of n = 2 over n = 1 for both groups of proton transfer reactions at relatively low water concentrations in acetonitrile. At high water concentrations, we have found that larger water solvates must also be involved in the solvation of the proton while the spectral features already observed for n = 2, H(+)(H(2)O)(2), remain almost unchanged at large n values up to at least 10 M of water.

Direct observation of power-law behavior in the asymptotic relaxation to equilibrium of a reversible bimolecular reaction

We report the first direct observation of power-law relaxation to equilibrium of the diffusion influenced reversible reaction, AB⇆A+B (B≫A). Our experimental findings confirm the predicted t−3/2 decay law relaxation of AB population a long time after the photoinitiation of the reaction. This t−3/2 relaxation of the excess-over-equilibrium population is similar to that found in diffusion influenced geminate recombination reactions.

The fractal nature of the surfaces of porous silicas as revealed in electronic energy transfer between adsorbates: Comparison of three donor/acceptor pairs

Electronic energy transfer between adsorbed rhodamine 6G (R6G, donor) and malachite green (MG, acceptor) was studied on nonporous silica, on silica with average pore diameter of 60 A (Si–60), on Si‐100 and on Si‐200. The decay profiles obey the Klafter–Blumen equation for the survival probability in fractal environments, with fractal dimensions D of 2.1, 2.3, 2.5, and 2.7, respectively, in good agreement with the results obtained with two other pairs, rhodamine‐B/MG and R6G/R6G (in a depolarization experiment). The invariance of D to changes in the critical Forster radius, suggests that the derived D values reflect structural scale invariance properties of the supports and in particular the surface fractal dimension as ‘‘seen’’ by surface/molecule interaction. This interpretation compares favorably with other surface studies of SiO2.

Nature of Interaction Between Basic Fibroblast Growth Factor and the Antiangiogenic Drug 7,7-(Carbonyl-bis[imino-N-Methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])bis-(1,3-naphthalene disulfonate)

PNU145156E (7,7-(carbonyl-bis[imino-N-methyl-4, 2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino]) -bis-(1, 3-naphthalene disulfonate)) is a naphthalene sulfonic distamycin A derivative that interacts with heparin-binding growth factors. Because PNU145156E inhibits tumor angiogenesis, it was selected for clinical development. Picosecond time-resolved fluorescence emission and anisotropy were used to characterize the binding of PNU145156E to the basic fibroblast growth factor (a protein associated with tumor angiogenesis). A decrease in PNU145156E fluorescence lifetime was observed as a function of human basic fibroblast growth factor (bFGF) concentration. Nonlinear least-squares fitting of the binding isotherm yielded Kd = 145 nM for a single class of binding sites. Time-resolved anisotropy gave Kd = 174 nM. Kd = 150 nM was independently verified by quantitative high-performance affinity chromatography. The displaced volume of the complex, calculated from its rotational correlation time, fitted a sphere of 1:1 stoichiometry. These results account for the formation of a tight yet reversible PNU145156E:bFGF complex. An evaluation of PNU145156E fluorescence lifetimes in various solvents has highlighted the forces involved in stabilizing the complex. These are mostly electrostatic-hydrophobic in nature, with a relatively low contribution from hydrogen bonding. Both polar and nonpolar groups are involved on the protein-binding site within a largely hydrophobic cleft. A potential binding trajectory, based on a combination of these results with site-directed chemical modification and known bFGF x-ray structure, is suggested.

Proton Dissociation and Solute-Solvent Interactions Following Electronic Excitation of Photoacids

Photoacids are an extremely useful class of probe molecules that are often used in modern proton—transfer research. This review summarizes some of the recent applications of photoacids in the research of proton-transfer reactions in aqueous solutions and in the study of polar-solvation by site-specific hydrogen-bonding interactions.

Nature of Interaction between Basic Fibroblast Growth Factor and the Antiangiogenic Drug 7,7-(Carbonyl-Bis[Imino-N-Methyl-4,2-Pyrrolecarbonylimino[N-Methyl-4,2-Pyrrole]-Carbonylimino])-Bis-(1,3-Naphtalene Disulfonate). II. Removal of Polar Interactions Affects Protein Folding

Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.