Noriaki Funasaki

Advances in physical chemistry and pharmaceutical applications of cyclodextrins

Cyclodextrins (CDs) attract much attention for industrial applications and academic research. A few experimental methods for determination of the binding constant between CD and a guest molecule were reviewed critically. A hydrophile–hydrophobe matching model for host–guest docking was proposed for estimation of the binding constant and the solution structure of the complex. Rather detailed solution structures of CD complexes were determined by proton NMR spectroscopy, aided by calculations of molecular mechanics and surface areas, and were used to analyze the binding constants. The binding constants of CDs with multi-site guests were analyzed on the basis of their solution structures. The working mechanisms and physicochemical predictions in a few pharmaceutical applications of CDs were proposed on the basis of detailed solution structures and accurate binding constants.

The dissociation constants of acid-base indicators on themicellar surface of dodecyldimethylamine oxide

The dissociation constants K of acid-base indicators (p-nitrophenol, bromocresol purple, chlorophenolred, bromophenol blue, and bromocresol green) in aqueous solutions of 20 mM dodecyldimethylamine oxide (DDAO), 1% Brij 35 (Brij), and 20 mM cetyltrimethylammonium bromide (CTAB) were measured spectroscopically and applicability of the equation, pK = pKi − (0.4343e0Ψ0/kT), to the micelle-solubilizate systems was determined. The pK value in DDAO, a weak electrolyte, increased with an increase in the degree of dissociation β of an indicator, while this value in Brij, a nonelectrolyte, and in CTAB, a strong electrolyte, remained unchanged regardless of β. Using the value of the surface potential Ψ0 of DDAO micelle which has already been determined potentiometrically, it was found that pKi in DDAO was independent of β, Ψ0, and the surface charge density of DDAO micelle and moreover, that it was not equal to that in water but almost equal to that in Brij. The latter finding strongly suggested that the solvent property of the micellar surface of nonionic DDAO is different from that of water and more like that of the Brij micelle surface. Thus the above equation is applicable to the micelle-solubilizate systems.

meso-Tetra(tert-butyl)porphyrin as a precursor of porphine

Abstract Treatment of meso -tetra( tert -butyl)porphyrin with sulfuric acid/1-butanol at 90°C over 15 min afforded porphine in an isolated yield of 74%. De- tert -butylation of the substituted porphyrin provides a rational access to porphine.

Thermodynamical properties of reaction intermediates during apoplastocyanin folding in time domain.

Two intermediates observed for the folding process of apoplastocyanin (apoPC) were investigated by using a photoinduced triggering system combined with the transient grating and transient lens methods. The thermodynamic quantities, enthalpy, heat capacity, partial volume, and thermal expansion volume changes during the protein folding reaction were measured in time domain for the first time. An interesting observation is the positive enthalpy changes during the folding process. This positive enthalpy change must be compensated by positive entropy changes, which could be originated from the dehydration effect of hydrophobic residues and/or the translational entropy gain of bulk water molecules. Observed negative heat capacity change was explained by the dehydration effect of hydrophilic residues and/or motional confinement of amino acid side chains and water molecules in apoPC. The signs of the volume change and thermal expansion volume were different for two processes and these changes were interpreted in terms of the different relative contributions of the hydration and the dehydration of the hydrophilic residues. These results indicated two-step hydrophobic collapses in the early stage of the apoPC folding, but the nature of the dynamics was different.

Gel filtration chromatographic study on the self-association of surfactants and related compounds

After a brief survey of gel filtration chromatography (GFC) and the self-association equilibrium, it is shown that GFC is a unique tool for investigating the self-association of many substances, such as surfactants, chlorpromazine hydrochloride (CPZ), Methylene Blue (MB) and a sulfobetaine derivative (CHAPS) of cholic acid. The hydrodynamic radius of an aggregate can be estimated with the gel of large pore size, such as Sephadex G-200. This method is utilized for the study of the sphere-rod transition of surfactant micelles. A precise value of monomer concentration can be determined from the centroid elution volume of a frontal chromatogram on Sephadex G-10. This important parameter in self-associating systems allows us to determine the micellar aggregation number as a function of the total surfactant concentration. The derivative chromatogram can be used to detect slight changes in self-association. From these results, it is found that nonionic surfactants form premicelles including dimer and that multiple equilibrium model for micelle formation is more appropriate than mass-action model. The Tanford theory with some modifications appears to fit the concentration dependence of the aggregation properties of surfactants forming small globular micelles and allows us to estimate the stepwise aggregation constant and the micelle size distribution function. The pronounced cooperativity in the self-association of surfactants forms the basis of mass action model and the concept of the cmc, though they are actually approximations even for surfactants. They would be more inadequate, though often used, for the self-association of drugs and bile salts. The stepwise aggregation constant of MB is almost independent of aggregation numbers, which may be expected from the stacking mode of aggregation. The main aggregates of CPZ in 154 mM sodium chloride solution are dimer and 38-mer and its dimerization constant is smaller than that of MB and larger than those of surfactants. The stepwise aggregation constant of CHAPS has the maximum at hexamer and oscillates, depending on odd or even aggregation numbers. Thus, the aggregation pattern of a substance is closely related with the chemical structure of the hydrophobic group of its molecule. The definition of cmc is discussed together with its significance.

Prediction of retention times in reversed-phase high-performance liquid chromatography from the chemical structure

The capacity factor, k′, on a Zorbax ODS column in methanol-water solvents was determined for alcohols and ethers with positional and geometric isomers by high-performance liquid chromatography. These log k′ values are almost linearly correlated with the molecular cavity surface areas, S, various molecular connectivity indices, e.g., the first-order index, 1χ, the logarithm of the octanol-water partition coefficient, P, and the logarithm of the aqueous solubility. The three parameters S, 1χ and log P are best correlated with log k′. The advantages and disadvantages of these parameters in the correlation with log k′ are considered. The present log k′ data can be used for prediction of log P and log k′ for other compounds including amino acids.

Proton NMR study of the myoglobin reconstituted with meso-tetra(n-propyl)hemin

Spectrophotometric titration of meso-tetra(n-propyl)hemin with sperm-whale apomyoglobin revealed their 1:1 complex formation. The purified reconstituted metmyoglobin bound with an equal molar amount of CN- and the second CN- ligation was not evidenced, suggesting that the hemin is not loosely attached to the globin surface, but incorporated into the heme pocket. The hyperfine-shifted proton NMR spectrum of the deoxy myoglobin revealed the proximal imidazole NH resonance at 85.1 ppm to indicate the formation of the Fe-N(His-F8) bond. The eight pyrrole protons of the hemin of myoglobin in the absence of external ligand were observed as a single peak at -16 ppm. This indicates the electronic symmetry of the hemin and the low-spin configuration of the heme iron. The pyrrole-proton NMR patterns of the cyanide and deoxy myoglobins were found to be remarkably temperature-dependent, which was consistently explained in terms of the free rotation of the prosthetic group. The NMR results suggest that introduction of meso-tetra(n-propyl)hemin totally disrupts the highly stereospecific heme-globin contacts, making the prosthetic group mobile in the heme cavity.

Electrochemistry of monoazahemin reconstituted myoglobin at an indium oxide electrode

Abstract Monoazahemin reconstituted myoglobin was prepared and its electrochemical behavior was studied in comparison with native myoglobin. For both myoglobins well-defined voltammograms were clearly obtained at highly hydrophilic surfaces of indium oxide electrodes. Although monoazahemin showed a more positive redox potential than hemin (measured in methanol), monoazahemin reconstituted myoglobin showed a more negative redox potential than native myoglobin in a 50 mM bis-Tris buffer solution (pH 6.5), suggesting that for both native and reconstituted myoglobins the heme environment including proximal histidine as an axial ligand of the redox center plays an important role in determining the redox potential. Also, a unique electrochemical response of cyano-monoazahemin reconstituted myoglobin was demonstrated.

Etiohemin as a prosthetic group of myoglobin

Sperm whale myoglobin was reconstituted with etioheme and the stoichiometric complex formation was confirmed. The proton NMR spectrum of the deoxy myoglobin exhibits an NH signal from the proximal histidine at 78.6 ppm, indicating heme incorporation into the heme pocket to form the Fe-N(His-F8) bond. The appearance of a single set of the heme-methyl NMR signals shows that etioheme without acid side-chains specifically interacts with the surrounding globin. The visible spectral data suggest retention of a normal iron coordination structure. The functional and NMR spectral properties of etioheme myoglobin are similar to those of mesoheme myoglobin, reflecting the absence of the electron-withdrawing heme vinyl groups.

Existence of critical demicellization concentration

Abstract The existence of the critical demicellization concentration (CDC) which had been predicted by Mysels has been demonstrated in the system of mixed fluorocarbon (NF) and hydrocarbon (STrS) surfactants in 0.05 M sodium chloride solution at 30°C. The composition of mixed micelles of NF and STrS was determined from the surface tension data on NF-STrS solutions above the critical micelle concentration (CMC). The region in which the surface tension remains unchanged with the micellar composition was observed. Both ends of this region, xA and xB (xA