Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Synthesis and characterization of drug–saccharide conjugates by enzymatic strategy in organic media

A facile and efficient strategy to prepare drug derivatives with saccharides was developed, and 12 drug–saccharide conjugates were obtained by selective enzymatic synthesis methods. When the transesterification of chlorphenesin vinyl esters with glucose was chosen as a model reaction, the influence of reaction conditions was optimized. Then, we successfully prepared a series of chlorphenesin–saccharide derivatives of three monosaccharides (glucose, mannose and galactose) and three disaccharides (maltose, lactose and sucrose). In order to study the relationship between the solubility of drug–saccharide conjugates and the structure of parent drugs, five hydrophobic drugs (chlorphenesin, mephenesin, guaifenesin, propranolol and clorprenaline) were chosen as substrates to synthesize drug–saccharide conjugates. The results indicated that the aqueous solublity of drug–saccharide derivatives was greatly improved, which was changed by different saccharides and the structure of parent drugs.     

Thermodynamics of the interaction of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in aqueous solutions at 298.15K

The Gibbs energy interaction parameters of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in water, g(ES), were obtained from electromotive force (emf) measurements of the electrochemical cell without liquid junction and containing two ion-selective electrodes (ISE): K-ISEmid R:RbCl(m(E))mid R:ISE-Cl and K-ISEmid R:RbCl(m(E)),saccharide (m(S))mid R:ISE-Cl, at 298.15K. The enthalpy interaction parameters of RbCl with these monosaccharides in water, h(ES), are determined according to the McMillan-Mayer theory from the measurements of the enthalpies of mixing of aqueous RbCl solutions with aqueous monosaccharide solutions, as well as the enthalpies of dilution of RbCl and monosaccharide solutions in pure water at 298.15K by a calorimetric method. Furthermore, the entropy interaction parameters, s(ES), can be evaluated through g(ES) and h(ES). The results suggest that the electrostatic interactions of these monosaccharides with RbCl in water are predominant compared with structural interactions, and these parameters are controlled primarily by the stereochemical structure of the monosaccharides in water.     

Activity coefficients for NaCl-monosaccharide (D-glucose, D-galactose, D-xylose, D-arabinose)-water systems at 298.15 K

Electrochemical cells with a sodium ion selectivity electrode (Na-ISE) versus a chloride ion selectivity electrode (Cl-ISE) as a reference electrode were used to determine the activity coefficients for NaCl-monosaccharide (D-glucose, D-galactose, D-xylose, and D-arabinose) systems in water at 298.15 K. A comparison of the results thus obtained was made with those determined by another electromotive force (emf) method. It is shown that agreement is excellent. The Gibbs free energy parameters of the interactions between these sugars and NaCl in water were evaluated together with the parameter C1(CHOH, exo), indicating the interaction of the exocyclic CHOH group of saccharide molecules and NaCl. The results suggested that the interactions of these monosaccharides with NaCl are controlled mostly by the dominant conformer of their molecules in water.     

Volumetric properties for the monosaccharide (D-xylose, D-arabinose, D-glucose, D-galactose)-NaCl-water systems at 298.15 K

Densities have been measured for monosaccharide (D-xylose, D-arabinose, D-glucose and D-galactose)-NaCl-water solutions at 298.15 K. These data have been used to determine the apparent molar volumes of these saccharides and NaCl in the studied solutions. Infinite-dilution apparent molar volumes for the saccharides (V0(phi,S)) in aqueous NaCl and those for NaCl (V0(phi,E)) in aqueous saccharide solutions have been evaluated, together with the standard transfer volumes of the saccharides (delta(t) V0S) from water to aqueous NaCl and of NaCl (delta(t) V0E) from water to aqueous saccharide solutions. It is shown that the delta(t) V0S and delta (t) V0E values are positive and increase with increasing co-solute molalities. Volumetric parameters indicating the interactions of NaCl with saccharides in water have been obtained, respectively, by using transfer volumes of the saccharides and NaCl, and the resulting values are in good agreement with each other within experimental error. The interactions between saccharides and NaCl are discussed in terms of the structural interaction model and the stereochemistry of the saccharide molecules in water.     

Lanthanide-saccharide chemistry: synthesis and characterisation of Ce(III)-saccharide complexes

A series of nine Ce(III) complexes has been synthesised with seven different monosaccharides (D-glucose, D-fructose, D-galactose, D-mannose, L-sorbose, D-ribose and D-xylose) and two different disaccharides (D-maltose and L-lactose), and these have been characterised with various analytical, spectral, magnetic and electrochemical techniques. The NMR studies have highlighted some interesting features about the metal-ion-binding pattern of the saccharides. Some additional coordination has been proposed along with the chelating groups in the saccharide molecules, based on the shifts in 13C NMR spectra. On the other hand, solution absorption studies and solid-state magnetic susceptibilities have indicated the contribution from the d-character to the spectral features to some extent.     

Highly sensitive, quick and simple quantification method for mono and disaccharides in aqueous media using liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS)

A highly sensitive, rapid LC-APCI-MS method for identification and quantification of mono and disaccharides in simple or complex aqueous phase has been developed. This original method is easy to use, no derivation and no post-column injection are needed. The separation is performed with a hydrophilic amino interaction (HILIC) column allowing high-throughput analysis with analysis times of 15 min for monosaccharides to 22 min for disaccharides. The development of the method carried out with 9 standard saccharides allowed to point out a dynamic range from 0.1-25.6 to 1-256 μg mL(-1) depending on the considered sugar. Next, the method was validated on saccharides at known concentrations in water and on 2 real samples: orange juice and aqueous phase obtained after enzymatic hydrolysis of sunflower seeds.     

A small diffusion pore in the outer membrane of Pseudomonas aeruginosa

The permeability properties of the outer membrane of Pseudomonas aeruginosa were re-examined, since the reported conclusions are conflicting [Decad, M. G. and Nikaido, H. (1976) J. Bacteriol. 128, 325-336; Caulcott, C. A., Brown, M. R. W. and Gonda, I. (1984) FEMS Microbiol. Lett. 21, 119-123]. On the basis of the experimental evidence to be described below we conclude that the exclusion limit of the outer membrane of P. aeruginosa is smaller than the size of uncharged disaccharides but larger than the size of hexose. This conclusion is based on the following evidence. Penetration of monosaccharides into the expanded periplasm was large and that of disaccharides was small, after the cells were plasmolyzed with 600 mosM NaCl. A significant amount of protein was released after osmotic down-shock of cells treated with the hypertonic monosaccharides but not of cells treated with the hypertonic saccharides larger than disaccharides. Centrifuged pellets of cells treated with hypertonic di, tri and tetrasaccharides weighed about 15-20% less than that of cells treated with the isotonic monosaccharide, suggesting that the osmotic pressure was exerted on the outer membrane causing dehydration and shrinking of the cells. By contrast, cells treated with the hypertonic pentose and hexoses weighed about 0.1% and 6% less, respectively, than cells treated with the isotonic saccharide, suggesting that pentose diffused through the outer membrane freely.     

Modeling and measurements of solid-liquid and vapor-liquid equilibria of polyols and carbohydrates in aqueous solution

The solubilities of five saccharides in water have been measured at various temperatures. This includes the monosaccharides xylose and galactose, and the disaccharides maltose monohydrate, cellobiose and trehalose dihydrate. A method that uses interaction energies and interaction parameters calculated with molecular mechanics methods has shown to give good predictions of the phase behavior of a variety of mixtures, including glycols and small saccharides in aqueous solution. The method is completely predictive, as the strength of the molecular interactions is determined with a theoretical method in the absence of any phase equilibrium data. For calculating solubilities, experimental values for the melting points and the heats of fusion of the compounds under study are, however, necessary. The solubilities of the five saccharides listed above, raffinose and meso-erythritol in water were calculated with this method. The calculated solubilities are in reasonably good agreement with experiment, and in the case of meso-erythritol, which is a polyalcohol (polyol), and galactose, the agreement between prediction and experiment is excellent. Also the vapor pressures of water over several polyols and saccharides in aqueous solution have been predicted with this method, giving results in excellent agreement with the experimental values.     

Hydration of oligosaccharides: anomalous hydration ability of trehalose.

The disaccharide trehalose extensively exists in anhydrobiotic organism and is considered to play an important role in preserving the integrity of biomembrane. However, the preserving mechanism remains unclear. In this report, we examine the hydration abilities of trehalose and several oligosaccharides composed of alpha-D-glucopyranosyl residues. The unfrozen water fraction per molecule was determined from differential scanning calorimetry measurements of their aqueous solutions. Further, the NMR relaxation time of the natural abundance 17O of water is measured for several saccharide solutions. These results indicate that trehalose has the highest hydration ability among the saccharides studied. In other words, trehalose can effectively lower the mobility of water molecules hydrogen-bonded with saccharides. It is thus reasonable that, among the disaccharides studied, trehalose exhibits the maximum stabilizing effect on the bilayer structure of lipid whose acyl chains are bonded with each other by the apolar interaction, because the apolar interaction is strengthened with the stabilization of the surrounding water structure.     

Thermodynamics of carbohydrate-lipid interactions.

Thermodynamic analyses of carbohydrate-lipid interactions were performed by investigating the effects of a series of carbohydrates, including monosaccharides, disaccharides, and trisaccharides, on the phase-transition properties of aqueous dispersions of 1,2-dipalmitoyl phosphatidylcholine (DPPC). The temperature of the lipid's main phase transition from the gel to liquid-crystalline phase is essentially unchanged in the presence of carbohydrate. The change in the free energy (delta G) of the transition is zero when a carbohydrate is added to aqueous dispersions of DPPC, while the enthalpy (delta H) and the entropy of the melting of DPPC are decreased. The thermodynamic information was used to examine carbohydrate-lipid interactions. Such interactions were elucidated according to our knowledge of the specific properties of carbohydrates in aqueous solutions and the previously proposed hydrophobic interaction involving hydrocarbon tails of the lipid in aqueous dispersions.     

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.     

Solubilization of a Lipophilic Compound in Highly Concentrated Saccharide Solutions Containing Protein

Solubilization of a lipophilic compound in highly concentrated saccharide solutions containing protein was studied by measuring the amount of the lipophilic compound solubilized, the surface hydrophobicity of protein, the line width of the water signal in ¹H-NMR spectra, and the unfreezable water content using a differential scanning calorimeter (DSC).

The solubilizing ability, which was shown by the amount of solubilized p-dimethylaminoazobenzene (DMAB), increased with increasing saccharide concentration in the aqueous system. The effects of different saccharides on the solubilizing ability of a saccharide and bovine serum albumin (BSA) mixture decreased in the following order: sucrose > maltose > fructose > glucose. The solubilizing ability of a saccharide and BSA mixture was higher about 4–5 times than that of saccharide only.

A good correlation was observed between the solubilizing ability of a saccharide and BSA mixture and the surface hydrophobicity of BSA. The line width of the water signal in ¹H-NMR spectrum and the unfreezable water content using DSC, that is, the bound water content in saccharide solution containing BSA increased with increasing saccharide concentration.

From these results, a large amount of DMAB solubilized in a highly concentrated saccharide solution containing BSA would be attributed to the hydrophobicity interaction between BSA and DMAB due to the surface hydrophobicity of BSA which increased with increasing bound water content.     

Effect of different monosaccharides and disaccharides on boar sperm quality after cryopreservation

The aim of the present study was to evaluate the cryoprotectant effect of different non-permeating sugars for boar sperm. Pooled semen from three boars was used for the experiments. In the first experiment, the sperm quality of boar sperm cryopreserved with an egg-yolk based extender supplemented with different monosaccharides (glucose, galactose or fructose) was compared to a control cryopreserved in lactose-egg yolk extender. In the second experiment, the effect of five disaccharides (lactose, sucrose, lactulose, trehalose or melibiose) on boar sperm cryosurvival was studied. Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37°C after thawing: percentages of sperm with intact plasma membrane (SIPM), sperm presenting high plasma membrane fluidity (HPMF), sperm with intracellular reactive oxygen substances production (IROSP) and apoptotic sperm (AS). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. Freezing extenders supplemented with each of the monosaccharide presented smaller cryoprotective effect than the control extender supplemented with lactose (P<0.05). However, from the three monosaccharides tested, glucose provided the best sperm quality after freezing-thawing. With respect to the disaccharides studied, samples frozen with the extender supplemented with lactulose exhibited in general the lowest sperm quality, except for the percentage of capacitated sperm, which was highest (P<0.05) in the samples cryopreserved with the trehalose extender. Our results suggest that disaccharides have higher cryoprotective effect than monosaccharides, although the monosaccharide composition of the disaccharides is also important, since the best results were obtained with those disaccharides presenting glucose in their composition.     

Hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues in subcritical water

The hydrolysis kinetics of trisaccharides consisting of glucose, galactose, and fructose residues with different glycosidic bonds, 1-kestose, d-melezitose, d-raffinose, and lactosucrose, in subcritical water were conducted over the temperature range of 150-230 degrees C and at a constant pressure of 10 MPa. The hydrolysis of trisaccharides in subcritical water proceeded consecutively, i.e., one cleavage of the two bonds antedated the other. The preceding cleavage was not expressed by the first-order kinetics, but by the kinetics considering the concentration of the acidic compounds, which were produced by the degradation of the constituent monosaccharides. The hydrolysis of the constituent disaccharides, except sucrose composed of the alpha-Glc-(1-->2)-beta-Fru bond, obeyed first-order kinetics. All of the rate constants of the hydrolytic kinetics were determined, and the values were found to depend on the type of bond.     

Microcalorimetric investigations of the metabolism of yeasts

Summary The anaerobic growth of the yeast Saccharomyces cerevisiae with six different mono- and disaccharides as energy source was investigated calorimetrically. With mixtures of monosaccharides and disaccharides or disaccharides with each other, biphasic thermograms were obtained. The diauxic growth is discussed in view of constitutive and inducible transport systems and degradation enzymes.     

Characterization of sugar diffusion coefficients in alginate membranes

The diffusivity of several monosaccharides and disaccharides in calcium alginate gels was determined using a specially designed diaphragm cell. The diffusion coefficients of the tested sugars are 4 to 18% smaller in alginate gel than in water and, with the exception of fructose, this difference increases with increasing sugar molecular weight. Also the position of the carbonyl group seems to be determined in the value of the diffusion coefficient - ketoses have lower diffusion coefficients than aldoses.     

Reduced protein adsorption at solid interfaces by sugar excipients

Sugar excipients are shown to reduce the adsorption of ribonuclease A, bovine serum albumin, and hen egg white lysozyme at the liquid-solid interface. The amount of protein adsorbed decreased as the concentration of the sugar increased. At the same sugar concentration, the ability of sugars to reduce protein adsorption followed the trend: trisaccharides > disaccharides > 6-carbon polyols > monosaccharides. This trend in adsorbed protein amounts among sugars was explained by stabilization of the protein native state in solution by the sugar excipients. The heat of solution of the amorphous saccharide was found to correlate with the amount of protein adsorbed.     

Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study

A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mM NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mM NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis.     

Influence of water activity and aqueous solvent ordering on enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase

Effects of the water activity (a(w)) and the solvent ordering, as determined by the activity coefficient of water, were investigated on the enzyme kinetics of alcohol dehydrogenase, lysozyme, and beta-galactosidase in various aqueous solutions. The water activity and the solvent ordering were adjusted by addition of electrolytes (NaCl, KCl, CsCl, etc.) or nonelectrolytes (sugars, alcohols, urea, etc.) at various concentrations. Although the enzyme kinetics were strongly dependent on a(w), a(w) was not a complete determinant of the enzyme behavior in aqueous solutions. Enzyme kinetics were also dependent on the solvent ordering. At a fixed a(w), all the enzyme kinetic parameters tested had a good correlation with the solvent ordering parameter as represented by the parameter alpha, an index of the deviation of the water state from the ideal solution, determined from the activity coefficient of water in solutions. Solvent ordering was expected to affect the enzyme kinetics through its effect on the hydrophobic interaction between the enzyme and the substrate and also on the thermal fluctuation.