Hideki Uchiyama

Chemical sulfation of preparations of chondroitin 4- and 6-sulfate, and dermatan sulfate. Preparation of chondroitin sulfate E-like materials from chondroitin 4-sulfate

Abstract A solution of the tributylammonium salts of chondroitin 4- or 6-sulfate, or dermatan sulfate in N,N -dimethylformamide was treated with 2.0–8.0 mol/hydroxyl group of pyridine-sulfur trioxide at 0° for 1 h. The progress of the sulfation was studied by chondroitinase ABC digestion and liquid chromatography. The results suggested that sulfation proceeded homogeneously according to the order of reactively of the hydroxyl groups. Various chondroitin polysulfates, which resemble natural chondroitin sulfate E with respect to the disaccharide unit composition, were prepared from chondroitin 4-sulfate.

Preparation and properties of fluorescent glycosamino-glycuronans labeled with 5-aminofluorescein

The uronic acid residues of all known glycosaminoglycuronans reacted with 5-aminofluorescein to yield fluorescent glycosaminoglycuronan derivatives, which showed fluorescence characteristics identical to those of fluorescein or 5-acetamidofluorescein. The fluorescent products could be purified by chromatography on Octyl-Sepharose; three preparations of labeled chondroitin 6-sulfate having different degrees of substitution, and a labeled heparin were obtained. Fluorescent hyaluronic acid containing labeled and unlabeled molecules was digested with testicular hyaluronidase to give fluorescent oligosaccharides. Fluorescent chondroitin 6-sulfate was treated with chondroitinase AC to give a nonfluorescent disaccharide and minor proportion of fluorescent octasaccharide. Fluorescent heparin retained its anticoagulant activity, which was similar to that of the starting heparin; its half-life in circulating rabbit blood was 36 min (by fluorometry) and 45 min (by clotting-time assay).

Preparation and properties of biologically active fluorescent heparins

Hog mucosal heparin (N-sulfate, 0.84 mol; O-sulfate, 1.55 mol; N-acetyl, 0.12 mol; anticoagulant activity assayed by the method of U.S. Pharmacopeia, 161 USP units/mg) or its partially N-desulfated heparin (N-sulfate, 0.71 mol; O-sulfate, 1.47 mol; N-acetyl, 0.12 mol; anticoagulant activity, 117 USP units/mg/ was reacted with 5-isothiocyanatofluorescein in 0.5 M carbonate buffer (pH 8.5) at 35 degrees C for 6 h to yield the corresponding N-fluoresceinylthiocarbamoyl heparins (lambdaem 516 nm, lambdaex 491 nm; degree of substitution 0.006 and 0.013, respectively, anticoagulant activity, 174 and 140 USP units/mg, respectively). The fluorescent heparin (degree of substitution, 0.006; 174 USP units/mg) was injected into rabbits intravenously. The half-life of the fluorescent heparin determined by fluorometry was 24 min, that determined by the clotting time assay was 39 min. The time-course of concentration and the half-life of the fluorescent heparin and of the starting heparin obtained by the clotting time assay were virtually identical.

Chemical change involved in the oxidative-reductive depolymerization of heparin.

A solution of hog intestinal heparin (average M(r) 12,000, anti-clotting activity 168 USP units/mg) in 0.2 M phosphate buffer (pH 7.2), was incubated in the presence of Fe2+ for 20 h at 50 degrees under an O2 atmosphere to yield oxidative-reductively depolymerized heparin (ORD heparin, average M(r) 3,000, anti-clotting activity 34 USP units/mg). Chemical analysis of the ORD heparin showed a 22, 26, and 14% loss of hexosamine, uronic acid, and N-acetyl group, respectively, but no remarkable loss of both total and N-sulfate groups. 1H and 13C NMR spectroscopic analysis indicated no decrease in the amount of L-iduronic acid 2-sulfate, but a marked loss of nonsulfated uronic acid (73 and 39% loss of D-glucuronic acid and L-iduronic acids, respectively, the sum of which corresponds to the chemically determined loss of total uronic acid). The results indicated that the ORD reaction of heparin proceeds essentially by destruction of monosaccharide units, except L-iduronic acid 2-sulfate residues, due to oxygen-derived free radicals, followed by secondary hydrolytic cleavage of the resulting unstable residues.

Reactivity toward chemical sulfation of hydroxyl groups of heparin

Abstract The pyridinium salt of hog-mucosal heparin was desulfated by heating in 3:6:1 (v/v) 1,4-dioxane-dimethyl sulfoxide-methanol at 90° for 72 h, with the intermittent addition of pyridinium chloride (5 mol/mol of disaccharide). After N-resulfation, the desulfated polysaccharide (tributylammonium salt) was treated with 5–20 mol of pyridine-sulfur trioxide in N,N-dimethylformamide per mol equiv. of hydroxyl group at −10, 0, or 20° for 1 h to undergo O-sulfation. The sulfated products were quantitatively analyzed for 6-sulfate and 2-sulfate esters in 2-amino-2-deoxy- d -glucose and l -idosuronic acid units, respectively, by 13C- and 1H-n.m.r. spectroscopy and for total sulfate content by a chemical method. The new procedure gave 97% of desulfation of heparin, i.e., 100% N- and 6-desulfation of 2-amino-2-deoxy- d -glucose and 91.5% 2-desulfation of l -idosiduronic acid units. The O-resulfation proceeded according to the order of reactivity of the hydroxyl groups: HO-6 in GlcN ⪢ HO-2 in IdoA > other available hydroxyl groups (HO-3 in GlcN, HO-3 in IdoA, and HO-2 or HO-3 in GlcA).

Separation of heparin into fractions with different anticoagulant activity by hydrophobic interaction chromatography

Hog mucosal heparin purified on Sephadex G-100 (anticoagulant activity assayed by the method of the United States Pharmacopoeia, 179 units/mg) was separated by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B into two groups, one with high affinity and another with low affinity for the gels. The former group was further separated into three fractions differing in hydrophobicity. The anticoagulant activities of the fractions with higher hydrophobicity ranged from 210 to 254 units/mg, whereas that of the fraction with lower hydrophobicity was 100 units/mg. The difference in antithrombin III-activation potency was much more prominent. The data obtained from affinity chromatography of these fractions on antithrombin III-Sepharose also substantiated the observed difference in anticoagulant activity. Analytical data of the fractions revealed a characteristic difference in both N-acetyl content and molecular size. While the N-acetyl content (mol/mol of hexosamine) and Kav value (on Ultrogel AcA44) of the fraction with the lowest hydrophobicity were 0.12 mol and 0.48, those of the fractions with higher hydrophobicity were 0.15-0.17 mol and 0.35-0.23, respectively.

Gas chromatographic determination of microamounts of carbaryl and 1-napththol in natural water as sources of water supplies

A method for the clean-up and quantitative determination of Carbaryl and its hydrolysis product, 1-naphthol, in natural waters is described. After extraction of a water sample with methylene chloride, the two compounds were separated from possible organochlorine pollutants such as Endrin, gamma-BHC, p,p-DDT, pentachlorophenol and polychlorinated biphenyl, and their heptafluorobutyryl derivatives obtained. Determination by electron-capture gas chromatography at the 2.5-10 ppb level, using 11 of water samples, was carried out.

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on Phenyl-Sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate.

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 M ammonium sulfate containing 10mM hydrochloric acid showed that: The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.

Hydrophobic-interaction chromatography of glycosaminoglycuronans: The contribution of N-acetyl groups in heparin and heparan sulfate to the affinity for hydrophobic gels, and variety of molecular species in beef-kidney heparan sulfate

Contribution of N-acetyl groups in heparin and heparan sulfate to their affinity for hydrophobic gels was examined by use of a series of semi-synthetic, N-acetylated, hog-intestinal heparins, a whale-intestinal heparin, and a beef-kidney heparan sulfate. Chromatography on Phenyl-Sepharose CL-4B in 3.8-1.0M ammonium sulfate-10mM hydrochloric acid indicated that an increasing N-acetyl content, which is correlated to a decreasing N-sulfate content, results in a marked increase in the affinity for the gels. The variety of molecular species in beef-kidney heparan sulfate, previously fractionated by conventional chromatographic procedures, was demonstrated by separating further, by hydrophobic-interaction chromatography, the polysaccharide into several fractions composed of molecular species distinctly different in N-acetyl and sulfate content, and in molecular size.

Hydrophobic interaction chromatography of mucopolysaccharides : Examination of fundamental conditions for fractionation of heparin on hydrophobic gels

Abstract For hydrophobic interaction chromatography of mucopolysaccharides, some fundamental chromatographic conditions were examined mainly on a combination of Phenyl-Sepharose CL-4B gel and heparin. Every one of the conditions, such as column dimensions, amount of heparin applied, flow-rate, electrolyte and acidity of elution medium, and temperature, influenced the distribution of heparin among the fractions separated on the gel. The solutions of 4.0 M –1.0 M ammonium sulphate in water or in 0.01 M hydrochloric acid were excellent as elution media. Serious temperature effects were generally observed on the interaction between mucopolysaccharides and different types of hydrophobic gel. Commercially available hydrophobic gels of two types were examined: (1) hydrophobic gels without any ionizable function—Phenyl- and Octyl-Sepharose CL-4B gels and Benzyl- and Octyl-Agarose gels, and (2) hydrophobic gels with some ionizable groups, such as isoureide and primary amino groups—Alkyl-Agarose and ω-Aminoalkyl-Agarose gels.