Kinzo Nagasawa

Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol.

A solvolytic desulfation of glycosaminoglycuronan sulfates was developed by treatment of their pyridinium salts with dimethyl sulfoxide containing 10% of water or methanol at 80-100 degrees. Chemical and physical studies showed that the solvolytic desulfation is a useful method applicable to all the known glycosaminoglycuronan sulfates without producing depolymerization or unfavorable chemical changes in the polysaccharide molecules. An almost completely desulfated, N-acetylated heparin (S: 0.12%) was obtained by treatment of an N-desulfated and N-acetylated heparin with dimethyl sulfoxide containing 10% of methanol for 2 h at 100 degrees.

Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol

A solvolytic N-desulfation of heparin was developed by treatment of its pyridinium salt with dimethyl sulfoxide containing 5% of water or methanol for 1.5 h at 50 degrees. Chemical and chromatographic studies showed that the solvolytic desulfation is a useful method for N-desulfation of heparin without depolymerization of the heparin molecule. The partially N-desulfated heparins were also obtained by treatment with dimethyl sulfoxide containing 5% of water at 20 degrees, and their anticoagulant activity is related to the degree of N-desulfation.

Peptide inhibitors of angiotensin I-converting enzyme in digests of gelatin by bacterial collagenase.

Peptide inhibitors of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) were produced by digesting gelatin with bacterial collagenase. The inhibitors were isolated from the digests with a combination of alcohol fractionation, treatment with Amberlite CG-50 column, gel filtration through Sephadex G-25, and Dowex 50 column and paper chromatography. Nine peptide fractions were purified to apparent homogeneity judging by thin-layer and ion-exchange column chromatography, and amino acid composition. Amino acid sequences of the peptides were determined: 2 were found to be mixtures of peptides and the sequence of another was only partially determined. Six of the peptides were potent inhibitors of the converting enzyme, while the other three were less active. 6 peptides were substrates for the enzyme. The enzyme released a dipeptide, Ala-Hyp from one peptide and was strongly inhibited by this dipeptide. The remainder of the parent peptides was a less effective inhibitor.

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.

Reaction between carbohydrates and sulfuric acid : Part I. Depolymerization and sulfation of polysaccharides by sulfuric acid

Abstract The molecular weight and sulfur content of the reaction products obtained by treatment of amylopectin, cellulose, dextran, chitin, alginic acid, chondroitin 6-sulfate, and chitosan with concentrated sulfuric acid for 2 h at 0° were determined. All the polysaccharides were sulfated and depolymerized according to their structure. Analyses of sulfated and depolymerized products of cellulose, chitin, and chondroitin 6-sulfate revealed that no structural change in the constitutional monosaccharides occurred during reaction with concentrated sulfuric acid. Drastic depolymerization and sulfation occurred when solid chitin was dissolved in concentrated sulfuric acid, followed by gradual depolymerization and sulfation; the temperature during the reaction markedly influenced the degree of depolymerization.

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.

A new method for the determination of N-sulfate in heparin and its analogs

Abstract A new method for the determination of N-sulfate in heparin and its analogs is described. The method is based on the determination of inorganic sulfate liberated by deamination with nitrous acid. The accuracy, simplicity, and validity of this method are evaluated by comparing it with previous methods.

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.

Preparation, by chemical degradation of hyaluronic acid, of a series of even- and odd-numbered oligosaccharides having a 2-acetamido-2-deoxy-d-glucose and a d-glucuronic acid residue, respectively, at the reducing end

Abstract A series of even-numbered hyaluronate oligosaccharides (di- to octadeca-) having a 2-acetamido-2-deoxy- d -glucose residue at the reducing end was prepared by treatment of sodium hyaluronate with dimethyl sulfoxide containing 10% of 0.1 m hydrochloric acid for 16 h at 95°. The mixture of the even-numbered oligosaccharides obtained was converted with 0.15 m sodium carbonate for 6 h at 40° into a series of odd-numbered oligosaccharides (mono- to pentadeca-) having a d -glucuronic acid residue at the reducing end. Reaction of the hyaluronate tetrasaccharide with saturated calcium hydroxide gave β- d -Glc p A-(1→3)-β- d -Glc p NAc-(1→3)- d - arabo -trihydroxyglutaric acid besides the expected trisaccharide.