Diagnosis of Maroteaux-Lamy syndrome by the use of radiolabelled oligosaccharides as substrates for the determination of arylsulphatase B activity.

The kinetic parameters (Km and V) of human arylsulphatase B (4-sulpho-N-acetylgalactosamine sulphatase) activity in cultured skin fibroblasts were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo chondroitin 4-sulphate and dermatan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, were desulphated up to 4400 times faster than the minimum arylsulphatase-B-specific substrate, namely the monosaccharide N-acetylgalactosamine 4-sulphate. Aglycone structures that influence substrate binding and/or enzyme activity were an adjacent-residue C-6 carboxy group and a second but internal N-acetylgalactosamine 4-sulphate residue. Arylsulphatase B activity in fibroblast homogenates assayed with O-(beta-N-acetylgalactosamine 4-sulphate)-(1----4)-O-D-(beta-glucuronic acid)-(1----3)-O-D-N-acetyl[1-3H] galactosaminitol 4-sulphate derived from chondroitin 4-sulphate as substrate clearly distinguished Maroteaux-Lamy-syndrome patients from normal controls and other mucopolysaccharidosis patients. We recommend the use of the above trisaccharide substrate for both postnatal and prenatal diagnosis of Maroteaux-Lamy syndrome.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

The disaccharides formed by deaminative cleavage of N-deacetylated glycosaminoglycans.

Chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and keratan sulphate were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0, and the resulting products were reduced with NaB3H4. This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues, which were converted into 3H-labelled 2,5-anhydro-D-mannitol (AManR) or 2,5-anhydro-D-talitol (ATalR) residues respectively. The end-labelled disaccharides, composed of D-glucuronic acid (GlcA), L-iduronic acid (IdoA) or D-galactose (Gal) and one of the anhydrohexitols, were identified as follows: both chondroitin 4-sulphate and chondroitin 6-sulphate gave GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4), IdoA----ATalR (4-SO4) and GlcA(2-SO4)----ATalR(6-SO4); dermatan sulphate gave IdoA----ATalR(4-SO4), GlcA----ATalR(4-SO4), GlcA----ATalR(6-SO4)----IdoA(2-SO4)ATalR(4-SO4) and IdoA----ATalR (4,6-diSO4); keratan sulphate gave Gal(6-SO4)----AManR(6-SO4), Gal----AManR(6-SO4), Gal(6-SO4)----AManR and Gal----AManR. Several additional disaccharides were generated by treatment of the uronic acid-containing disaccharides with hydrazine to epimerize their uronic acid residues at C-5. A number of these disaccharides were found to be substrates for lysosomal sulphatases and glycuronidases. Methods were developed for the separation of all of the disaccharide products by h.p.l.c. The rate of N-deacetylation of chondroitin 4-sulphate by hydrazinolysis was significantly lower than the rate of N-deacetylation of chondroitin 6-sulphate or chondroitin. Dermatan sulphate was N-deacetylated at an intermediate rate. The relative amounts of disaccharides obtained from chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate under optimum hydrazinolysis/deamination conditions were comparable with the amounts of the corresponding products released from the polymers by chondroitinase treatment.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Synthesis of a mucin-type O-glycosylated amino acid, beta-Gal-(1----3)-[alpha-Neu5Ac-(2----6)]-alpha-GalNAc-(1----3)- Ser

Total synthesis of O-beta-D-galactopyranosyl-(1----3)-O-[(5-acetamido-3,5-dideoxy- D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2----6)]-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1----3 )-L- serine was achieved by use of the key glycosyl donor O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O- [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha-D- galactopyranosyl trichloroacetimidate and the key glycosyl acceptor N-(benzyloxycarbonyl)-L- serine benzyl ester in a regiocontrolled way.     

Synthesis of a fully protected derivative of O-(N-acetyl-alpha-D-neuraminyl)-(2----3)-O-beta-D-galactopyranosyl-(1- ---3)-O- [(N-acetyl-alpha-D-neuraminyl)-(2----6)]-O-(2-acetamido-2-deoxy-alpha- D-galactopyranosyl)-(1----3)-L-serine

N-(Benzyloxycarbonyl)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O-acetyl-beta-D - galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-O-(2-acetamido-4-O-acetyl-2- deoxy-alpha-D- galactopyranosyl)-(1----3)-L-serine benzyl ester was synthesized by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5- di-deoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl)onate]- (2----3)-O-(2,4,6- tri-O-acetyl-beta-D-galactopyranosyl)-(1----3)-O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D-galact o-2- nonulopyranosyl)onate-(2----6)]-4-O-acetyl-2-azido-2-deoxy-a lpha- and -beta-D-galactopyranosyl trichloroacetimidate as a key glycotetraosyl donor which, upon reaction with N-(benzyloxycarbonyl)-L-serine benzyl ester, afforded a 44% yield of a mixture of the alpha- and beta-glycosides in the ratio of 2:5.     

The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-(14)C]glucose and [(35)S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride-cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-(14)C]glucose were found to be 0.79+/-0.16 and 3.2+/-0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-(14)C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [(35)S]sulphate incorporation and were found to be in good agreement with those obtained from [U-(14)C]glucose labelling.     

Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern.

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion-exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 x 10(5), contained on average six oversulphated chondroitin sulphate chains (Mr 4 x 10(4)) bound on a polypeptide of Mr 2.8 x 10(4), and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), alpha-chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self-aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 4-sulphate (delta di-4S) and disulphated disaccharides (delta di-diS) [90% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 6-sulphate (delta di-diSD) and 10% 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid 2/3-sulphate)-D-galactose 4-sulphate (delta di-diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (delta di-triS) and small amounts of 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose 6-sulphate (delta di-6S) and 2-acetamido-2-deoxy-3-O-(alpha-L-threo-4-enopyranosyluronic acid)-D-galactose (delta di-OS). Trisulphated disaccharides contained sulphate groups at C-4 and C-6 of the galactosamine and at C-2 or C-3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.     

The biosynthesis in vitro of keratan sulphate in bovine cornea

1. Bovine corneas were incubated in vitro with [U-(14)C]glucose. 2. The glycosaminoglycans of corneal stroma were isolated and fractionated on cetylpyridinium chloride-cellulose columns. The major components were keratan sulphate (71%), chondroitin 4-sulphate (17%) and chondroitin 6-sulphate (4%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan biosynthesis of corneal stroma were separated on Dowex 1 (formate form) and the tissue content and cellular concentrations were determined. 4. The rates of synthesis of the intermediates of glycosaminoglycan biosynthesis in corneal stroma were determined. 5. The incorporation of radioactivity from [U-(14)C]glucose into the uronic acid and hexosamine components of the glycosaminoglycans present in corneal stroma were measured and the turnover rates of these polymers were calculated. It was found that keratan sulphate was turning over in about 723h and chondroitin 6-sulphate in 251h.     

Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.     

Synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp+ ++-(1----6)] - beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp- (1----6)]-D- GlcpNAc, a core glycoheptaose of a "bisected" complex-type glycan of glycoproteins

A synthesis of alpha-D-Manp-(1----3)-[beta-D-GlcpNAc-(1----4)]-[alpha-D-Manp++ +-(1----6)]- beta-D-Manp-(1----4)-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-( 1----6)]-D- GlcpNAc was achieved by employing benzyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(3,6-di-O-benzyl-2-deoxy-2 - phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O-benzyl-2-deoxy-6-O-p- methoxyphenyl-2-phthalimido-beta-D-glucopyranoside as a key glycosyl acceptor. Highly stereoselective mannosylation was performed by taking advantage of the 2-O-acetyl group in the mannosyl donors. The alpha-L-fucopyranosyl residue was also stereoselectively introduced by copper(II)-mediated activation of methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside.     

Total synthesis of globotriaosyl-E and Z-ceramides and isoglobotriaosyl-E-ceramide

Stereoselective, total synthesis of O-alpha-D-galactopyranosyl-(1----4) -O-beta-D-galactopyranosyl-(1----4)-O-beta-D-glucopyranosyl-(1----1)-N -tetracosanoyl-[2S,3R,4E (and 4Z)]-sphingenine and O-alpha-D -galactopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-O-beta-D -glucopyranosyl-(1----1)-N-tetracosanoyl-(2S,3R,4E)-sphin gen ine was achieved by using O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate, O-(2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl) -(1----4)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyranosyl)-(1----4)-2,3,6- tri-O-acetyl-alpha (and beta)-D-glucopyranosyl fluoride, and O-(2,3,4,6-tetra-O-acetyl-alpha-D -galactopyranosyl)-(1----3)-O-(2,3,6-tri-O-acetyl-beta-D-galactopyran osyl)-(1----4)-2,3,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate.     

Synthesis of oligosaccharides containing the X-antigenic trisaccharide (alpha-L-Fucp-(1----3)-[beta-D-Galp-(1----4)]-beta-D-GlcpNAc) at their nonreducing ends.

The "armed" methyl 2,3,4-tri-O-benzyl-1-thio-beta-L-fucopyranoside was reacted with "disarmed" phenyl O-(tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-6-O-benzyl-2- deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside in the presence of CuBr2-Bu4NBr complex to give phenyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----4)-O- [(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-(1----3])-6-O-benzyl-2-deoxy -2- phthalimido-1-thio-beta-D-glucopyranoside (6) as a novel glycosyl donor. The glycosylating capability of 6 was further examined using N-iodosuccinimide-triflic acid as a reagent. This led to the synthesis of a tetrasaccharide and a pentasaccharide incorporating the X-antigenic structure represented by 6.     

Feruloylated xyloglucan and p-coumaroyl arabinoxylan oligosaccharides from bamboo shoot cell-walls.

A novel feruloylated xyloglucan disaccharide and p-coumaroylated arabinoxylan trisaccharide were isolated from cell walls of growing bamboo (Phyllostachys edulis) shoots. On the basis of chemical and spectral data, their structures were determined to be O-(4-O-trans-feruloyl-alpha-D- xylopyranosyl)-(1----6)-D-glucopyranose and O-[5-O-(trans-p-coumaroyl)- alpha-L-arabinofuranosyl]-(1----3)-O-beta-D-xylopyranosyl-(1----4)-D- xylopyranose, respectively. This is the first reported evidence of a phenolic acid covalently associated with the cell wall hemicellulose, xyloglucan.     

Total synthesis of the modified ganglioside de-N-acetyl-GM3 and some analogs.

Methyl[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosid]onat e was used for the glycosylation of benzyl O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)- and benzyl O-(2,3-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl- 2-O-pivaloyl-beta-D-glucopyranoside to give benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2-- --3)-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(21) and benzyl O-[methyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5- dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonate]-(2----6) -O-(2,3-di- O-benzyl-beta-D-galactopyranosyl)-(1----4)-3,6-di-O-benzyl-2-O-pivaloyl- beta-D-glucopyranoside (18), respectively, accompanied by the beta-linked isomers 22 and 19, respectively. Compounds 18, 21, and 22 were converted into the corresponding glycotriosyl donors which, upon coupling with (2S,3R,4E)-3-O-benzoyl-2-N-tetracosanoylsphingenine, afforded completely protected ganglioside analogs 39, 40, and 41, respectively. Deprotection of 40, 41, and 39 completed the synthesis of the modified ganglioside de-N-acetyl-GM3, a stereoisomer, and a regioisomer. The N-deprotected forms of 40 and 39, on successive treatment with methyl isocyanate and O-deprotection, gave the N-(N-methylcarbamoyl) analogs of GM3 and its regioisomer.     

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans.

The relative binding affinity of Zn2+ to several glycosaminoglycans was determined by gel-filtration chromatography. Binding was observed only between Zn2+ and heparin. No binding was observed between Zn2+ and chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate of hyaluronic acid. All of the glycosaminoglycans contained carboxy groups, but only heparin bound Zn2+. This observation suggests that, contrary to a previously proposed hypothesis, simple electrostatic interactions between the negatively charged carboxy groups of the glycosaminoglycans and the positively charged Zn2+ cannot explain the observed binding.     

Isolation of sulfited oligosaccharides from glycoproteins treated with alkaline sulfite

The oligosaccharide products resulting from treatment of mucin-type glycoproteins with alkali in the presence of the sulfite anion have been investigated. Treatment of fetuin and of tryptic glycopeptides from the human erythrocyte with this reagent resulted in the release of sulfited oligosaccharides identified as N-acetylsulfohexosamine (HexNAcSO3), alpha-NeuAc-(2----6)-HexNAcSO3, and alpha-NeuAc-(2----3)-Gal-(1----3 or 4)-[GlcNAc-(1----6)]-HexNAcSO3. In addition, 2.7 moles of sialic acid were released per mole of alpha-NeuAc-(2----6)-HexNAcSO3 from fetuin. The sulfohexosamine moiety is formed via unsaturated intermediates from a 3-O-substituted 2-acetamido-2-deoxy-D-galactosyl residue at the carbohydrate-peptide linkage site when this residue is not substituted at O-4 by another sugar residue. A reaction mechanism accounting for the release of the sulfited oligosaccharides from a 3-O- and 6-O-substituted hexosamine is proposed in which the oligosaccharide branch attached to O-6 is obtained as a specific fragment terminating in sulfohexosamine.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

Scammonins I and II, the resin glycosides of radix scammoniae from Convolvulus scammonia.

The ether-soluble resin glycoside ('jalapin') fraction obtained from scammony roots, on alkaline hydrolysis, gave a glycosidic acid, scammonic acid A, together with isobutyric, 2S-methylbutyric and tiglic acids. In addition, two kinds of resin glycosides, named scammonin I and II, were isolated and characterized, respectively, as (11S)-hydroxyhexadecanoic acid, 11-[( O-6-deoxy-4-O-(2(E)-methyl-1-oxo-2- butenyl)-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobut yl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta- D-glucopyranosyl]oxy)-, intramol. 1,3"'-ester and (11S)-hydroxyhexadecanoic acid, 11-[( O-beta-D-glucopyranosyl-(1----4)-O-6-deoxy-2-O-(2-methyl-1-oxobutyl)- alpha-L-mannopyranosyl-(1----2)-O-beta-D-glucopyranosyl-(1----2)-6-deoxy -beta-D - glucopyranosyl]oxy)-, intramol. 1,3"'-ester.