Syntheses of linear tetra-, hexa-, and octa-saccharide fragments of the i-blood group active poly-(N-acetyl-lactosamine) series. Blockwise methods for the synthesis of repetitive oligosaccharide sequences

N-Phthaloylation of lactosamine gave various glycosyl donors (beta-chloride, beta-trichloroacetimidate) and glycosyl acceptors (3',4'-diol). Coupling of the chloride with a methyl beta-D-glycoside led to the tetrasaccharide fragment, beta-D-Galp-(1----4)-beta-D-GlcpNac-(1----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAcOMe. Acetolysis of the protected tetrasaccharide, followed by treatment with hydrogen chloride, gave a tetrasaccharide chloride which was coupled with the methyl beta-glycoside of lactosamine. A hexasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]2-beta-D-Galp-(1----4)-bet a- D-GlcpNAcOMe, was thus obtained by this ("n + 1") method. A more efficient ("n + n") method was applied for the synthesis of an octasaccharide fragment, [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)]3-beta-D-Galp- (1----4)-beta-D-GlcpNAcOMe (38), where di- and tetra-saccharide intermediates having a 3,4-O-isopropylidene-beta-D-galactopyranosyl nonreducing terminal group and a benzyl beta-D-glycoside group were precursors, either as glycosyl donors (beta-trichloroacetimidates) or glycosyl acceptors (3,4-diols as nonreducing terminal groups). Thus, doubling the length of the repetitive oligosaccharide sequence could be efficiently accomplished at each glycosylation step.     

Synthesis of O-alpha-D-glucopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-O-alpha -D-xylopyranosyl-(1----4)-D-glucopyranose as a substrate analogue of alpha amylase

The tetrasaccharide a-D-Glcp-(1----4)-a-D-Xylp-(1----4)-a-D-Xylp-(1----4)-D- Glcp (1) has been synthesized, as a substrate analogue of alpha amylase, by silver perchlorate-catalyzed glycosylation of benzyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-a-D-xylopyranosyl)-beta-D- glucopyranoside (30) with 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D- glucopyranosyl)-a-D-xylopyranosyl chloride or by methyl triflate-promoted condensation of 30 with methyl 2,3-di-O-benzyl-4-O-(2,3,4,6-tetra-O-benzyl-a-D-glucopyranosyl)-1-thio- beta-D-xylopyranoside, followed by removal of protecting groups of the resulting tetrasaccharide derivative 40.     

Further definition of the size of the blood group-i antigenic determinant using a chemically synthesised octasaccharide of poly-N-acetyllactosamine type.

In earlier studies, the minimum structure which inhibited the binding of anti-i to an i-active glycoprotein was the linear trisaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-D-Gal. There was an increasing hierarchy of inhibitory activities in the linear tetrasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D -GlcNAc , its methyl beta-glycoside, and in the methyl beta-glycoside of the hexasaccharide. The linear octasaccharide methyl beta-glycoside in this series is approximately only half as active as the hexasaccharide methyl beta-glycoside. Analyses by high resolution 1H-n.m.r. of these two oligosaccharides indicated that they have similar conformations in solution, and there is no evidence for the occurrence of inter-molecular interactions which might partially hinder the binding of anti-i to the octasaccharide methyl beta-glycoside. These results are consistent with the size of the i antigen being in the region of a hexasaccharide. It is proposed that the methyl aglycon group of the hexasaccharide methyl beta-glycoside confers an above normal activity by presenting a hydrophobic area for additional contact in the vicinity of the antibody-combining site.     

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 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.     

Artificial carbohydrate antigens: a block synthesis of a linear, tetrasaccharide repeating-unit of the Shigella flexneri variant Y polysaccharide

Glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl bromide gave methyl 2,4-di-O-benzoyl-3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl) -alpha-L-rhamnopyranoside (4) in 93% yield. Conversion of 4 into the corresponding glycosyl bromide was accomplished with dibromomethyl methyl ether. Under Koenigs-Knorr conditions, this bromide reacted with 8-(methoxycarbonyl)octyl 2-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glycopyranosyl)- 3,4-di-O- benzyl-alpha-L-rhamnopyranoside, to provide the protected tetrasaccharide in 91% yield. Removal of blocking groups gave 8-(methoxycarbonyl)octyl O-alpha-L-rhamnopyranosyl-(1---- 3)-O-alpha-L-rhamnopyranosyl-(1---- 3)-O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1----2)-alpha-L- rhamnopyranoside. Together with previously synthesized tetrasaccharides of the Shigella flexneri Y O-antigen, this oligosaccharide has been used to study the conformation of O-antigens and to assist in the selection of S. flexneri, variant Y, specific monoclonal antibodies.     

A synthetic heptasaccharide reveals the capability of a monoclonal antibody to read internal epitopes of a polysaccharide antigen.

The binding of the synthetic heptasaccharide,beta-D-Galp-(1----3)-beta-D-Galp-(1----6)-beta-D-Galp-(1 ----6)-beta-D-Galp-(1----6)-beta-D-Galp-1-OCH3 (10) with two monoclonal IgAs of the X24 gene-family has been investigated. The ligand 10 was synthesized by silver triflate mediated coupling of O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----3)-O-(2,4,6,-t ri-O-benzoyl-beta-D-galactopyranosyl)-(1----3)-2,4,6-tri-O-benzoyl-alpha -D-galactopyranosyl chloride (5) to the benzoylated, all-beta-(1----6)-linked methyl galactotetraoside 13, having O-6(4) free, followed by debenzoylation of the formed, fully protected methyl galactoheptaoside. The blockwise synthesis of the nucleophile 13 from readily available monosaccharides, and the synthesis of 5 from the corresponding beta-1-O-benzoylated trisaccharide, is also described. Heptasaccharide 10 binds with the (1----6)-beta-D-galactan-specific monoclonal antibodies X-24 and J539 with essentially the same Ka-values (5.4 x 10(5) M-1 and 6.4 x 10(5) M-1, respectively) as does the methyl beta-glycoside of all-beta-(1----6)-linked galactotetraose 14 (5.7 x 10(5) M-1 and 5.9 x 10(5) M-1, respectively). Of the series of homologous oligosaccharides studied previously (di- through a hexa-saccharide), 14 was found to show the highest affinity of interaction with both these immunoglobulins. The beta-(1----3)-linked galactotriose, which forms the bulky terminus of 10, does not appear to bind to these IgA. Thus, the observation that the affinity of 10 is the same as that of 14 confirms that these immunoglobulins bind internal tetrasaccharide sequences of the antigenic (1----6)-beta-D-galactopyranan.     

Crystallization of the lectin IV of Griffonia simplicifolia and its complexes with the Lewis b and Y human blood group determinants

Single crystals of the lectin IV from Griffonia simplicifolia have been grown in the tetragonal crystal system. The space group is P4(2)2(1)2 with a = 78.95(5) A and c = 89.01(2) A, and there is one subunit of the dimeric glycoprotein in the crystallographic asymmetric unit. The crystals diffract to at least 2.5 A d spacings and are stable in the x-ray beam for 3 weeks. Crystals of the complex with the Lewis b (Leb) and Y human blood group determinants as the methyl glycosides, alpha-L-Fuc(1----2)beta-D-Gal(1----3)[alpha-L-Fuc(1----4)]beta-D-GlcNAc-OMe (where Fuc is fucose and -OMe is methoxy) and alpha-L-Fuc(1----2)beta-D-Gal(1----4)[alpha-L-Fuc(1----3)]-beta-D-GlcNAc- OMe, respectively, have also been grown and found to be isomorphous with the native lectin. Crystals have also been obtained with several derivatives of the Lewis b-OMe tetrasaccharide including that which has the 6-hydroxyl of the beta-D-GlcNAc unit replaced by iodine. In the latter case, the presence of the iodine atoms was established.     

Structural studies of the Escherichia coli O78 O-antigen polysaccharide

The structure of the O-antigen polysaccharide from Escherichia coli O78 has been investigated; methylation analysis, partial solvolysis with liquid hydrogen fluoride, and 2D-n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure.----3)-beta-D-GlcpNAc-(1----4)-beta-D-GlcpNAc- (1----4)-beta-D-Manp-(1----4)-alpha-D-Manp-(1----     

Isolation and structure determination of a diacetamidodideoxyuronic acid-containing glycan chain from the S-layer glycoprotein of Bacillus stearothermophilus NRS 2004/3a

A glycan isolated from the surface-layer glycoprotein of Bacillus stearothermophilus strain NRS 2004/3a was shown by 1H- and 13C-n.m.r. spectroscopy to have the tetrasaccharide repeating-unit ----4)-beta-ManpA2,3(NAc)2-(1----3)-alpha-GlcpNAc-(1----4)-beta- ManpA2,3(NAc)-(1----6)-alpha-Glcp(1----.     

Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.     

Structure of the group G streptococcal polysaccharide

The structure of the group-specific polysaccharide of group G Streptococcus was determined by means of methylation analysis and selective chemical degradations. The anomeric configurations and conformations of the sugar residues were studied by 1H- and 13C-n.m.r. spectroscopy. The tetrasaccharide repeating unit, ----3)-alpha-D-Galp-(1----2)-[alpha-L-Rhap-(1----3)-beta-D-GalpNAc - (1----4)]-alpha-L-Rhap-(1----, was determined.     

The application of 1H/13C inversely correlated NMR spectroscopy to the determination of acylation and glycosylation sites in the O-specific polysaccharide from Hafnia alvei 1187

An inversely correlated 1H/13C NMR spectrum defined the amino sugars acylated by acetyl or 3-hydroxybutyryl groups and revealed partial sequences and glycosylation sites in a tetrasaccharide repeating unit of the title polysaccharide, (----2DGlc alpha 1----3DGlcNAcyl alpha 1----4DGalNAc alpha 1----3DGalNAc beta 1----)n, where Acyl = 3-hydroxybutyryl.     

Structural variation in the antithrombin III binding site region and its occurrence in heparin from different sources

A tetrasaccharide possessing a biosynthetically permissible structural variability in and adjacent to the antithrombin III (ATIII) binding site has been isolated from heparin lyase depolymerized bovine lung heparin by using strong anion-exchange high-pressure liquid chromatography (SAX-HPLC). On the basis of two-dimensional 500-MHz 1H NMR experiments, including phase-sensitive correlated spectroscopy (COSY) and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY), and fast-atom bombardment mass spectrometry (FAB-MS), the primary structure of this tetrasaccharide was unambiguously established as delta UAp2S (1----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D-GlcNp2S3S6S (where delta UA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid). The 1H NMR ROESY experiment proved to be particularly valuable in offering sequence information. Heparins from a variety of species and tissue sources were examined by oligosaccharide mapping using SAX-HPLC and gradient polyacrylamide gel electrophoresis. Two of these heparins are used as anticoagulants; they are porcine intestinal mucosal heparin and bovine lung heparin. The predominant ATIII-binding site in porcine heparin contained an N-acetylated glucosamine residue. We now report the structure of the predominant ATIII-binding site in bovine heparin as----4)-alpha-D-GlcNp2S6S(1----4)-beta-D-GlcAp(1----4)-alph a-D- GlcNp2S3S6S(1----4)-alpha-L-IdoAp2S(1----4)-alpha-D-GlcNp 2S6S(1----. This study shows the presence of one or both types of ATIII-binding-site variants in all of the heparins that were examined.     

Comparative study of the mucin-type sugar chains of human chorionic gonadotropin present in the urine of patients with trophoblastic diseases and healthy pregnant women

Human chorionic gonadotropins (hCGs) highly purified from the urine of patients with trophoblastic diseases and of healthy pregnant women contain approximately four mucin-type sugar chains in one molecule. The structures of these sugar chains were studied comparatively by using a new sensitive method to obtain mucin-type sugar chains quantitatively as radioactive oligosaccharides from a small amount of glycoproteins. The mucin-type sugar chains of all hCGs include sialylated and nonsialylated Gal beta 1----3GalNAc and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc. In the case of normal hCG and hydatidiform mole hCG, oligosaccharides containing the tetrasaccharide core occupy approximately 10% of the total mucin-type sugar chains. The ratio of the tetrasaccharide containing oligosaccharides is increased prominently to approximately 60% in choriocarcinoma hCG. The proportion in invasive mole hCG was also increased, but less than the proportion of choriocarcinoma hCG.     

Structural studies of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum

The structure of the O-antigen of the lipopolysaccharide from an avirulent strain (M4S) of Pseudomonas solanacearum has been investigated by methylation analysis, n.m.r. spectroscopy, and N-deacetylation-deamination, followed by analysis and controlled Smith-degradation of the product. These studies demonstrate that the O-antigen is composed of a tetrasaccharide repeating-unit having the following structure: ----3)-alpha-D-GlcpNAc-(1----2)-alpha-L-Rhap-(1----2)-alpha- L-Rhap-(1----3)- alpha-L-Rhap-(1----.     

Structural variability of the neutral carbohydrate moiety of cow colostrum kappa-casein as a function of time after parturition. Identification of a tetrasaccharide with blood group I specificity

New neutral oligosaccharides from cow colostrum kappa-casein were identified and characterized by 500-MHz 1H-NMR spectroscopy. Their structures are Gal beta(1----3)GalNAc-ol, Gal beta(1----3)[GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Gal beta(1----4)GlcNAc beta(1----6)]GalNAc-ol, Gal beta(1----3)[Fuc alpha(1----3)[Gal beta(1----4)]GlcNAc beta(1----6)]GalNAc-ol. The tetrasaccharide and the cow colostrum kappa-caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow kappa-casein as a function of time after calving is studied.     

Structure of a neutral polymer isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O18

A neutral polymer (the putative O antigen) has been isolated from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup 018. From the results of spectroscopic and degradative studies, the repeating unit of the polymer was identified as a linear tetrasaccharide having the structure shown. ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----6)-alpha-D- GlcpNAc-(1----     

Efficient chemical synthesis of methyl beta-glycosides of beta-(1----6)-linked D-galacto-oligosaccharides by a stepwise and a blockwise approach

Bromoacetylation of methyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (1) followed by cleavage of the methoxyl group from the resulting 6-O-bromoacetyl derivative 2 with 1,1-dichloromethyl methyl ether gave 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl chloride (3). Reaction of 3 with 1, promoted by silver trifluoromethanesulfonate, afforded methyl O-(2,3,4-tri-O-benzoyl-6-O-bromoacetyl-beta-D-galactopyranosyl)-(1----6) -2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (12), bearing at O-6 of its non-reducing end-group the selectively removable bromoacetyl group. This was O-debromoacetylated and the disaccharide nucleophile 15 formed was again treated with 3, to give the analogous trisaccharide 18. This sequence of reactions was repeated to afford the analogous tetrasaccharide 20, showing the feasibility of stepwise construction of the title oligosaccharides. Similar reactions of 3 with 1,2,3,4-tetra-O-benzoyl-alpha- (7) and beta-D-galactopyranose (5) gave, respectively, O-(2,3,4-tri-O-benzoyl-6-O-bromoacetyl-beta-D-galactopyranosyl)-(1----6) -1,2,3,4-tetra-O-benzoyl-alpha- (14) and beta-D-galactopyranose (13). These could be separately converted into the same glycosyl halide, namely, alpha-(2,3,4-tri-O-benzoyl-6-O-bromoacetyl-beta-D-galactopyranosyl)-(1-- --6)-2,3,4-tri-O-benzoyl-alpha-D-galactopyranosyl chloride (16), by cleavage with 1,1-dichloromethyl methyl ether. The chloride 16 was treated with tri- and tetra-saccharide nucleophiles analogous to 15 to give, respectively, the corresponding pentasaccharide 23 and the hexasaccharide 25, demonstrating the possibility of the blockwise construction of higher beta-(1----6)-linked D-galacto-oligosaccharides. The disaccharide 12 was also obtained by the reaction of 1,2,3,4-tetra-O-benzoyl-6-O-bromoacetyl-beta-D-galactopryanose (6) with 1 in the presence of trimethylsilyl trifluoromethane-sulfonate. Similarly, the trisaccharide 18 and the tetrasaccharide 20 were obtained by the treatment of 13, respectively, with 1 and 15, showing that, as with their 1-O-acetyl counterparts, beta-1-benzoates of saccharides bearing at O-2 a group capable of neighboring-group participation can act under these conditions as glycosyl donors. Crystalline methyl beta-glycosides of (1----6)-beta-D-galacto-tetraose (22), -pentaose (24) and -hexaose (27) have been obtained for the first time, by deacylation (Zemplén) of their fully protected precursors.     

Identification by n.m.r. spectroscopy of oligosaccharides obtained by treatment of the hairy regions of apple pectin with rhamnogalacturonase

2D-N.m.r. methods have been used to determine the composition of a mixture of oligosaccharides obtained by enzymic degradation of the modified hairy (ramified) regions of apple pectin with a new rhamnogalacturonase. The structures of the oligosaccharides were based on the unit alpha-Rhap-(1----4)-alpha-GalA-(1----2)-alpha-Rhap-(1----4)- GalA. A-beta-Galp unit was 4-linked to approximately half of the terminal Rhap residues and to half of the (1----2)-linked Rhap residues. The sample contained a mixture of a tetrasaccharide, two pentasaccharides, and one hexasaccharide.