Chemical synthesis of the trisaccharide unit of the species-specific phenolic glycolipid from Mycobacterium leprae

O-(3,6-Di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, the haptenic trisaccharide of the Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) antigen, and related trisaccharides, were synthesized by allylation of O-2 of benzyl 4-O-benzyl-alpha-L-rhamnopyranoside using phase-transfer catalysis, methylation of the product, deallylation, and coupling to O-(2,4-di-O-acetyl-3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-2,3- di-O-methyl-L-rhamnopyranosyl bromide or related disaccharides. Anomeric mixtures of the trisaccharide derivatives were separated by preparative t.l.c., deacetylated, and hydrogenolyzed, to give the pure trisaccharides. It had already been demonstrated that only those trisaccharides containing an intact, terminal 3,6-di-O-methyl-beta-D-glucopyranosyl unit are effective in inhibiting the specific binding between PGL-I and anti-PGL-I immunoglobulin M antibodies in human lepromatous leprosy sera.     

Synthesis and immunoreactivity of neoglycoproteins containing the trisaccharide unit of phenolic glycolipid I of Mycobacterium leprae

The trisaccharide segment, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O-methyl- alpha-L-rhamnopyranosyl)-(1----2)-3-O-methyl-L-rhamnopyranose, of the Mycobacterium leprae-specific phenolic glycolipid I has been synthesized as its 8-(methoxycarbonyl)octyl glycoside and coupled to a carrier protein, to produce a leprosy-specific neoglycoprotein, the so-called natural trisaccharide-octyl-bovine serum albumin (NT-O-BSA). Special features of the synthetic strategy were the use of silver trifluoromethanesulfonate (triflate) to promote glycosylation, resulting in the rhamnobiose in high yield and absolute stereospecificity. The terminal 3,6-di-O-methyl-D-glucopyranosyl group was introduced after O-deallylation of the rhamnobiose. Removal of protecting groups yielded the trisaccharide hapten suitable for coupling to carrier protein. Poly(acrylamide)-gel electrophoresis of the neoglycoprotein demonstrated its purity, and subsequent immunoblotting with a monoclonal antibody directed to the terminal 3,6-di-O-methyl-beta-D-glucopyranosyl epitope of the native glycolipid demonstrated its antigenicity. Comparative serological testing in enzyme-linked immunosorbent assays of NT-O-BSA, the corresponding disaccharide-containing products, and another trisaccharide-containing neoglycoprotein, O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-(1----4)-O-(2,3-di-O- methyl-alpha-L-rhamnopyranosyl)-(1----2)-(3-O-methyl-alpha-L-rhamnopy ran osyl)- (1----4')-oxy-(3-phenylpropanoyl)-BSA (NT-P-BSA) [Fujiwara et al., Agric. Biol. Chem., 51 (1987) 2539-2547] against sera from leprosy patients and control populations showed concordance; the presence of the innermost sugar did not contribute significantly to sensitivity or specificity. The di- and tri-saccharide-containing neoantigens, on account of ready availability and solubility, provide greater flexibility than the native glycolipid for the serodiagnosis of leprosy.     

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, from cellobiose, of a trisaccharide closely related to the GlcNAc----GlcA----GlcN segment of the antithrombin-binding sequence of heparin

O-(2-Deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)- O-(beta-D- glucopyranosyluronic acid)-(1----4)-1,6-anhydro-2-deoxy-2-sulfamido-6-O-sulfo-beta-D-gl ucopyranose pentasodium salt (14) was synthesized as a heparin-related oligosaccharide. The glycosyl acceptor (derived from cellobiose) and a glycosyl donor, 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide, were coupled in the presence of mercuric bromide and molecular sieves 4A to afford a 69% yield of fully protected trisaccharide, namely, 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)-3-O-acetyl- 1,6-anhydro-2 - azido-2-deoxy-beta-D-glucopyranose (10), which was converted into the partially sulfated trisaccharide 14. Compound 10 also underwent acetolysis to afford the glycosyl acetate, for further elongation of the glycosyl chain.     

Synthesis of p-nitrophenyl 6(5)-O-benzyl-alpha-maltopentaoside, a substrate for alpha amylases

p-Nitrophenyl alpha-maltopentaoside, having a benzyl group on O-6 of the terminal (nonreducing) D-glucosyl group was prepared by use of a reductive ring-opening reaction. Highly regioselective reduction of p-nitrophenyl O-(2,3-di-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl)-(1----4)- tris[O-(2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl)-(1----4)]-2,3,6-tri- O- benzoyl-alpha-D-glucopyranoside by dimethylamine-borane and p-toluenesulfonic acid, followed by debenzoylation, gave p-nitrophenyl O-(6-O-benzyl-alpha-D-glucopyranosyl)-(1----4)-tris[O-alpha-D-glucopyran osyl- (1----4)]-alpha-D-glucopyranoside. An experiment was done on the mode of action of human pancreatic and salivary alpha amylases on this derivative. The compound is suitable as a substrate for the assay of alpha amylase when used with glucoamylase and alpha-D-glucosidase as coupling enzymes.     

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.     

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.     

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.     

Carbohydrate epitope structural elucidation by 1H-NMR spectroscopy of a new Mycobacterium kansasii phenolic glycolipid antigen.

The complete primary structure of the carbohydrate moiety of a new phenolic glycolipid antigen namely PheGl K-IV from Mycobacterium kansasii was successfully established from only one- and two-dimensional 1H-NMR data. Among the scalar two-dimensional techniques, correlated spectroscopy with a 45 degree mixing pulse and phase-sensitive double-quantum-filtered correlated spectroscopy were selected, combined with two-dimensional dipolar techniques (nuclear Overhauser effect). These techniques using milligram of quantities native PheGl K-IV allowed the following monoacetylated tetrasaccharide to be proposed for its carbohydrate part: 4-O-Me-alpha-Manp-(1----3)-4-O-Ac-2-O-Me-alpha-Fucp-(1----3) -2-O-Me-alpha-Rhap- (1----3)-2,4-di-O-Me-alpha-Rhap. The PheGl K-IV shares, with the other phenolic glycolipids isolated from M. kansasii (K-I, K-II), a common core assigned to the lipid aglycone glycosylated by the monoacetylated trisaccharide part. It differs in the structure of the distal monosaccharide residue.     

Synthesis of a peripheral trisaccharide sequence of lutropin, a pituitary glycoprotein hormone; use of chitobiose as a key starting material

A chitobiose derivative, methyl O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-3,6 - di-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranoside, was derived from the corresponding N-acetyl derivative and this was converted into the glycosyl bromide (5). Glycosidation reaction between 5 and methyl 3,4,6-tri-O-benzyl-alpha-D-mannopyranoside in the presence of silver trifluoromethanesulfonate gave a beta-D-linked trisaccharide derivative. Replacement of the N,N-phthaloyl group by acetyl groups resulted in a product that was converted into methyl O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-O -(2- acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----2)-3,4,6- tri-O- benzyl-alpha-D-mannopyranoside (11) by use of a few reaction steps. The 4(3)-hydroxyl group of 11 was methanesulfonylated, and the product subjected to SN2 replacement with acetate anion, to give the D-galactosamine-containing trisaccharide derivative (12). After basic hydrolysis of 12, the 4(3)-hydroxyl group was sulfated, and all benzyl groups were removed by hydrogenolysis, giving methyl O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-(1----4)-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----2)-alpha-D-mannopyranosid e monosodium salt, the methyl alpha-glycoside derivative of the peripheral trisaccharide sequence of the pituitary glycoprotein hormone lutropin.     

Structural elucidation and antigenicity of a novel phenolic glycolipid antigen from Mycobacterium haemophilum

The structure of a novel antigenic glycolipid that distinguishes the opportunistic pathogen Mycobacterium haemophilum from all other mycobacteria was established by a series of degradation reactions leading to products that were analyzed by gas/liquid chromatography-mass spectrometry. The complete structure of the oligosaccharide unit was determined as 2,3-di-O-CH3-alpha-L-Rhap(1----2)3-O-CH3-alpha-L-Rhap(1----4 )-2,3-di-O-CH3-alpha-L-Rhap(1----. The lipid portion of the phenolic glycolipid was composed of two component phenolphthiocerols differing by two methylene groups, as determined by analysis of their per-O-trideuteriomethylated derivatives. The diol unit of the phenolphthiocerols has a threo relative configuration. The absolute stereochemistry of the asymmetric centers of the phenolphthiocerols is uncertain, but the centers are probably 3R, 4S, 9R, and 11R as found for phthiocerol A from Mycobacterium tuberculosis. The hydroxyl functions of the branched glycolic chain are esterified to a complex mixture of multi-methyl branched mycocerosic acids, C27, C30, C32, C34, and C37 with molecular weights (as methyl esters) of 424, 466, 494, 522, and 564, respectively. The stereochemistry of the methyl branches of the mycocerosates have R absolute configuration. The glycolipid is highly antigenic and appears to be specific for M. haemophilum. There are intriguing similarities between the product from M. haemophilum and the well-known phenolic glycolipid I of Mycobacterium leprae, a matter that is discussed.     

Structural characterization of a new anticoagulant fucan sulfate from the brown seaweed Ecklonia kurome.

Methylation analysis of a fucose-containing, sulfated polysaccharide (C-II), which was isolated from the brown seaweed Ecklonia kurome and has a potent anticoagulant activity, showed the presence of 3-O- and 3,4-O-disubstituted fucopyranosyl residues in addition to small proportions of nonreducing, terminal fucofuranosyl and fucopyranosyl groups, and 2,3-di-O- and 2,3,4-tri-O-substituted fucopyranosyl and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-II gave several neutral oligosaccharide fractions in small proportions and two high-molecular-weight acidic fractions in large proportions. Methylation analysis of the low-sulfated acidic fraction showed that the proportion of 3-O-linked fucosyl residues increases and that of 3,4-O-disubstituted decreased as compared to C-II. Methylation and g.l.c.-m.s. analysis of the neutral oligosaccharide fractions showed the presence of Fuc-(1----3)-Fuc and a fucosyl trisaccharide, in addition to small proportions of Gal-(1----4)-Fuc, Fuc-(1----2)-Fuc, Fuc-(1----4)-Fuc, Fuc-(1----2)-Gal, and Fuc----Gal----Fuc. Methylated C-II was also desulfated by methanolysis, followed by remethylation with (2H3)methyl iodide, and most of (2H3)methyl groups were linked to O-4 of the 3-O-linked fucosyl residues. These results suggested a highly branched, new type of fucan sulfate containing a backbone of (1----3)-linked L-fucosyl residues having sulfate groups mainly attached to C-4.     

Synthesis of p-nitrophenyl beta-glycosides of (1----6)-beta-D-galactopyranosyl-oligosaccharides

Sequential tritylation, benzoylation, and detritylation of p-nitrophenyl beta-D-galactopyranoside gave p-nitrophenyl 2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (2). Reaction of 2 with 2,3,4,6-tetra-O-benzoyl-alpha-D-galactopyranosyl bromide gave p-nitrophenyl O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----6) -2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (4) in 94% yield. Deprotection with sodium methoxide then gave the crystalline p-nitrophenyl O-(beta-D-galactopyranosyl)-(1----6)-beta-D-galactopyranoside (5). Condensation of 2 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide (3) readily yielded the protected disaccharide p-nitrophenyl O-(2,3,4-tri-O-benzoyl-6-O-bromoacetyl-beta-D-galactopyranosyl)-(1----6) -2,3,4-tri-O-benzoyl-beta-D-galactopyranoside (6) from which the bromoacetyl groups could be selectively removed. Condensation of the resulting material with tetra-O-benzoyl-alpha-D-galactopyranosyl bromide then yielded p-nitrophenyl O-(2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl)-(1----6)-O-(2,3,4 -tri-O-benzoyl-beta-D-galactopyranosyl)-(1----6)-2,3,4-tri-O-benzoyl-bet a-D -galactopyranoside, (8), which was converted into the crystalline trisaccharide p-nitrophenyl O-(beta-D-galactopyranosyl)-(1----6)-O-beta-D-galactopyranosyl)-(1----6) -beta -D-galactopyranoside (9) by treatment with sodium methoxide. Preliminary experiments on the interaction of p-(bromoacetamido)phenyl and p-isothiocyanatophenyl glycoside derivatives of some of these galacto-saccharides with monoclonal anti-(1----6)-beta-D-galactopyranan antibodies have been conducted.     

Linkage of phenolic acids to cell-wall polysaccharides of bamboo shoot

Hydrolysis of bamboo shoot cell walls with Driselase (a fungal enzyme preparation) gave xyloglucan and arabinoxylan oligosaccharides containing ferulic and p-coumaric acids, respectively. The structures of two oligosaccharides containing phenolic acids are here determined to be O-(4-O-trans-feruloyl-alpha-D-xylopyranosyl)-(1----6)-D-glucopy rano se and O-[5-O-(trans-p-coumaroyl(-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D-xylopyranosyl-(1----4)-D-xylopyranose, on the basis of n.m.r. spectroscopy, methylation analysis, and f.a.b.-m.s. The possible role of phenolic acid substituents in cell-wall architecture is discussed.     

A novel mannose containing phenolic glycolipid from Mycobacterium kansasii

Using high-performance liquid chromatography, a new kind of phenolic glycolipid quantitatively minor, called phenolic glycolipid-II, was isolated from a lipidic fraction of Mycobacterium kansasii. The structure was determined by fast atom bombardment-mass spectrometry and proton nuclear magnetic resonance spectroscopy, as: 2,4-di-O-Me-alpha-D-Manp(1----3) 4-O-Ac-2-O-Me-alpha-L-Fucp(1----3)2-O-Me- alpha-L-Rhap(1----3) 2,4-di-O-Me-alpha-L-Rhap 1----phenolphthiocerol dimycocerosate. Phenolic glycolipids I and II differ only by their distal monosaccharide hapten which is 2,6-dideoxy-4-O-Me-alpha-D-arabinohexopyranosyl and the 2,4-di-O-Me-alpha-D-mannopyranosyl, respectively. This sugar appears to be characteristic and apparently unique in the Mycobacterium genus. Moreover, phenolic glycolipids I and II constitute with the lipooligosaccharides two classes of antigens of M. kansasii.     

Diglycosyl phenol phthiocerol diester of Mycobacterium leprae

A diglycosyl phenol phthiocerol diester that had not been previously detected was isolated from M. leprae-infected armadillo tissues. Spectroscopy methods allowed the elucidation of its structure. The diglycoside was a 2,3-di-O-methylrhamnopyranosyl (alpha 1----2)3-O-methylrhamnopyranosyl (alpha 1-linked to the phenolic hydroxyl of phthiocerol dimycocerosates). It differs from the major phenolic glycolipid (PGL I) only by the absence of the terminal 3,6-di-O-methylglucopyranosyl unit. The diglycoside could be an intermediate in the synthesis of the latter antigen or a degradative product in the host detoxification process.     

Acetylation at O-2 of arabinofuranose residues in feruloylated arabinoxylan from bamboo shoot cell-walls.

Hydrolysis of bamboo shoot cell-walls with Driselase (a fungal enzyme preparation) gave an arabinoxylan trisaccharide with ferulic and acetic acids as ester groups. The structure of this oligosaccharide was determined to be O-[2-O-acetyl-5-O-[E)-feruloyl)-alpha-L-arabinofuranosyl]-(1----3) -O-beta- D-xylopyranosyl-(1----4)-D-xylopyranose, on the basis of spectroscopy and methylation analysis.     

Synthesis of trisaccharides containing 3-deoxy-D-manno-2-octulosonic acid residues related to the KDO-region of enterobacterial lipopolysaccharides

Glycosylation of methyl (allyl 7,8-O-carbonyl-3-deoxy-alpha-D-manno-2-octulo-pyranosid)o nate with an alpha-(2----4) linked per-O-acetylated KDO-disaccharide bromide derivative under Helferich conditions afforded a 2:1 mixture of the alpha- and beta-linked trisaccharide derivatives in 50% yield. Removal of the protecting groups gave sodium O-[sodium (3-deoxy-alpha-D-manno-2-octulopyranosyl)onate]-(2----4)-O-[ sodium (3-deoxy-alpha- and -beta-D-manno-2-octulopyranosyl)onate]-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate. Radical copolymerization of the allyl glycosides afforded artificial antigens, suitable for defining antibody specificities directed against the KDO-region of enterobacterial lipopolysaccharides.     

Globo-A--a new receptor specificity for attaching Escherichia coli

Uropathogenic Escherichia coli strains designated as ONAP, based on their O negative A positive agglutination of human P1 erythrocytes, were shown to prefer the globo-A glycolipid as a receptor structure. The dependence on both the A terminal and the globoseries chain was confirmed by agglutination of human AP1, but not Ap or OP1 erythrocytes and by binding to the globo-A glycolipid on TLC plates. Neither Gal alpha 1----4Gal beta nor the A trisaccharide GalNAc alpha 1----3(Fuc alpha 1----2)Gal beta alone functioned as receptors. The bacteria thus appeared to recognize an epitope resulting from the combination of the terminal and internal structures.     

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.