Structure of the O-antigen of Francisella tularensis strain 15.

The O-specific polysaccharide, obtained by mild acid degradation of the lipopolysaccharide of Francisella tularensis strain 15, contained 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc), 4,6-dideoxy-4-formamido-D-glucose (D-Qui4NFm), and 2-acetamido-2-deoxy-D-galacturonamide (D-GalNAcAN) in the ratios 1:1:2. Tri- and tetra-saccharide fragments were obtained on treatment of the polysaccharide with anhydrous hydrogen fluoride and partial hydrolysis with 0.1 M hydrochloric acid, respectively. On the basis of 1H- and 13C-n.m.r. spectroscopy of the polysaccharide and the saccharides, it was concluded that the O-antigen had the structure: ----4)-alpha-D-GalpNAcAN-(1----4)-alpha-D-GalpNAcAN-(1----3) -beta-D-QuipNAc-(1----2)-beta-D-Quip4NFm-(1----. This O-antigen is related in structure to those of Pseudomonas aeruginosa O6, immunotype 1, and IID 1008, and Shigella dysenteriae type 7.     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chains of P. aeruginosa O11 (Lányi) lipopolysaccharides

Lipopolysaccharides were isolated from the phenol layer on aqueous phenol extraction of cells of Pseudomonas aeruginosa O11 (Lányi classification), strains 170021 and 170040. On mild acid degradation of the lipopolysaccharides, with the subsequent gel-filtration on Sephadex G-50, neutral O-specific polysaccharides made up of 6-deoxysugars alone were obtained. Two 2-acetamido-2,6-dideoxy-L-galactose (LFucNAc), 2-acetamido-2,6-dideoxy-D-glucose (DQuiNAc) and L-rhamnose (LRha) residues were found to be the components of the strain 170021 polysaccharide repeating units; those of strain 170040 contained the same monosaccharides, but, instead of 2-acetamido-2,6-dideoxy-D-glucose residue, that of 2-acetamido-2,6-dideoxy-D-galactose (DFucNAc) was present. On the basis of the 13C nuclear magnetic resonance data, methylation analysis and three successive Smith degradations the following structures were determined for the polysaccharide repeating units: strain 170021----2) LRha(alpha 1----3)LFucNAc(alpha 1----3)LFucNAc(alpha 1----3)DQuiNAc(beta 1----; strain 170040,----2)LRha(alpha 1----3)LFucNAc-(alpha 1----3)LFucNAc(alpha 1----3)DFucNAc(beta 1----; differing from one another by configuration of C-4 of 2-acetamido-2,6-dideoxy-D-hexopyranose only.     

Antigenic polysaccharides of bacteria. 17. The structure of O-specific polysaccharide chain of Pseudomonas aeruginosa X (Meitert) lipopolysaccharide

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of P. aeruginosa X (Meitert classification) lipopolysaccharide. On the basis of non-destructive analis using 1H, 13C NMR spectroscopy and Klyne's rule calculation, as well as chemical methods (acid hydrolysis, methylation, Smith degradation), it was established that the polysaccharide is built up of disaccharide repeating units of the following structure: ----4)-alpha-L-Rha-(1----3)-beta-D-ManNAc-(1----.     

Structure of the O-specific polysaccharide of Vibrio fluvialis serovar 3]

O-Specific polysaccharide composed of L-rhamnose and 2-acetamido-2-deoxy-D-mannose was obtained on mild acid degradation of the V. fluvialis lipopolysaccharide. On the basis of the 13C-NMR data and methylation studies, the following structure was suggested for the polysaccharide repeating unit: ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1---- This structure was confirmed by calculations using known glycosidation effects on 13C chemical shifts.     

Synthesis of a branched pentasaccharide sequence of "bisected" structure of N-glycoproteins

For the synthesis of the threefold-branched pentasaccharide, O-alpha-D-mannopyranosyl-(1----3)-O-[(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1----4)]-O-[alpha-D-mannopyranosyl-(1----6)]-O-beta-D- mannopyranosyl-(1----4)-2-acetamido-2-deoxy-D-glucopyranose (20), which is a part of the structure of the N-glycoproteins, the disaccharide 4-O-(4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl) -1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranose was synthesized as a key compound by use of the silver silicate-catalyst procedure. After elimination of the 4-O-acetyl group, a 2-acetamido-2-deoxy-beta-D-glucopyranosyl group was attached according to the phthalimido method. Further elimination of the allyl groups allowed the linkage of two alpha-D-mannopyranosyl groups in the presence of mercury salt. A deblocking sequence consisting of four steps gave 20.     

Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype A1) organisms

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A1 organisms was purified and characterized by chemical analysis and NMR spectroscopy. The polymer has the structure----3)-O-(2-acetamido-2-deoxy-4-O-acetyl-beta-D-mannopyranos yluronic acid)-(1----4)-O-(2-acetamido-2-deoxy-beta-D-mannopyranose)-(1----. The polysaccharide was immunogenic (able to evoke production of antibodies) for sheep but not for rabbits. Immuno electron-microscopy studies using the Protein A-gold technique showed the polysaccharide to be peripherally located on the bacterial surface. Reduction, oxidation and de-O-acetylation of the polymer did not appear to alter its immunological precipitability with specific antiserum, but all three treatments destroyed its ability to adhere to sheep erythrocytes at neutral pH. De-N-acetylation of the polymer destroyed both immunological precipitability and erythrocyte adherence.     

A facile synthesis of nitrophenyl oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyrano syl unit at their nonreducing end.

A facile approach towards the synthesis of 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyra nos ide, 2-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-2-acetamido-2-deoxy-alpha-D-galactopyranoside, 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-alpha-D-mannopyranoside, and 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)-(1----6)-beta-D-galactopyranoside has been accomplished through the development and use of methyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-1-thio-beta-L-fucopyranoside as the glycosyl donor.     

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.     

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.     

Total synthesis of sialosylcerebroside, GM4

Described are total syntheses of O-[sodium (5-acetamido-3,5-dideoxy-D -glycero-alpha-D-galacto-2-nonulopyranosyl)onate]-(2----3)-O -beta-D -galactopyranosyl-(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingen ine,O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl+ ++)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine, O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-beta-D-gal act opyranosyl -(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingenine, and O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2-nonulopyranosyl++ +)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D -galacto-2-nonulopyranosyl)onate]-(2----3)-2,3,4,6-tetra-O-a cetyl-D -galactopyrano-syl trichloroacetimidate and O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-2,4,6-tri-O-ace tyl-D-galactopyranosyl trichloroacetimidate as key glycosyl donors, and (2S,3R,4E)-3 -O-benzoyl-2-N-tetracosanoylsphingenine as a key glycosyl acceptor.     

Antigenic polysaccharides of bacteria of the genus Shigella. Determination of the structure of polysaccharide chains of Shigella boydii type 9 lipopolysaccharide and detection of unusually high molecular weight glycolipid

Specific acidic polysaccharide has been isolated from the Shigella boydii type 9 antigenic lipopolysaccharide after mild hydrolysis followed by chromatography on Sephadex G-50. The polysaccharide consists of D-glucose, D-glucuronic acid, 2-acetamido-2-deoxy-D-glucose, and L-rhamnose. From the results of methylation analysis, partial acid hydrolysis and 13C NMR data the structure of the repeating unit of the polysaccharide was deduced as follows: [----4)DGlcp(alpha 1----4)DGlcAp(beta 1----3)DGlcNAcp(alpha 1----3)LRhap(alpha 1----]n. The lipopolysaccharide from Sh. boydii 9 was fractionated by gel chromatography on the Sephadex G-200 column in a buffer containing sodium deoxycholate into three fractions. PAGE-SDS of the fractions obtained, 13C NMR- and chromato-mass-spectrometry data indicated that the three fractions contained the O-specific polysaccharide as the only carbohydrate component. The substance from the most high-molecular weight fraction contained unusually long O-specific chains (60,000 dalton). In the fat acid composition this fraction differed from other lipopolysaccharides by absence of beta-hydroxymyristic acid.     

Antigenic bacterial polysaccharides. 23. The structure of the O-specific polysaccharide chain of Salmonella arizonae 059 lipopolysaccharide

The O-specific polysaccharide of Salmonella arizonae O59 (Arizona 19) is composed of D-galactose, N-acetyl-D-glucosamine, and N-acetyl-L-fucosamine (FucNAc, 2-acetamido-2,6-dideoxy-L-galactose) in the ratio 1:1:1. The computerized calculation of the 13C NMR spectrum of the polysaccharide, based on the monosaccharide composition, spectra of the free monosaccharides and glycosydation effects, together with the chemical analysis (methylation and Smith degradation) showed that the polysaccharide is built up of trisaccharide repeating units of the following structure: ----3)-alpha-L-FucNAcp(1----3)-beta-D-GlcNAcp-(1----2)-beta- D-Galp-1(----. The molecular basis of serological interrelations between S. arizonae O59 and Pseudomonas aeruginosa O7 (Lányi) is discussed.     

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.     

Structural elucidation of two capsular polysaccharides from one strain of Bacteroides fragilis using high-resolution NMR spectroscopy.

The capsule of Bacteroides fragilis is unusual in that it consists of two distinct capsular polysaccharides. Using a combination of high-resolution NMR spectroscopy, theoretical calculations, and as few chemical procedures as required, the structure of both polysaccharide antigens (polysaccharides A and B) was elucidated. Using the above procedures, it was possible to obtain the complete structures using minimal quantities of polysaccharides A and B (8 and 5 mg, respectively). Only small amounts of each subjected to chemical analysis were not recoverable. Polysaccharide A is composed of the following repeating unit: [----3)alpha-D-AATp(1----4)[beta-D-Galf(1----3)]alpha-D- GalpNAc(1----3)beta-D-Galp(1----], where AAT is 2-acetamido-4-amino-2,4,6-trideoxygalactose. A pyruvate substituent having the R configuration spans O-4 and O-6 of the beta-D-galactopyranosyl residue. Polysaccharide B is composed of the following repeating unit: [----4)alpha-L-QuipNAc(1----3)beta-D-QuipNAc(1----4)[alpha-L - Fucp(1----2)beta-D-GalpA(1----3)beta-D-GlcpNAc(1----3)]alpha -D-Galp(1----]. A 2-aminoethylphosphonate substituent is situated on O-4 of the N-acetyl-beta-D-glucopyranosyl residue.     

An efficient synthesis of ganglioside GM3: highly stereocontrolled glycosylations by use of auxiliaries

An efficiently stereocontrolled total synthesis of GM3 alpha-D-Neup5Ac-(2----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----1) -Cer was achieved by employing both methyl 5-acetamido-4,7,8,9-tetra-O-benzyl-2-bromo-2,3,5-trideoxy-3- phenylthio-D-erythro-beta-L-gluco-2-nonulopyranosonate for the key sialylation step, and O-[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O- acetyl-beta-D-galactopyranosyl-(1----4)-3,6-di-O-acetyl-2-O-pivaloyl- alpha-D-glucopyranosyl trichloroacetimidate and fluoride for the key coupling step with a ceramide derivative. These two steps were significantly altered and improved in comparison with our previous synthesis that had been executed without use of stereocontrolling auxiliaries. GM3 was obtained in 4.5% overall yield in 19 steps starting from allyl O-(2,6-di-O-acetyl-3,4-O-isopropylidene-beta-D-galactopyranosyl)-(1----4 )-2,3,6-tri-O-acetyl-beta-D-glucopyranoside.     

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.     

Structural determination of the capsular polysaccharide of Neisseria meningitidis group I: a two-dimensional NMR analysis

The capsular polysaccharide antigen of Neisseria meningitidis group I was isolated by Cetavlon precipitation and purified by ion-exchange chromatography. The structure of the I polysaccharide was determined largely by comprehensive proton and carbon-13 nuclear magnetic resonance studies in which both one-dimensional and two-dimensional experiments were carried out directly on the I polysaccharide. The I polysaccharide is composed of the repeating unit----4)alpha-L-GulpNAcA(1----3)[4-OAc]beta-D-ManpNA-cA(-- --in which the former residue adopts the 4C1 (L) conformation and the latter residue adopts the 4C1 (D) conformation. The one-bond coupling between the anomeric carbon and proton (1J13C,H) of the 2-acetamido-2-deoxy-beta-D-mannuronopyranosyl residue is not consistent with its beta-D configuration. This anomalous value of 1J13C,H for this residue is due to through-space anisotropy effects on its anomeric proton, generated by the proximity of the carboxyl group of the neighboring 2-acetamido-2-deoxy-alpha-L-guluronopyranosyl residue. The O-acetyl substituents of the I polysaccharide are essential for its antigenicity to group I polysaccharide-specific antibodies.     

Purification and determination of the structure of capsular polysaccharide of Vibrio vulnificus M06-24.

Virulence of Vibrio vulnificus has been strongly associated with encapsulation and an opaque colony morphology. Capsular polysaccharide was purified from a whole-cell, phosphate-buffered saline-extracted preparation of the opaque, virulent phase of V. vulnificus M06-24 (M06-24/O) by dialysis, centrifugation, enzymatic digestion, and phenol-chloroform extraction. Nuclear magnetic resonance spectroscopic analysis of the purified polysaccharide showed that the polymer was composed of a repeating structure with four sugar residues per repeating subunit: three residues of 2-acetamido-2,6-dideoxyhexopyranose in the alpha-gluco configuration (QuiNAc) and an additional residue of 2-acetamido hexouronate in the alpha-galactopyranose configuration (GalNAcA). The complete carbohydrate structure of the polysaccharide was determined by heteronuclear nuclear magnetic resonance spectroscopy and by high-performance anion-exchange chromatography. The 1H and 13C nuclear magnetic resonance spectra were completely assigned, and vicinal coupling relationships were used to establish the stereochemistry of each sugar residue, its anomeric configuration, and the positions of the glycosidic linkages. The complete structure is: [----3) QuipNAc alpha-(1----3)-GalpNAcA alpha-(1----3)-QuipNAc alpha-(1----]n QuipNAc alpha-(1----4)-increases The polysaccharide was produced by a translucent phase variant of M06-24 (M06-24/T) but not by a translucent, acapsular transposon mutant (CVD752). Antibodies to the polysaccharide were demonstrable in serum from rabbits inoculated with M06-24/O.     

Structural elucidation of the O-specific polysaccharide of the phenol-phase soluble lipopolysaccharide of Vibrio anguillarum

The structure of the O-specific polysaccharide of the phenol-soluble cellular lipopolysaccharide of Vibrio anguillarum has been investigated. The studies involved the use of methylation analysis, partial hydrolysis with 48% hydrogen fluoride, Smith degradation, oxidation with chromium trioxide, and comprehensive proton and carbon-13 nuclear magnetic resonance studies, in which one- and two-dimensional experiments were carried out. As a result of these studies it is proposed that the O-specific polysaccharide of Vibrio anguillarum is composed of a regular heteropolymer, i.e., a main chain of (1----4)-linked 3-acetamido-3,6-dideoxy-beta-L-glucose residues alternately substituted through O-2 with side chain residues of 2-acetamido-2,6-dideoxy-alpha-D-glucose, which seem to be substituted through either O-3 or O-4 with propionyl groups (R), as in the following structure. (Formula: see text)     

A regio- and stereo-controlled synthesis of beta-D-Glcp NAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D-GlcpNAc 6SO3- (1----3)-D-Galp, a linear acidic glycan fragment of keratan sulfate I

A stereocontrolled synthesis of beta-D-GlcpNAc6SO3-(1----3)-beta-D-Galp6SO3-(1----4)-beta-D- GlcpNAc6SO3- (1----3)-D-Galp, was achieved by use of benzyl O-(2-acetamido-3,4 di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-beta-D- glucopyranosyl)-(1----3)-O-(2,4-di-O-tert-butyldiphenylsilyl-beta- D- galactopyranosyl-(1----4)-O-(2-acetamido-3-O-benzyl-2-deoxy-6-O-p-methox yphenyl - beta-D-glucopyranosyl)-(1----3)-2,4,6-tri-O-benzyl-beta-D-galactopyranos ide as a key intermediate, which was in turn prepared by employing two glycosyl donors, 3,4-di-O-benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D- glucopyranosyl trichloroacetimidate and O-(3,6-di-O-acetyl-2,4-di-O-benzyl-beta-D-galactopyranosyl)-(1----4)-3-O - benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate, and a glycosyl acceptor, benzyl 2,4,6-tri-O-benzyl-beta-D-galactopyranoside.