Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

The Shigella flexneri O-antigenic polysaccharide chain. Nature of the biological repeating unit

The sequence of monosaccharides in the biological repeating tetrasaccharide unit of Shigella flexneri variant Y O-antigenic polysaccharide chain was determined by subjecting three oligosaccharides of the polysaccharide, obtained by phage-Sf6-mediated enzymatic hydrolysis, to methylation analysis and proton nuclear magnetic resonance spectroscopy. The smallest saccharide was shown to be a tetrasaccharide with the structure alpha-L-Rhap-(1-2)-L-Rha. The next saccharide, an octasaccharide, was shown to be a dimer of the tetrasaccharide with the L-Rha residues linked alpha 1.3. The longest saccharide was shown to be a decasaccharide with the following structure: alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-beta-D-GlcpNAc-(1-2)-alpha-L-Rhap-(1-2)-alpha-L-Rhap++ + +-(1-3)-alpha-L-Rhap-(1-3)-beta-D-GlcpNAc-(1-2)-alpha-L-R hap-(1-2)-L-Rha. Thus the decasaccharide differed from the octasaccharide and tetrasaccharide by having the alpha-L-Rhap-(1-2)-L-Rhap disaccharide added in the terminal non-reducing end of the saccharide chain. This shows that the alpha-L-Rhap-(1-2)-alpha-L-Rhap-(1-3)-alpha-L-Rhap-(1- 3)-D-GlcpNAc tetrasaccharide is the biological repeating unit of the O chain and that the repeating units are joined through a beta-D-GlcpNAc-(1-2)-L-Rhap linkage. Inhibition experiments utilizing the enzyme-linked immunosorbent assay (ELISA) with S. flexneri Y lipopolysaccharide/S. flexneri Y rabbit antiserum showed that the decasaccharide was the best inhibitor (threefold as active as the octasaccharide and sixtyfold as active as the tetrasaccharide); this supports the postulated structure of the biological repeating unit.     

Molecular recognition of oligosaccharide epitopes by a monoclonal Fab specific for Shigella flexneri Y lipopolysaccharide: X-ray structures and thermodynamics

The antigenic recognition of Shigella flexneri O-polysaccharide, which consists of a repeating unit ABCD [-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-beta-D-GlcpNAc-(1-->], by the monoclonal antibody SYA/J6 (IgG3, kappa) has been investigated by crystallographic analysis of the Fab domain and its two complexes with two antigen segments (a pentasaccharide Rha A-Rha B-Rha C-GlcNAc D-Rha A' and a modified trisaccharide Rha B-Rha C-GlcNAc D in which Rha C* is missing a C2-OH group). These complex structures, the first for a Fab specific for a periodic linear heteropolysaccharide, reveal a binding site groove (between the V(H) and V(L) domains) that makes polar and nonpolar contacts with all the sugar residues of the pentasaccharide. Both main-chain and side-chain atoms of the Fab are used in ligand binding. The charged side chain of Glu H50 of CDR H2 forms crucial hydrogen bonds to GlcNAc of the oligosaccharides. The modified trisaccharide is more buried and fits more snugly than the pentasaccharide. It also makes as many contacts (approximately 75) with the Fab as the pentasaccharide, including the same number of hydrogen bonds (eight, with four being identical). It is further engaged in more hydrophobic interactions than the pentasaccharide. These three features favorable to trisaccharide binding are consistent with the observation of a tighter complex with the trisaccharide than the pentasaccharide. Thermodynamic data demonstrate that the native tri- to pentasaccharides have free energies of binding in the range of 6.8-7.4 kcal mol(-1), and all but one of the hydrogen bonds to individual hydroxyl groups provide no more than approximately 0.7 kcal mol(-1). They further indicate that hydrophobic interactions make significant contributions to binding and, as the native epitope becomes larger across the tri-, tetra-, pentasaccharide series, entropy contributions to the free energy become dominant.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins.

Sugar-beet pulp was de-esterified and submitted to 72 h hydrolysis by 0.1 M HCl at 80 degrees C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n(>/= 2) repeats of the dimer -->4)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1--> were isolated from the hydrolysate by ion-exchange and gel-permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O-3 of a galacturonic acid (GalA) residue, as shown for the two pentamers beta-D-GlcpA-(1-->3)-alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-alpha-D-GalpA-(1-->2)-L-Rhap and alpha-D-GalpA-(1-->2)-alpha-L-Rhap-(1-->4)-[beta-D-GlcpA-(1-->3)]-alpha-D-GalpA-(1-->2)-L-Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

A concise synthesis of two isomeric pentasaccharides, the O repeat units from the lipopolysaccharides of P. syringae pv. porri NCPPB 3364T and NCPPB 3365

A concise synthesis of two isomeric pentasaccharides, alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap (A) and alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-GlcpNAc-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap (B), the O repeats from the lipopolysaccharides of Pseudonomonas syringae pv. porri NCPPB 3364T and 3365 was achieved via assembly of the building blocks, allyl 3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (1), 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (2), allyl 4-O-benzoyl-3-O-chloroacetyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (10). Coupling of 1 with 2 followed by deallylation and trichloroacetimidate formation gave the disaccharide donor 5, while condensation of 6 with 7, followed by dechloroacetylation, offered the disaccharide acceptor 9. Then, 5 was coupled with 10 to obtain the trisaccharide 11, and subsequent deallylation and trichloroacetimidate formation furnished the trisaccharide donor 13. Coupling of 9 with 13, followed by deprotection, afforded pentasaccharide 19, while condensation of 9 with 5, followed by deallylation and trichloroacetimidate formation, gave the tetrasaccharide donor 16, whose coupling with 10 and subsequent deprotection yielded another pentasaccharide 22.     

Facile synthesis of a pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C

A pentasaccharide mimic of a fragment of the capsular polysaccharide of Streptococcus pneumoniae type 15C beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[alpha-D-Galp-(1-->2)-beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->OCH2CH2N3) (1) was synthesized in a regio- and stereoselective manner. The 2-azidoethyl-spacered pentasaccharide mimic 1 can be used to construct a neoglycoconjugate antigen.     

Synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap,the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942

An efficient synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942 and its isomer beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->4)-[beta-L-Xylp-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap was achieved via sequential assembly of the building blocks, allyl 2,3-O-isopropylidene-alpha-L-rhamnopyranoside (2), allyl 3,4-O-isopropylidene-alpha-L-rhamnopyranoside (3), allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and 2,3,4-tri-O-benzoyl-beta-L-xylopyranosyl trichloroacetimidate (12). The process was carried out in a regio- and stereoselective manner using glycosyl trichloroacetimidates as donors and unprotected or partially protected rhamnopyranosides as acceptors in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf).     

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.     

Analysis of the interaction between lectins and tetra- and tri-saccharide mimics of the Sd(a) determinant by surface plasmon resonance detection

The binding properties of a spacer-linked synthetic Sd(a) tetrasaccharide beta-D-GalpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (1), two tetrasaccharide mimics beta-D-Galp-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (2) and beta-D-GlcpNAc-(1-->4)-alpha-Neu5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (3), and two trisaccharide mimics beta-D-GalpNAc-(1-->4)-3-O-(SO(3)H)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (4) and beta-D-GalpNAc-(1-->4)-3-O-(CH(2)COOH)-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->O)-(CH(2))(5)-NH(2) (5) with lectins from Dolichos biflorus (DBL), Maackia amurensis (MAL), Phaseolus limensis (PLL), Ptilota plumosa (PPL), Ricinus communis 120 (RCL120) and Triticum vulgaris (wheat germ agglutinin, WGA) have been investigated by surface plasmon resonance (SPR) detection. MAL, PPL, RCL120 and WGA did not display any binding activity with compounds 1-5. However, DBL and PLL, both exhibiting GalNAc-specificity, showed strong binding activity with compounds 1, 4 and 5, and 1, 3, 4 and 5, respectively. The results demonstrate that SPR is a very useful analysis system for identifying biologically relevant oligosaccharide mimics of the Sd(a) determinant.     

Conformational analysis of two xylose-containing N-glycans in aqueous solution by using 1H NMR ROESY and NOESY spectroscopy in combination with MD simulations

The conformational behavior of the synthetic hexa- and heptasaccharide methyl beta-glycosides alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-beta-D-GlcpNAc-(1 --> OMe and alpha-D-Manp-(1 --> 6)-[alpha-D-Manp-(1 --> 3)-][beta-D-Xylp-(1 --> 2)-]beta-D-Manp-(1 --> 4)-beta-D-GlcpNAc-(1 --> 4)-[alpha-L-Fucp-(1 --> 6)-]beta-D-GlcpNAc-(1 --> OMe, representing the xylosylated and the xylosylated alpha-(1 --> 6)-fucosylated core structures of N-glycans in alpha(D)-hemocyanin of the snail Helix pomatia, respectively, were investigated by 1H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. 1H and 13C chemical shifts of the oligosaccharides were assigned using 1H-(1)H COSY, TOCSY, and NOESY, and 1H-(13)C HMQC techniques. Experimental 2D 1H cross-peak intensities from one series of NOESY and one series of ROESY experiments of the two oligosaccharides were compared with calculated values derived from MD trajectories using the CROSREL program, yielding information about the conformation of each glycosidic linkage of the methyl glycosides. The flexibility of the linkages was described by generalized order parameters and internal rotation correlation times. Analysis of the data indicated that several conformations are likely to exist for the alpha-D-Man-(1 --> 6)-beta-D-Man, the alpha-L-Fuc-(1 --> 6)-beta-D-GlcNAc, and the alpha-D-Man-(1 --> 3)-beta-D-Man linkage, whereas the beta-D-Xyl-(1 --> 2)-beta-D-Man-(1 --> 4)-beta-D-GlcNAc-(1 --> 4)-beta-D-GlcNAc fragment occurs in one rigid conformation. No significant differences were found between the corresponding structural elements in both methyl glycosides. NOESY and ROESY experiments proved to be suitable for providing the experimental data required, however, due to more overlap within the ROESY spectra, reducing the accuracy of the analysis, NOESY spectral analysis is preferred.     

N-acetylmuramic acid as capping element of alpha-D-fucose-containing S-layer glycoprotein glycans from Geobacillus tepidamans GS5-97T

Geobacillus tepidamans GS5-97(T) is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Fucp-(1-->](n=approximately 20), with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an alpha-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a beta-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core -->2)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1--> to carbon 3 of beta-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97(T).     

The surface layer (S-layer) glycoprotein of Geobacillus stearothermophilus NRS 2004/3a. Analysis of its glycosylation.

Geobacillus stearothermophilus NRS 2004/3a possesses an oblique surface layer (S-layer) composed of glycoprotein subunits as the outermost component of its cell wall. In addition to the elucidation of the complete S-layer glycan primary structure and the determination of the glycosylation sites, the structural gene sgsE encoding the S-layer protein was isolated by polymerase chain reaction-based techniques. The open reading frame codes for a protein of 903 amino acids, including a leader sequence of 30 amino acids. The mature S-layer protein has a calculated molecular mass of 93,684 Da and an isoelectric point of 6.1. Glycosylation of SgsE was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [-->2)-alpha-L-Rhap-(1-->3)-beta-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->](n = 13-18), with a 2-O-methyl group capping the terminal trisaccharide repeating unit at the non-reducing end of the glycan chains. The glycan chains are bound via the disaccharide core -->3)-alpha-l-Rhap-(1-->3)-alpha-L-Rhap-(L--> and the linkage glycose beta-D-Galp in O-glycosidic linkages to the S-layer protein SgsE at positions threonine 620 and serine 794. This S-layer glycoprotein contains novel linkage regions and is the first one among eubacteria whose glycosylation sites have been characterized.     

Structure of the O-specific polysaccharide of Proteus vulgaris O45 containing 3-acetamido-3,6-dideoxy-D-galactose

An O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O45 and studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H,13C HSQC and HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established:-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-D-GalpNAc-(1-->4)-alpha-D-GalpA-(1-->3)-beta-D-GlcpNAc-(1-->2)-beta-D-Fucp3NAc4Ac-(1-->where Fuc3NAc4Ac is 3-acetamido-4-O-acetyl-3,6-dideoxygalactose. A cross-reactivity of anti-P. vulgaris O45 serum was observed with several other Proteus lipopolysaccharides, which contains Fuc3N derivatives.     

Efficient synthesis of a heptasaccharide,the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642

alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-Xylp-(1-->2)-][beta-D-Xylp-(1-->4)-]alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642 was synthesized as its methyl glycoside via 3-O-selective glycosylation of methyl alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (9) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (8), followed by dixylosylation with 2,3,4-tri-O-benzoyl-alpha,beta-D-xylopyranosyl trichloroacetimidate (12) and subsequent deacylation.     

Primary structure of four human milk octa-, nona-, and undeca-saccharides established by 1H- and 13C-nuclear magnetic resonance spectroscopy.

The structures of two octasaccharides, one nonasaccharide, and one undecasaccharide, isolated from human milk, have been investigated by 1H- and 13C-nuclear magnetic resonance spectroscopy. The structures of these oligosaccharides are: beta-D-Galp-(1----4)-[alpha-L-Fucp- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-[alpha-L-Fucp+ ++- (1----3)]-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-D-Glc; beta-D-GALp-(1----3)-[alpha-L-Fucp-(1----4)]-beta-D-GlcpNAc-(1---- 3)-beta-D - Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----3)-beta -D-Galp- (1----4)-D-Glc; beta-D-Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1---- 6)-(alpha - L-Fucp-(1----2)-beta-D-Gal-(1----3)-[alpha-L-Fucp-(1----4)]- beta-D-GlcpNAc- (1----3))-beta-D-Galp-(1----4)-D-Glc; and alpha-L-Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3) -beta-D- Galp-(1----4)-[alpha-L-Fucp-(1----3)]-beta-D-GlcpNAc-(1----6)-[alp ha-L- Fucp-(1----2)-beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)]-beta-D -Galp- (1----4)-D-Glc. The two octasaccharides have been previously isolated from human milk as a mixture, and in a pure form from new-born feces, but the n.m.r. data were not provided. These two octasaccharides display the di-Lewis X and the composite Lewis A-Lewis X antigenic determinant, previously described as neo-antigens of adenocarcinoma cell lines.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Structural studies of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide

The structure of the O-specific side-chains of the Escherichia coli O2 lipopolysaccharide has been investigated, different 1H- and 13C-n.m.r. techniques being the main methods used. It is concluded that they are composed of pentasaccharide repeating-units having the following structure, in which D-Fuc3NAc is 3-acetamido-3,6-dideoxy-D-galactose. ----4)-beta-D-GlcpNAc-(1----3)-alpha-L-Rhap-(1----2)-alpha-L-Rh ap-(1----3)-beta-L-Rhap-(1----2 increases 1 alpha-D-Fucp3NAc.