Rudolf Christian

Artificial antigens. Synthesis of polyacrylamide copolymers containing 3-deoxy-d-manno-2-octulopyranosylonic acid (KDO) residues

Starting from an anomeric mixture of methyl (allyl 4,5,7,8-tetra-O-acetyl-3-deoxy-alpha- and -beta-D-manno-2-octulopyranosid)onates, the glycosides sodium (allyl 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosid)onate, sodium O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-[allyl 3-deoxy-alpha-D-manno-2-octulopyranosid]onate and sodium (allyl 3-deoxy-7-O-beta-D-ribofuranosyl-beta-D-manno-2-octulopyranosid)++ +onate were prepared in several steps. Radical copolymerization of the allyl glycosides with acrylamide afforded linear macromolecular antigens containing mono- and di-saccharide residues corresponding to the KDO-region of Salmonella minnesota rough-form lipopolysaccharide and to partial structures of the capsular polysaccharide from Escherichia coli K 23, respectively. The copolymers were substituted by KDO-residues in a ratio of 1:18 +/- 2 (based on acrylamide) and had molecular masses of 60-100 kdaltons.

A nuclear magnetic resonance spectroscopic investigation of Kdo-containing oligosaccharides related to the genus-specific epitope of chlamydia lipopolysaccharides

The 1H- and 13C-NMR parameters, chemical shifts and coupling constants, for the pentasaccharide of the genus-specific epitope of Chlamydia lipopolysaccharide and related di-, tri-, and tetra-saccharides have been measured and assigned completely using 1D and 2D techniques, and their structures have been confirmed. NOE experiments indicated the preferred conformation of the pentasaccharide and the component oligosaccharides. The 3JH,H demonstrate a change in conformation by rotation of the C-6-C-7 bond of the side chain of the (2----8)-linked Kdo (unit b) in alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcN-(1--- -6)- GlcNol, alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1- ---O)- allyl, and alpha-Kdo-(2----8)-alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl relative to that preferred in alpha-Kdo-(2----4)-alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, alpha-Kdo-(2----8)-alpha-Kdo-(2----O)-allyl, alpha-Kdo-(2----4)-alpha-Kdo-(2----O)-allyl, and alpha-Kdo-(2----6)-beta-GlcNAc-(1----O)-allyl, irrespective of the size of the aglycon, e.g., allyl or beta-D-GlcN residues. The conformational results have been substantiated by computer calculations using the HSEA approach.

The diacetamidodideoxyuronic-acid-containing glycan chain of Bacillus stearothermophilus NRS 2004/3a represents the secondary cell-wall polymer of wild-type B. stearothermophilus strains.

The diacetamidodideoxymannuronic-acid-containing glycan of Bacillus stearothermophilus NRS 2004/3a with the repeating unit structure [-->4)-beta-D-ManpA2,3(NAc)2-(1-->6)-alpha-D-Glcp-(1-->4)-beta-D-+ ++ManpA2,3 (NAc)2-(1-->3)-alpha-D-GlcpNAc-(1-->], was examined to identify its linkage to the bacterial cell wall. In a previous paper it was suggested that this glycan is covalently linked to the surface layer (S-layer) glycoprotein of that organism. By improved chromatographic techniques (gel permeation over Sephacryl S-1000 SF; C4 reversed-phase HPLC) the diacetamidodideoxyuronic-acid-containing material was completely separated from the S-layer glycoprotein. This implicates only low, if any, specific affinity between these cell-wall components. To obtain sufficient amounts for the chemical characterization of its linkage region, the identical diacetamidodideoxyuronic-acid-containing material was isolated from sonicated cells of that organism by a purification procedure different to that for preparation of S-layers. This method allowed collection of the intact molecule including its linkage region. From the combined results of the chemical characterization and 600 MHz NMR spectroscopy it is proposed that the diacetamidodideoxyuronic-acid-containing glycan chain, consisting of approximately six tetrasaccharide repeating units, is directly linked via a pyrophosphate bridge to carbon 6 of muramic acid residues of the peptidoglycan sacculus. About 20-25% of the muramic acid residues are substituted with these polysaccharide chains. Thus, the diacetamidodideoxyuronic-acid-containing glycan represents a secondary cell-wall polymer of B. stearothermophilus NRS 2004/3a.

Synthesis of both epimeric 2-deoxy-n-acetylneuraminic acids and their behaviour towards CMP-sialate synthetase-a comparison with 2-β-methylketoside of n-acetylneuraminic acid☆

Methyl-2-β-chloro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminate 2 respectively methyl 1-2,3-didehydro-4,7,8,9-tetra-O-acetyl-N-acetylneuraminate 3 were transformed by catalytic hydrogenation or by reduction with tributyltin hydride to the corresponding 2-deoxy derivatives 4 and 5, which gave after saponification the acids 6 and 7. Both epimers are not inhibitors of CMP-sialate synthetase (EC 2.7.7.43), whereas the 2-β-methylketoside of Neu5Ac behaves as a competitive inhibitor of the activation of Neu5Ac (N-acetylneuraminic acid).

On the side-chain conformation of N-acetylneuraminic acid and its epimers at C-7, C-8, and C-7,8☆

The side-chain conformation of N-acetylneuraminic acid and analogs has been studied by n.m.r. spectroscopy. The results of the 1H-, 13C-n.m.r.-, and 1H-nuclear-Overhauser-enhancement measurements were used to distinguish between different local-minima conformations suggested by hard-sphere calculations. Attempts were made to correlate the major conformation determined for each compound with the behavior towards activation with N-acetylneuraminic acid-CMP-synthetase.

Structure of a rhamnan from the surface-layer glyco-protein of Bacillus stearothermophilus strain NRS 2004/3a

The structure of a glycan from the surface-layer glycoprotein of Bacillus stearothermophilus strain NRS 2004/3a has been studied by 1H- and 13C-n.m.r. spectroscopy. The results indicate the glycan to be a polymer of the trisaccharide repeating-unit ----2)-alpha-L-Rhap-(1----2)-alpha-L-Rhap-(1----3)-beta-L-++ +Rhap-(1----.

Zur struktur der 3-desoxyoctulosonsäure- (KDO-) region des lipopolysaccharids von Salmonella minnesota Re 595

Spectroscopic data (1H- and 13C-N.m.r.) indicate that the tetrasaccharide αdOclAp(2→4)αdOclAp(2→6)βGIcNp(1→6)GlcN is formed upon hydrazinolysis of the lipopolysaccharide from Salmonella minnesota Re 595.

Characterization of the S-Layer Glycoproteins of Two Lactobacilli

Surface layer (S-layer) glycoproteins have been described on many archaeobacteria. Among eubacteria their presence has only been reported for the Bacillaceae (for a review see Messner and Sleytr, 1992). Lactic bacteria often produce extracellular polymers such as slimes and capsules (for example those described by Nakajima et al., 1990 and Racine et al., 1991). Most of these polymers are polysaccharides but a few reports claim the presence of glycoproteins in lactic streptococci (Macura and Townsley, 1984) and lactobacilli (Garcia-Garibay and Marshall, 1991). These observations have encouraged us to extend our survey on glycosylated S-layer proteins to lactobacilli.

A pyrophosphate bridge links the pyruvate-containing secondary cell wall polymer of Paenibacillus alvei CCM 2051 to muramic acid.

AbstractThe peptidoglycan, the secondary cell wall polymer (SCWP), and the surface layer (S-layer) glycoprotein are the major glycosylated cell wall components of Paenibacillus alvei CCM 2051. In this report, the complete structure of the SCWP, its linkage to the peptidoglycan layer, and its physicochemical properties have been investigated. From the combined evidence of chemical and structural analyses together with one- and two-dimensional nuclear magnetic resonance spectroscopy, the following structure of the SCWP-peptidoglycan complex is proposed:[(Pyr4,6)-β-D-Manp NAc-(1→4)-β-D-Glcp NAc-(1→3)]ñ11-(Pyr4,6)-β-D-Manp NAc-(1→4)-α-D-Glcp NAc-(1→O)-PO2-O-PO2-(O→6)-MurNAc-Each disaccharide unit is substituted by 4,6-linked pyruvic acid residues. Under mild acidic conditions, up to 50% of them are lost, leaving non-substituted ManNAc residues. The anionic glycan chains constituting the SCWP are randomly linked via pyrophosphate groups to C-6 of muramic acid residues of the peptidoglycan layer. 31P NMR reveals two signals that, as a consequence of micelle formation, experience different line broadening. Therefore, their integral ratio deviates significantly from 1:1. By treatment with ethylenediaminetetraacetic acid, sodium dodecyl sulfate, and sonication immediately prior to NMR measurement, this ratio approaches unity. The reversibility of this behavior corroborates the presence of a pyrophosphate linker in this SCWP-peptidoglycan complex.In addition to the determination of the structure and linkage of the SCWP, a possible scenario for its biological function is discussed.

GLYCAN STRUCTURE OF THE S-LAYER GLYCOPROTEIN OF BACILLUS SP. L420-91

AbstractPreliminary taxonomic characterization of isolate L420-91 has revealed that this organism is closely related to the speciesBacillus aneurinolyticus. The bacterium is covered by a squarely arranged crystalline surface layer composed of identical glycoprotein subunits with an apparent molecular mass in the range of 109 kDa. A total carbohydrate content of approximately 3.5% (wt/wt) was determined in the purified surface layer glycoprotein. Glycopeptides were obtained after exhaustive Pronase digestion and purification including gel filtration, ion exchange chromatography and HPLC. From the combined evidence of composition analysis, Smith degradation and nuclear magnetic resonance spectroscopy experiments we propose the following structure for the glycan chain of the surface layer glycoprotein: