Semimicro Determination of Sugar Configuration by Measurement of Circular Dichroism of Alditol Acetates

Bebault, Gwendolyn M.; Berry, Joffre M.; Choy, Y.M.; Dutton, Guy G.S.; Funnell, Norman A.; Hayward, L. D.; Stephen, Alistair M.

doi:10.1139/v73-046

Cited by 55 publications

(6 citation statements)

References 5 publications

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“…Amino sugars were released by hydrolysis with 6.1 M HCl at 105°C for 4 h, followed by repeated drying of the hydrolysates over KOH and P201, and were identified by PC (solvent B), HPAEC, GLC (of the alditol acetates), HVE, autoanalysis, and enzymic assay [17]. The absolute configurations of galactose, 2-amino-2-deoxygalactose, and rhamnose were also determined by GLC of the acetylated but-Zyl glycosides [18] and the assignment for rhamnose was confirmed by the CD spectrum of the alditol acetate [19]. Degradative methods.…”

Section: Methodsmentioning

confidence: 99%

Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4

Cox

Taylor

Anderson

et al. 1995

European Journal of Biochemistry

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Like several other strains of Burkholderia (Pseudomonas) cepacia, the reference strain for serogroup 04 in the French typing scheme [Heidt, A., Monteil, H. & Richard, C. (1983) J. Clin. Microbiol. 18, 738–740] produces a lipopolysaccharide containing two distinct polymers. Attempts to separate the polymers chromatographically were unsuccessful, but the periodate‐resistant major polymer could be isolated by application of the Smith degradation technique to the mixture. By means of chemical and NMR spectroscopic analyses, the following structure could be assigned to the repeating unit of the major polymer: →3)‐α‐d‐Galp ‐(1→3)‐β‐d‐Galp ‐(1→3)‐β‐d‐Galp NAc‐(1→. The following structure of the repeating unit of the minor polymer was established from similar studies of its degradation product, resulting from the oxidation of L‐rhamnose (Rha), and of the original mixture: →3)‐α‐d‐Galp NAc‐(1→3)‐β‐d‐Galp NAc‐(1→4)‐α‐L‐Rhap‐(1→. Individually, the polymers have recently been found in related strains of B. cepacia. The minor polymer was identified as the O‐antigen in serotype A of a Canadian typing scheme [Beynon, L. M. & Perry, M. B. (1993) Biochem. Cell Biol. 71, 417–420], and the major polymer in serotype C of a Japanese typing scheme [Paramonov, N. A., Shashkov, A. S., Knirel, Y. A., Soldatkina, M. A. & Zakharova, I. Y. (1994) Bioorg. Khim. 20, 984–993]. In the case of the O4 strain studied here, both polymers were produced under a variety of growth conditions.

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Section: Methodsmentioning

confidence: 99%

Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4

Cox

Taylor

Anderson

et al. 1995

European Journal of Biochemistry

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“…The specific rotation of the 0-specific polysaccharide was determined with a Bendix polanmeter (model 143A). The circular dichroism of rhamnitol pentaacetate [31] was determined by Dr P. M. Scopes, Westfield College, University of London. Gas-liquid chromatography/mass spectrometry was carried out by staff of the Physico-Chemical Measurements Unit, Harwell, or by Dr J.…”

Section: Physical Methodsmentioning

confidence: 99%

The Lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505

Tahara¹,

Wilkinson²

1983

European Journal of Biochemistry

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Structural studies have been carried out on the 0-specific fraction from the lipopolysaccharide of Pseudomonas aeruginosa NCTC 8505, Habs serotype 03. The 0-specific polysaccharide has a tetrasaccharide repeating-unit containing residues of L-rhamnose (Rha), 2-acetamido-2-deoxy-~-glucose (GlcNAc), 2-acetamido-2-deoxy-~-galacturonic acid (GalNAcA), and 2,4-diacetamid0-2,4,6-trideoxy-i~-glucose (BacNAc,). The following structure has been assigned to the repeating-unit :The parent lipopolysaccharide is a mixture of S, R, and SR species, and its high phosphorus content is partly due to the presence of triphosphate residues, as found for other lipopolysaccharides from P. aeruginosa. In addition to phosphorus, heptose, a 3-deoxyoctulosonic acid, and amide-bound alanine, the core oligosaccharide contains glucose, rhamnose, and galactosamine (molar proportions 3 : 1 : 1). The rhamnose and part of the glucose are present as unsubstituted pyranoside residues: other glucose residues are 6-substituted. We have previously reported general analyses of the lipopolysaccharide from P. aeruginosa NCTC 8505 [19], belonging to Habs serogroup 03, and identified the amino sugars present in the 0-specific fraction [20,21]. Here we describe the results of further studies of these products, including evidence for the structure of the 0-specific polysaccharide. MATERIALS AND METHODS Isolation and Fractionation of LipopolysaccharideMethods used for the batch culture of Pseudomonas aeruginosa NCTC 8505, the preparation of cell walls, and the Abbreviations. BacNAc,, 2,4-diacetamido-2,4,6-trideoxygluccse (NN-diacetylbacillosamine); GalNAcA, 2-acetamido-2-deoxygakacturonic acid; GlcNAc, 2-acetamido-2-deoxyglucose; Rha, rhamnose.

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“…Quantitative data (corrected) were obtained by the latter method, using acid-treated standards and inositol as the internal standard. L-Rhamnose was identified by gas-liquid chromatography of the acetylated oct-2-yl glycosides [14] and by the circular dichroic spectrum of the alditol acetate [15]. For the release of amino sugars, samples were hydrolysed with 6.1 M HCl for 4 h at 105°C under nitrogen.…”

mentioning

confidence: 99%

Structural studies of the putative O-specific polysaccharide of Serratia marcescens O9

Oxley

Wilkinson

1987

Eur J Biochem

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A polymeric fraction containing the putative 0-antigen has been isolated from the lipopolysaccharide of the reference strain (CDC 4534-60) for serogroup 0 9 of Serratia marcescens. The major component of the fraction was a polymer with a disaccharide repeating-unit of L-rhamnose (Rha) and 2-acetamido-2-deoxy-~-galactose (GalNAc) with the following structure: -+3)~-GalpNAc(~1+3)~-Rhap(crl+. Evidence for the presence in the fraction of a similar, minor polymer containing 4-substituted rhamnose residues was provided by the NMR spectra, methylation analysis, and Smith degradation.Although the serotyping of heat-stable (0) antigens is widely used for the characterisation of clinical isolates of Serratia marcescens, the organism has a reputation [l] for producing a complex array of surface carbohydrates, and extensive cross-reactions have been reported by some workers [2, 31. As part of a systematic study of the 0-antigens of S. marcescens [4 -lo], we have isolated and studied the lipopolysaccharide of the 0 9 reference strain (CDC 4534-60). Serological cross-reactions do not seem to be a major problem with 0 9 strains, although the reference strain apparently contains antigenic factors absent from another 0 9 strain, CDC 2870-67 [3, 111. Here we report the structure of the major polymer from the reference strain, and the presence of a minor polymer in the lipopolysaccharide preparation. MATERIALS AND METHODS Growth of bacteria, and extraction and fractionation of lipopolysaccharideSerratia marcescens 0 9 (CDC 4534-60) was grown for 16 hat 30°C in nutrient broth (20 1, Oxoid no. 2) with aeration at 20 1 min-l. The cells (170 g wet weight) were disintegrated mechanically, and the lipopolysaccharide (1.08 g) was isolated from the purified cell walls (4.66 g) as in previous studies [lo].The water-soluble products of mild hydrolysis (1 YO acetic acid, 100°C, 2.25 h) were fractionated on Sephadex G-50. The polymeric fraction obtained was further eluted from a column of DEAE-Sepharose with water or 0.1 M NaC1, and the products were recovered by dialysis and freeze-drying. pyridinelwater (1 31514, by vol.), or B, ethyl acetateipyridinei acetic acidiwater (51511 /3, by vol.). High-voltage paper electrophoresis was carried out at pH 5.3, and the detection reagents used on chromatograms and electrophoretograms were those listed previously [lo]. Gas-liquid chromatography of alditol acetates was carried out using a packed column with OV-275 as the stationary phase. A fused silica capillary column (25 m) of BPlO (S.G.E.) was used for chromatography of methylated alditol acetates, and mass spectra were obtained using a Finnigan model 1020B instrument fitted with this column. NMR spectra (lH and 13C) were recorded for samples in D20 (containing 10% dimethyl sulphoxide in the case of fraction SD2) with a Bruker WH-400 spectrometer. 'H spectra were recorded at about 80°C with 4,4-dimethyl-4-silapentane-1-sulphonate as an external standard. 3C spectra were recorded at about 50°C with tetramethylsilane as an external standard. Polyacry...

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Semimicro Determination of Sugar Configuration by Measurement of Circular Dichroism of Alditol Acetates

Cited by 55 publications

References 5 publications

Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4

Structures of the two Polymers Present in the Lipopolysaccharide of Burkholderia (Pseudomonas) Cepacia Serogroup O4

The Lipopolysaccharide from Pseudomonas aeruginosa NCTC 8505

Structural studies of the putative O-specific polysaccharide of Serratia marcescens O9

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