Guy G.S. Dutton

Applications of Gas-Liquid Chromatography to Carbohydrates :Part I

Publisher Summary This chapter explains that many developments have taken place in the applications of gas–liquid chromatography to carbohydrates. The chapter discusses a classical paper on the application of trimethylsilyl derivatives of carbohydrates in gas–liquid chromatography. The discovery that these derivatives are readily formed and that they are volatile revolutionized the separation and analysis of carbohydrate mixtures. The chapter reviews the determination of carbohydrates in biological materials by gas–liquid chromatography and the use of this method in the analysis of plant materials. In examining the structure of a polysaccharide, it is convenient to consider the methods involved under the three main headings: (1) quantitative analysis, ( 2 ) methylation, and (3) periodate oxidation. Each of these aspects of polysaccharide chemistry may be aided by the application of gas–liquid chromatography, either qualitative or quantitative, or both. Thus, separations impossible by other techniques may often be achieved, and analytical data obtained in a fraction of the time demanded by other methods.

Synthesis of 4-O-β-D-mannopyranosyl-L-rhamnopyranose

Abstract The title disaccharide ( 16 ) has been synthesized in 50% overall yield by way of condensation of 4,6-di- O -acetyl-2,3- O -carbonyl-α- D -mannopyranosyl bromide 5 with methyl 2,3- O -isopropylidene-α- L -rhamnopyranoside ( 1 ) in chloroform solution, in the presence of silver oxide. The disaccharide was characterized as the crystalline isopropyl alcoholate of methyl 4- O -β- D -mannopyranosyl-α- L -rhamnopyranoside ( 11 ) and as 1,2,3-tri- O acetyl-4- O - (2,3,4,6-tetra- O -acetyl-β- D -mannopyranosyl)-α- L -rhamnopyranose ( 15 ). Methyl β- D -mannopyranoside isopropyl alcoholate 7 was readily obtained in 85% yield via the reaction of bromide 5 with methanol. Reduction of 2,3-di- O -methyl- L -rhamnose with sodium borohydride, followed by acetylation, may result in the formation of an appreciable proportion of a boric ester, namely 1,5-di- O -acetyl-4-deoxy-2,3-di- O -methyl- L -rhamnitol-4-yl dimethyl borate, depending on the procedure used.

Chemical and n.m.r.-spectroscopic investigation of the capsular polysaccharide of klebsiella serotype k41

Abstract The structure of the repeating unit of the capsular polysaccharide from Klebsiella type 41 has been investigated by methylation analysis of the original and the carboxyl-reduced polymer, uronic acid degradation, Smith degradation, and graded acid hydrolysis. Proton- and 13 C-n.m.r. spectroscopy of the original polysaccharide and of the fragments obtained by various methods confirmed some structural features and allowed determination of the anomeric configuration of the glycosidic linkages. This polysaccharide is shown to have the following heptasaccharide repeating-unit: This is the first polysaccharide antigen K of the Klebsiella series found to have seven sugar residues in its repeating unit, and to contain a galactose residue in its furanose form.

The use of bacteriophage depolymerization in the structural investigation of the capsular polysaccharide from Klebsiella serotype K3.

The structure of the repeating unit of the capsular polysaccharide from Klebsiella serotype K3 has been established from the results of n.m.r. (1H and 13C) spectroscopy and methylation analysis of P1, the pyruvic acetal-bearing pentasaccharide obtained on depolymerization of the polysaccharide with a bacteriophage-borne endogalactosidase, reduced deacetalated P1, and the native polysaccharide. The data permit the assignment of the following structure to the repeating unit: (formula see text)

Structural investigation of the capsular polysaccharide of Klebsiella serotype k23

Abstract Klebsiella K23 capsular polysaccharide has been investigated by the techniques of hydrolysis, methylation, Smith degradation-periodate oxidation, and base-catalysed degradation, either on the original or the carboxyl-reduced polysaccharide. The structure was found to consist of a tetrasaccharide repeating-unit, as shown below. The anomeric configurations of the sugar residues were determined by 1 H-and 13 C-n.m.r. spectroscopy on the original and degraded polysaccharides.

Proton and carbon-13 nuclear magnetic resonance studies on methyl (methyl d-galactosid)uronates and their per-O-acetyl derivatives

Abstract The 1H- and 13C-n.m.r. spectra of the anomeric methyl (methyl d -galactosid)uronates, as well as the 1H-n.m.r. spectra of their acetyl derivatives, were analyzed. The spectra of the unacetylated d -galactopyranosiduronates showed good correlation with those of the corresponding anomeric d -galactopyranuronic acids and their methyl esters, and with those of the anomeric methyl d -galactopyranosides. From the values of the chemical shifts and coupling constants, it was concluded that the anomeric methyl (methyl d -galactopyranosid)uronates and their corresponding peracetates are in the 4C1( d ) conformation. The chemical shifts in the 13C-n.m.r. spectra show good correlation with those of the methyl d -galactosides. The signals of the furanose derivatives appear at fields lower than those of the corresponding pyranose compounds.

The capsular polysaccharide from Klebsiella serotype K54; location of the O-acyl groups, and a revised structure

The capsular polysaccharide from Klebsiella type K54, containing both O-formyl and O-acetyl groups, has been investigated by using the techniques of methylation analysis (by gas-liquid chromatography), periodate oxidation-Smith degradation, and both 1H- and 13C-n.m.r. spectroscopy. Degradation of the native polysaccharide with a bacteriophage-induced glucosidase generated a formylated, as well as a formylated and acetylated, tetrasaccharide, whereas similar depolymerization of the deacetylated polysaccharide yielded a single tetrasaccharide; the corresponding, O-acylated octasaccharides were also isolated and characterized. These oligosaccharides, utilized in chemical and spectroscopic studies in order to determine the location of the O-acyl substituents in the repeating sequence, indicated formylation at 0-4 of each lateral D-glucosyl group and acetylation at 0-2 of alternate L-fucosyl residues. A new structure for the repeating unit in the polysaccharide is proposed.

Structural investigation of the capsular polysaccharide of Klebsiella serotype K17.

Klebsiella K12 capsular polysaccharide has been investigated by the techniques of methylation, Smith degradation-periodate oxidation, uronic acid degradation, and partial hydrolysis, in conjunction with 1H-n.m.r. spectroscopy at 100 and 220 MHz, and 13C-n.m.r. spectroscopy at 20 MHz. The structure has been found to consist of the hexasaccharide repeating-unit shown, having a D-galactofuranosyl residue at the branch point. In this series, a D-galactofuranosyl residue has previously only been found in the polysaccharide from Klebsiella K41.

Preparation of oligosaccharides by bacteriophage degradation of polysaccharides from Klebsiella serotypes K21 and K32

Abstract Depolymerization of bacterial, capsular polysaccharides by phage enzymes is a convenient method of preparing oligosaccharides that correspond to one, or several, repeating unit(s). Thus, the capsular polysaccharide from Klebsiella K21 yields a linear pentasaccharide, and that from Klebsiella K32, a linear tetrasaccharide. Both oligosaccharides contain acetal substituents, but, whereas the 4,6- O -(1-carboxyethylidene)- D -galactosyl residue in the K21 structure is relatively acid-stable, the corresponding 3,4- O -(1-carboxyethylidene)- l -rhamnosyl residue in K32 is extremely acid-labile. Phage degradation may, therefore, be the only way by which an oligosaccharide corresponding to an intact repeating-unit may be obtained in such circumstances.

The structure of Chorisia speciosa gum

Abstract The purified, gum exudate from Chorisia speciosa (Palo borracho) was studied. It contains, in moles per mole, l -arabinose, ∼1; l -rhamnose, 2; d -mannose, 1; d -galactose, 8; and d -glucuronic acid, 3; and a trace of d -xylose. The results from methylation analysis and selective, alkaline degradation, combined with the characterization of the oligosaccharides resulting from partial hydrolysis with acid, made possible the assignment of a tentative “average structure”.