Partially ethylated alditol acetates as derivatives for elucidation of the glycosyl linkage-composition of polysaccharides

The use of partially ethylated alditol acetates for the analysis by gas-liquid chromatography of the components of polysaccharides, and the glycosidic linkages of these components, is described. The derivatives are prepared by procedures analogous to those for the synthesis of partially methylated alditol acetates. Derivatization requires two successive ethylations and more-strenuous conditions of hydrolysis and reduction than for the methyl analogs. The partially ethylated alditol acetates are formed in nearly quantitative yield and give single, sharp peaks on gas chromatography. Retention-time data, relative to two internal standards, are given for 79 glycosidic linkage-isomers of mannose, galactose, glucose, arabinose, xylose, rhamnose, and fucose, on four g.l.c. columns. One of these columns is a newly developed, highly polar, capillary column. Direct comparisons of these retention times to retention times of partially methylated alditol acetates are made. The ethyl analogs are eluted sooner that the corresponding methyl derivatives, and the amount of this shift in elution time is dependent upon the number of alkyl groups in the derivative. This change in elution time allows separation of many polysaccharide components by g.l.c. that are not separable as their partially methylated alditol acetates. Others, separated as their O-methyl derivatives, are coeluted as their partially ethylated alditol acetates. The two derivatives thus provide excellent complementary procedures because of their differential chromatographic separation and because of the similarity of their preparation.     

Chemical-ionization mass spectrometry of methylated hexitol acetates

Many of the stereoisomers of methylated hexitol acetates having the same arrangement of O-methyl and O-acetyl groups yield markedly different chemical-ionization (c.i.) mass spectra. For example, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol and 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylgalactitol yield clearly distinguishable c.i. mass spectra. The observed differences are reproducible. Hence, chemical-ionization mass spectrometry, when combined with analytical gas chromatography and electron-impact (e.i.) mass spectrometry, is of value in identifying the methylated alditol acetates obtained during methylation analysis of polysaccharides and other complex carbohydrates. The c.i. mass-spectral data that differentiate many of the possible partially methylated glucitol, galactitol, and mannitol acetates are presented. In addition, examples are presented that demonstrate the ability of c.i. mass spectrometry to characterize previously unidentified methylated hexitol acetates.     

A method of purification of partially methylated alditol acetates in the methylation analysis of glycoproteins and glycopeptides

A method for methylation analysis of intact glycoproteins is described. Starting with intact glycoprotein, the oligosaccharides are methylated, hydrolyzed, reduced, and acetylated. The partially methylated alditol acetates are then separated from noncarbohydrate contaminants on a silica gel G column. Partially methylated hexitol acetates are eluted from the column with petroleum ether:ethyl acetate (1:1, $\text{v}\text{v}$) and partially methylated N-acetylhexosaminitol acetates are subsequently eluted with methanol. Analysis by gas-liquid chromatography/mass spectrometry of the partially methylated alditol acetates shows no interfering contaminants. This method circumvents the need to make pronase glycopeptides and avoids the pitfalls of other methylation procedures.     

Quantitative determination of partially methylated alditol acetate of amino sugar by gas chromatography-mass spectrometry

Using doubly labeled N-acetyllactosamine. Hakomori's procedures to prepare partially methylated alditol acetates and their subsequent analysis by gas chromatography-mass spectrometry were followed from a quantitative aspect. It was found that both galactose and glucosamine were nearly quantitatively converted to PMAAs. Preferential loss of PMAA of glucosamine occurred on a column for gas chromatography when the amount of the PMAA injected onto a column was less than a certain level. Above this level, PMAAs of both galactose and glucosamine were eluted from the column with equal yields. However, in mass spectrometry with monitoring of total ions, the molar response factor of PMAA of glucosamine was found to be about 25% higher than that of PMAA of galactose. Based on these findings, methylation analysis of an oligosaccharide from Taka-amylase A composed of one glucosamine and five mannose residues could be carried out quantitatively.     

Conformations of acyclic sugar derivatives : Part II. Determination of the conformations of alditol acetates in solution by the use of 250-MHz n.m.r. spectra

The p.m.r. spectra of all the fully acetylated pentitols and several fully acetylated hexitols have been analysed. Computation by iterative analysis and recourse to 250-MHz spectra were required in several cases. The vicinal coupling constants were used to determine the conformations of these compounds in solution. The planar zigzag conformation was found to be predominant only in those configurations (arabino, manno) which do not possess parallel 1,3-interactions between acetoxyl groups on alternate carbon atoms. The other compounds were found to be mixtures of two or more conformers, none of which has the planar zigzag conformation, except in the case of hexa-O-acetylallitol. The conformations of alditols in the crystalline state and of the alditol acetates in solution are compared.     

Structural features of the mucilage from the stem pith of kiwifruit (acitinidia deliciosa_: part II,structure of the neutral oligosaccharides

Graded acid hydrolysis of the mucilage from the stem pith of Actinidia deliciosa gave a series of neutral oligosaccharides that were fractionated by gel filtration. The methylated alditol derivatives were isolated by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and g.l.c.—m.s. of the methylated alditol acetates. The results suggest the glucuronomannan core of the mucilage is substituted with oligosaccharides composed of (1→3)-linked β-d-galactose residues that are partially substituted through positions 2 and 6 with Araf, Arap, Fucp, and Galp. A tentative structure for the mucilage is proposed.     

Rapeseed hull pectin

A pectin isolated from rapeseed, hulls by extraction with aqueous ammonium oxalate, had a degree of esterification of 83% and contained residues of hexuronic (mainly D-galacturonic) acid (76%), D-galactose (2–3%), L-arabinose (8–9%), D-xylose (2%), L-rhamnose (2–3%), and L-fucose (1%). Partial acid hydrolysis of the derived pectic acid furnished 2-O-(α-D-galactopyranosyluronic acid)-L-rhamnose, 4-O-(α-D-galactopyranosyluronic acid)-D-galacturonic acid and the polymer-homologous tri- and tetrasaccharides, and 4-O-(glucopyranosyluronic acid)-L-fucose. The cleavage products from the methylated pectin were examined by g.l.c. and the partially methylated alditol acetates from the methylated carboxyl-reduced polysaccharide by g.l.c.-mass spectrometry. Parallel methylation studies on lemon-peel pectin have established a close similarity between the two pectins.     

Methylation analysis of cell wall glycoproteins and glycopeptides from Chlamydomonas reinhardii

Cell wall glycoproteins from Chlamydomonas reinhardii and the glycopeptides produced by the action of thermolysin were subjected to standard methylation analysis. GC-MS of the methylated alditol acetates revealed short oligosaccharides some of which show branching. O-glycosidically linked galactofuranosyl residues are present. The asymmetric distribution of the major O-glycosidic linkages is also reported.     

Synthesis and mass fragmentographic analysis of partially O-methylated 2-N-methylglucosamines.

A series of partially O-methylated N-methylglucosamines was synthesized by limited-time methylation of methyl-2-N-methylacetamido-2-deoxyglucopyranoside by Kuhn's procedure, followed by acid hydrolysis. These partially O-methylated N-methylglucosamines were separated satisfactorily by gas chromatography on a column of OV-17 on Gas-chrom Q as amino alditol acetates and identified from their mass spectra. For specific analysis of the methylated aminosugar derivatives, a mass fragmentographic method was established. Methylated aminosugars can be successfully determined in amounts as low as about 1 ng by this method.     

A comparison of some hydrolytic and gas chromatographic procedures used in methylation analysis of the carbohydrate units of glycopeptides

The polysaccharide secreted by Klebsiella aerogenes type 54 strain A3 was isolated, methylated, the ester carboxyl-reduced, and the product partially hydrolyzed. The resulting, partially O-methylated oligosaccharides were reduced and ethylated, and the mixture of products was fractionated by l.c. The l.c. fractions containing per-O-alkylated oligosaccharide-alditols were analyzed by e.i.-m.s. Pure per-O-alkylated oligosaccharide-alditols were also analyzed by ¹H-n.m.r. spectroscopy. The products obtained by base-catalyzed degradation and subsequent ethylation of the per-O-methylated polysaccharide were fractionated by l.c. The main product isolated was analyzed by e.i.-m.s., c.i.-m.s., and ¹H-n.m.r. spectroscopy. The results of these studies, in conjunction with results of analytical methods commonly used in the elucidation of polysaccharide structures, unambiguously characterized the primary glycosyl structure of the polysaccharide. Base-labile substituents, previously reported to be present in the polysaccharide, were not studied. Structure 1 revises, and complements, previously reported structures.

     

Synthesis of 2,5,6- and 3,5,6-tri-O-methyl-D-galactose

Starting from methyl β-D-galactofuranoside, 3,5,6-tri-O-methyl-D-galactose (9) and 2,5,6-tri-O-methyl-D-galactose (16) were synthesized. The alditol acetates were prepared from 9 and 16, and their behavior in g.l.c. was compared. Mass spectra of the alditol acetates from 9 and 16 showed that these compounds gave fragmentations as expected. The alditol acetate from 16 was also prepared by an alternative route.     

Structural features of the mucilage from the stem pith of kiwifruit (actinidia deliciosa): part I,structure of the inner core

The mucilage found in the stem pith of Actinidia deliciosa contains d-glucuronic acid, d-mannose, l-fucose, l-arabinose, and d-galactose in the molar ratios 1.0:1.5:2.0:4.0. The native, carboxyl-reduced, and partially acid-hydrolysed polysaccharides were subjected to methylation analysis. Partial acid hydrolysis of the methylated, carboxyl-reduced glucuronomannan core produced a series of methylated oligosaccharides which, as their alditol derivatives, were isolated by reverse-phase h.p.l.c. and characterised by e.i.- and f.a.b.-m.s. The data suggest that the polysaccharide contains a →4)-β-d-GlcpA-(1→2)-α-d-Manp-(1→ backbone with most of the d-mannosyl and d-glucosyluronic acid residues substituted through positions 3 with oligosaccharides containing l-arabinose, α-l-fucose, and β-d-galactose.     

Unusual partially 3-O-methylated alpha-galactan from mushrooms of the genus Pleurotus

Indistinguishable partially 3-O-methylated galactans were isolated from the edible basidiomycetes Pleurotus eryngii and Pleurotus ostreatoroseus. They were obtained via successive aqueous extraction, freeze-thawing, precipitation with Fehling solution of soluble material, and ultrafiltration. Mono- and bidimensional 13C and 1H-nuclear magnetic resonance spectroscopy (HMBC, HETEROTOCSY, COSY, and HMQC), and methylation analysis were used to determine their structures. They were linear, partially 3-O-methylated, (1-->6)-linked alpha-d-galactans containing Gal and 3-Me-Gal, in a 3:1 molar ratio (GC-MS of alditol acetates).     

A comprehensive procedure for preparation of partially methylated alditol acetates from glycoprotein carbohydrates.

Various steps involved in the preparation of partially methylated alditol acetates (PMAAs) from glycoprotein-derived carbohydrates were improved to obtain the derivatives in a rapid manner with excellent yields. Carbohydrates were permethylated in dimethyl sulfoxide (DMSO), using a fine suspension of sodium hydroxide and methyl iodide (CH3I). The fine suspension of NaOH was prepared conveniently from commercially available 50% aqueous NaOH in DMSO by sonication and washing the precipitate with DMSO. Methylation of ovalbumin and fetuin glycopeptides using the fine suspension of NaOH and CH3I was complete within 5 min, and the methylation reaction did not generate any nonsugar artifacts. Methylated carbohydrates without any purification were hydrolyzed in a mixture of volatile organic acids, which permitted rapid removal of the acids from samples by evaporation. Acetylation of partially methylated alditols with acetic anhydride for 2-4 h at ambient temperature using 4-N,N'-dimethylaminopyridine as a catalyst and the reaction was free from generating nonsugar reaction artifacts. The reaction time course for methylation, hydrolysis, and acetylation was determined to obtain optimum reaction conditions for preparation of the PMAAs. The procedure facilitated rapid identification and quantitation of PMAAs due to diminished reaction artifacts and the quality of the chromatogram depended only on the purity of starting material and the reagents used for the methylation analysis. Utility of these simple methods for rapid methylation analysis was demonstrated in the characterization of oligosaccharides isolated in small amounts using a carbohydrate analyzer.     

Nitrous acid deamination of methylated amino-oligosaccharide glycosides

G.l.c.-mass spectrometry has been used to characterize the products of N-deacetylation-nitrous acid deamination of per-O-methylated derivatives (8–11) of methyl 2-acetamido-2-deoxy-3-O-β-D-galactopyranosyl-α-D-glucopyranoside(1), methyl (2) and benzyl (3) 2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-β-D-glucopyranosides, and methyl 2-acetamido-2-deoxy-6-O-β-D-galactopyranosyl-α-D-glucopyranoside (4). 2,5-Anhydrohexoses have been converted into alditol trideuteriomethyl ethers, alditol acetates, and aldononitriles. The importance of side reactions that lead to the formation of 2-deoxy-2-C-formylpentofuranosides is discussed.     

Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species

The extracellular anionic polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) contains l-mannose, l-rhamnose, d-glucose, and d-glucuronic acid in the molar ratios 1.0:4.5:3.1:2.3. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide indicated terminal non-reducing rhamnose and mannose, (1→4)-linked rhamnose, (1→3)- and (1→3,1→4)-linked glucose, and (1→4)-linked glucuronic acid to be present in the ratios 1.0:0.8:2.1:2.2:2.0:2.2. Partial acid hydrolysis and base-catalysed β-elimination gave a series of oligosaccharides that were isolated as their alkylated alditol derivatives by reverse-phase h.p.l.c. and characterised by f.a.b.-m.s., e.i.-m.s., and ¹H-n.m.r. spectroscopy. The repeating unit 1, excluding O-acyl groups, is proposed.

     

Structure of the oligosaccharide chains in human alpha 1-protease inhibitor.

Two glycopeptides were obtained from alpha 1-protease inhibitor after extensive pronase digestion and chromatography on Bio-Gel P-10 and concanavalin A-Sepharose. these glycopeptides were characterized by compositional analysis and sequential exoglycosidase digestion followed at each step by methylation analysis. The partially methylated alditol acetates obtained were resolved by gas chromatography and identified by mass spectrometry. The proposes structures of the oligosaccharide moieties of the glycopeptides are given below. (formula: see text) The relative amounts of the two glycopeptides isolated from concanavalin A-Sepharose suggest that each protein molecule contains four carbohydrate chains; one large chain (A) and three small chains (B).     

Localization of partially methylated flavonol glucosides in Chrysosplenium americanum. I. Preparation and some properties of a trimethyl flavonol glucoside antibody

5,2′,5′-Trihydroxy-3,7,4′-trimethoxyflavone-2′-O-glucoside, a major flavonoid constituent of Chrysosplenium americanum Schwein. ex Hooker was conjugated to bovine serum albumin (BSA) by the diazo reaction in good yield and with a molar ratio of 11.5:1. Antibody raised against the latter conjugate had a titer value of 1:1600 and was found to be specific for the 2′-O-glucosides of tri- and tetramethoxyflavones. Some cross reactivity (about 55%) was observed against the pentamethoxyflavone-5′-O-glucoside; but almost none with the parent hydroxyflavone, quercetin, or any of its partially methylated (3,7,4′-tri- or 3,7,3′,4′-tetramethyl-) derivatives. The specificity of antibodies raised against the 2′-O-glucosides of Chrysosplenium makes them useful for the intracellular localization of these natural constituents.     

Comparative study of the asparagine-linked oligosaccharide structures of normal and acute-phase rat plasma thiostatin

Rat plasma thiostatin is a 68 kDa glycoprotein with kinin donor and cysteine proteinase inhibitor properties. Thiostatin is an acute-phase plasma protein (APPP) with dramatically elevated plasma levels in response to inflammatory stimuli. APPPs have been shown to possess altered glycan structures in inflammation. This study compares the carbohydrate structure of normal thiostatin with that expressed during the acute-phase response. Thiostatin from both normal and acute-phase plasma was purified by carboxymethyl-papain Sepharose 4B affinity chromatography. Sugar composition analysis by gas chromatography and the Warren method yielded similar mean values for both proteins on a mole sugar per mole protein basis (normal/acute phase): fucose, 2.4/1.7; mannose, 7.5/8.0; galactose, 11.2/10.6; and sialic acid, 14.2/13.0. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot identified a homogeneous 68–70 kDa molecular species for normal and acute-phase thiostatin. Inter-sugar linkage analysis was carried out for permethylated oligosaccharides released by hydrazinolysis. Gas chromatography yielded the following partially methylated alditol acetates relative to 1.0 mole of 1,3,6-tri-O-linked mannose (mean normal/mean acute phase): galactose: 1,3-di-O-, 1.44/1.01; 1,6-di-O-, 1.02/0.68; mannose: 1,2-di-O-, 1.64/1.42; 1,2,4-tri-O-, 0.24/0.13; 1,3,6-tri-O-, 1.0/1.0; 2-deoxy-2-N-methylacetamidoglucose: 1,4-di-O-, 1.42/1.12. These analytical studies indicated that corresponding carbohydrate structures are present in normal and acute-phase thiostatin. Crossed affinoimmunoelectrophoresis (CAIE) further confirmed the structural similarity between the glycan moieties.     

Host-Pathogen Interactions: XIII. Extracellular Invertases Secreted by Three Races of a Plant Pathogen Are Glycoproteins Which Possess Different Carbohydrate Structures

The invertase present in the culture fluid of races 1, 2, and 3 of Phytophthora megasperma Drechs. var. sojae A. A. Hildebrand (Pms) were purified until they gave but a single band, whether stained for protein or carbohydrate, after isoelectric focusing in flat bed gels. The sugar compositions of multiple preparations of the purified invertases from each race of this fungal pathogen were determined by quantitative gas chromatography of their alditol acetates. The invertases are composed of about 25% carbohydrate. Mannose and glucosamine make up more than 97% of the carbohydrate portions of the invertases of all three Pms races analyzed, but the ratio of mannose to glucosamine is clearly not the same in each race. The glycosyl linkage compositions of the glucosamine-containing mannans of multiple preparations of the Pms invertases were determined by GC-MS analysis of the partially methylated alditol acetate derivatives. The results of these analyses demonstrate clear quantitative differences between the glycosyl components of the different Pms races. The existence of race-specific carbohydrate structures in the differentially virulent Pms races suggests that these carbohydrates may be involved in determining the specificity of hostpathogen interactions.