Identification and quantitation of alditol acetates of neutral and amino sugars from mucins by automated gas-liquid chromatography

An automated GLC method has been described that allows easy identification and quantitation on a single column of the alditol acetates of the commonly occurring neutral and amino sugars found in epithelial mucins on a new high-temperature stable column. Due to various losses that occur during the derivatization procedure, a constant, termed LF, must be entered into the calculation of the amount of each sugar present. When this factor is applied, the correlation between colorimetric and GLC results is very good.     

Capillary gas chromatography of methylhexitol acetates obtalned upon methylation of N-glycosidically linked glycoprotein oligosaccharides

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.     

Thermolysis of derivatives of amino sugars

Amino groups influence the course of the pyrolysis of carbohydrate derivatives and result in substantial dehydration and charring. N-Phenylglycosylamines and their acetates rearrange with almost quantitative loss of water or acetic acid and retention of the arylamino groups. The sharp dehydration reaction contrasts with the behaviour of the corresponding phenyl glycosides on thermolysis, which results in cleavage of the phenolic groups and subsequent breakdown of the glycosyl moiety. Thermal analysis and chemical data indicate that charring of other derivatives of amino sugars is due to intermediate formation of glycosylamines from thermal reaction of the amino group with glycosidic centres.     

Human umbilical cord hyaluronate. Neutral sugar content and carbohydrate-protein linkage studies.

Paper chromatography of neutral sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neutral sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced eith NaBH4 -PdCl2, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular wieght to about one-half and an appreciable decrease in the specific rota tion of hyaluronate, suggesting a separation of the two antiparallel chains o the double helical hyaluronate. The umbilical cord hyluronate containe contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

A simple and convenient synthetic protocol for O-isopropylidenation of sugars using bromodimethylsulfonium bromide (BDMS) as a catalyst

Various O-isopropylidene derivatives of sugars and acyclic sugars were obtained in very good yields on reaction with acetone at room temperature with a catalytic amount of bromodimethylsulfonium bromide (BDMS). These O-isopropylidene derivatives can also be prepared in good yields on reaction with 2,2-dimethoxypropane (DMP) in acetonitrile using the same catalyst in shorter reaction times. Some of the advantages of this method are high effectiveness, a nonaqueous workup procedure, economic viability, and good yields.     

Human umbilical cord hyaluronate neutral sugar content and carbohydrate-protein linkage studies

Paper chromatography of neural sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neural sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced with NaBH₄-PdCl₂, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular weight to about one-half and an appreciable decrease in the specific rotation of hyaluronate, suggesting a separation of the two antiparallel chains of the double helical hyaluronate. The umbilical cord hyaluronate contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.     

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.     

On arabinose as a component of brain hyaluronate Confirmation by chromatographic,enzymatic and chemical ionization-mass spectrometric analyses

The controversy about the presence of the pentose arabinose in brain hyaluronate was reinvestigated using modern analytical technics. The purified bonive brain hyaluronate contained the neutral sugars: arabinose, 0.18%; glucose; 0.05%; and fucose, 0.22%. The confirmation of the presence of arabinose was obtained by paper and thin layer chromatography of the neutral sugars in deionized hyaluronate hydrolysate. Gas-liquid chromatography of the aldononitrile peracetate of the pentose isolated by preparative paper chromatography gave a single distinct peak, corresponding to standard arabinose on three columns packed with three different phases. Chemical ionization data and mass spectrum of the aldononitrile peracetate drivative agreed with those of the authentic arabinonitrile tetracetate. Analysis of the isolated pentose with the help of the enzymes l-arabinose isomerase and l-ribulose kinase, which are specific for their substrates, further established its identity as l-arabinose.     

Microquantitative analysis of neutral and amino sugars as fluorescent pyridylamino derivatives by high-performance liquid chromatography

A method for the quantitation of picomole amounts of neutral and amino sugars in glycoconjugates was developed. Glycoconjugates were hydrolyzed with a mixture of equal amounts of 4 M trifluoroacetic acid and 4 M hydrochloric acid, and the free amino groups were acetylated. Sugars were coupled with 2-aminopyridine. After the excess reagents were removed by gel-permeation high-performance liquid chromatography, the fluorescent pyridylamino derivatives of sugars were separated and quantified by high-performance liquid chromatography on a reversed-phase column. This method allowed the determination of 0.01-10 nmol of sugars. About 100 pmol of several glycoconjugates were analyzed by the present method, with satisfactory results.     

Quantitative analysis of total membrane-bound sugars and amino sugars as alditol acetates by combined thin-layer chromatography,gas-liquid chromatography,and radiogas chromatography

A sensitive method (0.4 μg of hexoses routinely detectable) for quantitative determinations of sugars and amino sugars in biological material, particularly in membranes, is described. The method consists of a combination of thin-layer chromatography (tlc), gas-liquid chromatography (glc), and radiogas chromatography (rgc), using a highly thermostable phase (Silar 10 c) for the analysis of the specific alditol acetates. In this method, the losses incurred during hydrolysis and preparation for glc are assessed by comparison with the specific recoveries of added radiolabeled internal standards.     

Thermal stabilization of antithrombin III by sugars and sugar derivatives and the effects on nonenzymatic glycosylation

A variety of neutral and acidic sugars and related compounds were evaluated in terms of their effect on the midpoint, T_d, of the thermal denaturation curve of antithrombin III. The objectives were to determine which structural features of these molecules are responsible for their stabilizing properties and to identify more efficient stabilizers which combine the effects of lyotropic anions such as citrate with those of the polyols in a single molecule. The presence of one or more carboxylate groups in a sugar molecule invariably increased its stabilizing potency, whereas the number and position of hydroxyl groups appeared to have no influence on the molecules' stabilizing ability. Several compounds were shown to be effective in preserving antithrombin III activity during pasteurization for 10 h at 60°C. However, the presence of reducing sugars invariably resulted in a decrease in activity following pasteurization, in spite of their ablity to increase T_d. In fact, when antithrombin III was pasteurized in the presence of 2 M glucose and 0.5 M citrate, it steadily losts its ability to inhibit thrombin even though T_d under the conditions was 10°C higher than in citrate alone where activity was preserved. This effect was shown to be coincident with the covalent incorporation of glucose into the protein molecule.     

Simultaneous assay of neutral sugars and amino sugars by an automatic sugar analyzer: applications to glycoproteins.

The simultaneous assay of neutral sugars and amino sugars commonly found in glycoproteins is described. The automatic sugar analyzer used for the determination is based on the ion-exchange chromatography of sugar-borate complexes on a strong anion-exchange resin. The sugars are identified with the orcinol/sulfuric acid reagent. While less than 40 nmol of mannose, fucose, galactose, glucose, xylose, or arabinose is sufficient for analysis at least 200 nmol mannosamine, glucosamine, or galactosamine is required; acidic monosaccharides cannot be determined. The technique of sugar analysis is applied to structural studies on natural compounds, e.g. the monosaccharide composition of lichenan and the carbohydrate moiety of the glycoproteins ovomucoid and Collocalia mucoid.     

The separation of enantiomeric sugars by chromatographic methods

This paper has reviewed the number of chromatographic methods by which one may determine the absolute configuration of sugars. Both indirect methods (converting the enantiomeric pair into diastereomers) and direct methods (using chiral stationary phases) have been discussed. Resolving reagents for the indirect methods include chiral hydroxy compounds, chiral amines, and chiral thiols; with subsequent separation of the diastereomers either by gas-liquid chromatography or by high pressure liquid chromatography. Direct methods discussed have exclusively utilized chiral substitution of organopolysiloxane phases for the separation of enantiomeric sugars as volatile derivatives by gas-liquid chromatography.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Synthesis and cytotoxicity of (R)‐ and (S)‐ricinoleic acid amides and their acetates

An environment‐friendly, free of solvent, process for the synthesis of (R )‐ and (S )‐ricinoleic acid amides has been developed. Starting from methyl ricinoleates and pyrrolidine or ethanolamine, the corresponding amides were obtained with yields ranging from 83–88%. Among 12 synthesized derivatives of ricinoleic acid, including the starting methyl esters, amides, and their acetates, nine compounds were obtained and tested for the first time. Studies on ricinoleic acid derivatives cytotoxicity showed that methyl esters were the least cytotoxic compounds and modification of their structure resulted in increasing cytotoxicity of the obtained products against both cancer cells and normal lymphocytes. Both enantiomers of the ethanolamine‐derived amides showed the most promising anticancer potential.     

Fast atom bombardment mass spectrometry of glycosphingolipids. Glycosphingolipids containing neutral sugars

Natural and synthetic glycosphingolipids containing neutral sugars have been analyzed by positive and negative ion fast atom bombardment mass spectrometry. Basic structural characterization including saccharide size and sequence and ceramide composition is possible on the basis of the fragment ions observed. The degree of fragmentation could be increased by using higher sample concentrations and lower fast atom beam energies. Commercially available synthetic compounds that had been presumed to be pure were shown to contain homologous fatty acids. Mixtures of glycosphingolipids such as those obtained from Gaucher's spleen and from human erythrocytes can be characterized and quantitated.     

Biosynthesis of nucleotide sugars by a promiscuous UDP-sugar pyrophosphorylase from Arabidopsis thaliana (AtUSP)

Nucleotide sugars are activated forms of monosaccharides and key intermediates of carbohydrate metabolism in all organisms. The availability of structurally diverse nucleotide sugars is particularly important for the characterization of glycosyltransferases. Given that limited methods are available for preparation of nucleotide sugars, especially their useful non-natural derivatives, we introduced herein an efficient one-step three-enzyme catalytic system for the synthesis of nucleotide sugars from monosaccharides. In this study, a promiscuous UDP-sugar pyrophosphorylase (USP) from Arabidopsis thaliana (AtUSP) was used with a galactokinase from Streptococcus pneumoniae TIGR4 (SpGalK) and an inorganic pyrophosphatase (PPase) to effectively synthesize four UDP-sugars. AtUSP has better tolerance for C4-derivatives of Gal-1-P compared to UDP-glucose pyrophosphorylase from S. pneumoniae TIGR4 (SpGalU). Besides, the nucleotide substrate specificity and kinetic parameters of AtUSP were systematically studied. AtUSP exhibited considerable activity toward UTP, dUTP and dTTP, the yield of which was 87%, 85% and 84%, respectively. These results provide abundant information for better understanding of the relationship between substrate specificity and structural features of AtUSP.     

Chemotaxis towards sugars by Bacillus subtilis.

Many sugars and derivatives were tested in the capillary assay for their attraction of Bacillus subtilis. The major attractants were 2-deoxy-D-glucose, D-fructose, gentiobiose, D-glucose, maltose, D-mannitol, D-mannose, N-acetylglucosamine, alpha-methyl-D-glucoside, beta-methyl-D-glucoside, N-acetylmannosamine, alpha-methyl-D-mannoside, D-sorbitol, L-sorbose, sucrose, trehalose and D-xylose. Only glucose chemotaxis was completely constitutive. Competition experiments were carried out to determine the specificities of chemoreceptors. There were 25 instances of no influence of two sugars on each other's taxis, 92 instances of one sugar interfering non-reciprocally with chemotaxis towards another and 49 instances of two sugars reciprocally competing. However, in most of the last instances, other sugars were identified that interfered with chemotaxis towards one member of the pair but not the other. Thus, nearly all sugars and related compounds appear to be detected by their own chemoreceptors, but many secondary interactions exist.     

High-performance liquid chromatography of N,O-acylated sialic acids

A rapid, isocratic high-performance liquid chromatographic method for the analysis of N-acetylneuraminic acid, N-glycolylneuraminic acid, and their O-acetylated derivatives is described. Separation of sialic acids and of other monosaccharides as sugar-borate complexes is achieved on an anion-exchange resin. The sialic acids elute as individual peaks after the other sugars tested. The method allows quantitative determination, for example, of amounts of N-acetylneuraminic acid as small as 10 nmol. On cation-exchange resin sialic acids cannot be differentiated, but can be separated from neutral and amino sugars, allowing the determination of as little as 3 nmol of total sialic acids.     

Application of nitrous acid deamination of hexosamines to the simultaneous GLC determination of neutral and amino sugars in glycoproteins

A method is described for simultaneous gas chromatographic analysis of neutral sugars and hexosamines in glycoproteins. Sugars are hydrolyzed with the aid of Dowex 50-X2 (H⁺) resin and the resin bound glucosamine and galactosamine are deaminated with NaNO₂ to the coresponding neutral 2,5-anhydrohexoses. Hexoses and 2,5-anhydrohexoses are then quantitated as the corresponding alditol acetates. Application of the procedure to several different glycoproteins is presented.