A multiple-column approach to the methylation analysis of plant cell-walls

A procedure has been developed for the improved analysis of the neutral-sugar glycosidic linkages in plant cell-walls, utilising capillary g.l.c. and columns of three different phases for the separation of the products of methylation analysis. Retention coefficients are reported for a wide range of partially methylated alditol acetates on columns of CP-Sil88 and a bonded phase (BP-1) equivalent to OV-1. Using these phases and SP-1000, all cases of co-chromatography can be resolved. Computers were used to process the large amounts of data produced, to identify peaks and to assist in merging the results obtained using the three phases.     

Quantitative analysis by various g.l.c. response-factor theories for partially methylated and partially ethylated alditol acetates

Results are presented which demonstrate that the molar flame-responses of partially methylated partially ethylated alditol acetates should be calculated on an effective carbon response (e.c.r.) basis. The relative responses of 2,3,4,6-tetra-O-ethyl-D-glucitol 1,5-diacetate, 2,3,6-tri-O-ethyl-D-glucitol 1,4,5-triacetate, hexa-O-ethyl-D-glucitol, hexa-O-methyl-D-glucitol, α-D-galactopyranose pentaacetate were measured and compared to the predicted values from three theories: equal molar response, equal weight response, effective carbon response. The observed values agree very well (±0?6%) with the e.c.r.-calculated values. The other theories of relative response can result in as much as 100% error in quantitation. The e.c.r-calculated relative response-factors for all commonly found partially methylated partially ethylated alditol acetates are presented, and their use is suggested for accurate quantitation.     

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.     

Glucuronic acid conjugates of bilirubin-IXα in normal bile compared with post-obstructive bile. Transformation of the 1-O-acylglucuronide into 2-, 3-, and 4-O-acylglucuronides

Structures have been determined for bilirubin-IXalpha conjugates in freshly collected bile of normal rats, dogs and man and in post-obstructive bile of man and rats. The originally secreted conjugate has been characterized as azopigment (I), i.e. a 1-O-acyl-beta-d-glucopyranuronic acid glycoside. Conversion of the acetylated methyl ester of azopigment (I) into methyl 2,3,4-tri-O-acetyl-1-bromo-1-deoxy-beta-d-glucopyranuronate (V) indicates the pyranose ring structure for the carbohydrate and a C-1 attachment for the bilirubin-IXalpha acyl group. Alternative procedures for deconjugation of azopigment (I) and its derivatives are also described. In post-obstructive bile, the 1-O-acylglucuronide is converted into 2-, 3- and 4-O-acylglucuronides via sequential intramolecular migrations of the bilirubin acyl group. The following approach was utilized. (1) The tetrapyrrole conjugates were cleaved to dipyrrolic aniline and ethyl anthranilate azopigments, and the azopigments were separated as the acids or methyl esters. (2) The isomeric methyl esters were characterized by mass spectral analysis of the acetates and silyl ethers. (3) The free glycosidic function was demonstrated by 1-oxime and 1-methoxime derivative formation. (4) The position of the dipyrrolic O-acyl group was determined for the methyl esters by protecting the free hydroxyl groups of the glucuronic acid moieties as the acetals formed with ethyl vinyl ether and by further conversion of the carbohydrates into partially methylated alditol acetates. These were analysed by using g.l.c.-mass spectrometry. The relevance of the present results with regard to previous reports on disaccharidic conjugates is discussed. Details of procedures for the formation of chemical derivatives for g.l.c. and mass spectrometry have been deposited as Supplementary Publication SUP 50081 (15 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5.     

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.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside

Described herein is the synthesis of eight positional isomers of methylated and acetylated or benzoylated methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside. The compounds were generated simultaneously from methyl 2-(acetylmethylamino)-2-deoxy-beta-D-glucopyranoside by sequential partial methylation and benzoylation and isolated in pure form by high-performance liquid chromatography (HPLC). The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography (GLC) retention indices on three different capillary columns.     

An alternative method for the rapid synthesis of partially O-methylated alditol acetate standards for GC-MS analysis of carbohydrates

Mixtures of partially O-methylated alditol acetate standards (PMAAs) of Glc, Gal, and Man were synthesized rapidly. Methylation of methyl glycosides was carried out in the presence of BaO/Ba(OH)(2) x 8H(2)O giving rise to mixtures of partially methylated glycosides (PMGs), whose degree of methylation was monitored by TLC. The batch containing the largest mixture of methyl ethers was converted into partially O-methylated alditol acetate derivatives (PMAAs), via successive hydrolysis, reduction, and acetylation, and then subjected to GC and GC-MS analysis. Detailed data on retention times, TIC, and EIMS are now provided.     

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.     

On the structure of cytolipin R, a ceramide tetrahexoside hapten from rat lymphosarcoma

Cytolipin R, a ceramide tetrahexoside isolated from rat lymphosarcoma, was studied by sequential hydrolysis with specific glycosidases which revealed the anomeric configurations of the glycosidic bonds. Sugar linkages were established by combined gas-liquid chromatography and mass spectrometry of the partially methylated alditol acetates prepared after permethylation and hydrolysis of the intact lipid. Results indicated the structure of cytolipin R to be N-acetylgalactosaminyl(beta1-->3)galactosyl(alpha1-->3) galactosyl(beta1-->4)glucosyl ceramide. Cytolipin K (globoside I) differs in having a -galactosyl(alpha1-->4)galactosyl- internal linkage, and this difference must account for the immunological differences between cytolipin K and cytolipin R.     

Separation of neutral oligosaccharide alditols from human meconium using high-pH anion-exchange chromatography

Neutral reduced oligosaccharides are in general not sufficiently retained to achieve adequate separation and reproducible chromatography using high-pH anion-exchange chromatography. We describe a method to increase the retention using two columns in series. This method has been applied to the separation of oligosaccharides purified from human meconium glycoproteins, obtained as their alditols after alkaline-borohydride release of oligosaccharides. The neutral oligosaccharide alditols were significantly retained upon two CarboPac PA-100 columns, connected in series, and eluted in 80 mM sodium hydroxide between 4 and 10 min. Three sialylated alditols studied were substantially retained and could be eluted in a gradient of 0–500 mM sodium acetate—80 mM sodium hydroxide between 10 and 45 min. The elution patterns were based on their size, charge and linkage, such that oligosaccharide alditol isomers could be separated.     

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.     

Purification and structures of branched blood-group-B-active glycosphingolipids from human erythrocyte membranes

Three different variants of complex, branched, highly blood-group-B-active glycosphingolipids (B-III, B-IV, and B-V) have been isolated from human erythrocytes by means of partition of their membranes in n-butanol/phosphate buffer, subsequent removal of nonpolar lipids and proteins by several steps of phase distribution, acetone or sodium acetate precipitation, peracetylation and repeated fractionation of all crude extracts by silicic acid and ion exchange column chromatography. Finally, peracetylated B-glycolipid fractions were purified to homogeneity by preparative silica gel high-performance thin-layer chromatography. Their structures were elucidated by gas chromatographical sugar analysis, by combined gas chromatography/mass spectrometry of partially methylated alditol acetates for the identification of glycosidic linkages, and by fast atom bombardment and electron impact mass spectrometry of the undegraded, permethylated substances in order to establish the molecular mass, sugar sequence, type of oligosaccharide chain, position of hexosyl branching points, number of N-acetyllatosamine units, as well as sphingosine and fatty acid patterns of the ceramide residues. 360-MHz 1H nuclear magnetic resonance spectroscopy in (2H)dimethylsulfoxide of deuterium-exchanged native B-III and B-IV identified all carbohydrate components, their sites of attachment, the anomeric nature of their glycosidic linkages and the sequential arrangement within the oligosaccharide chain. Furthermore, it established the nature of branching points within the carbohydrate sequence, and assigned the different typical saccharide branches to either the position 2 versus 3, or position 3 versus 6 of the 2,3-disubstituted or 3,6-disubstituted galactoses. The nature of the anomeric linkages and branching points of B-V was based upon the series of NMR data obtained from the B-I--B-IV analogues. All results thus establish the following structures: (formula; see text)     

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.     

Authentic standards for the reductive-cleavage method. The positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydroribitol

The eight positional isomers of methylated and benzoylated 1,5-anhydroribitol were generated simultaneously from 1,5-anhydroribitol by sequential partial methylation and benzoylation, and the four meso isomers and two enantiomeric pairs of isomers so-formed were isolated in chemically pure form by high-performance liquid chromatography. The corresponding acetates were obtained by debenzoylation of the pure isomers and acetylation. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

Purification and methylation analysis of cell wall material from Solanum tuberosum

Cell wall material (CWM) of potatoes was prepared by sequentially extracting the wet ball-milled tissue with 1 % aq. Na deoxycholate, PhOHHOAcH₂O and 90 % (v/v) aq. DMSO. The purity of the CWM (e.g. absence of residual starch) was established by carbohydrate analysis using different acid hydrolysis conditions and by methylation studies. The partially methylated alditol acetates from the CHCl₃MeOH soluble fraction (S) of the methylated CWM were separated into 15 main peaks by GLC. Fourteen of these peaks were carbohydrate derivatives and the identity of most of these was established by MS. Reduction of the hydrolysate of S with NaBD₄ was used to identify the carbohydrate derivatives present in peaks 7 and 11 above. The occurrence of 4-linked galacturonosyl residues in the methylated polymers was established after reduction of S with LiAlH₄ and LiAlD₄. The main glycosidic linkages present in the non-cellulosic polysaccharides of the wall in descending order of concentration are: 4-linked galactose, 4-linked galacturonic acid, 5-linked arabinose and 4,6-linked glucose. The major branch points are those through 0–6 of glucose and 0–4 of rhamnose. Arabinose, galactose and xylose residues constituted the non-reducing ends. Graded acid hydrolysis of the CWM made it possible to assess the relative strengths of some of the glycosidic linkages. The general structural features of the CWM are discussed in the light of these results.     

Authentic standards for the reductive-cleavage method: the positional isomers of partially methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol

Described herein is the synthesis of the eight positional isomers of methylated and acetylated or benzoylated 1,5-anhydro-L-arabinitol. The compounds were generated simultaneously from 1,5-anhydro-L-arabinitol by sequential partial methylation and benzoylation and isolated in pure from by high-performance liquid chromatography. The desired acetates were obtained by debenzoylation and acetylation of the pure isomers. Reported herein are the 1H NMR spectra of the benzoates and the electron-ionization mass spectra of the acetates and the tri-O-methyl derivative. Also reported for the acetates and the tri-O-methyl derivative are their linear temperature-programmed gas-liquid chromatography retention indices on three different capillary columns.     

Structures of gangliosides from bovine adrenal medulla.

Five gangliosides, accounting for over 95% of the total ganglioside fraction, were isolated from bovine adrenal medulla by preparative thin-layer chromatography and the carbohydrate structures determined by a combination of periodate oxidation and permethylation techniques. Partially methylated alditol acetates were generated from the neutral sugars of the fully methylated glycolipids and identified by gas-liquid chromatography. Substitution on N-acetylgalactosamine was determined by methanolysis of the permethylated ganglioside, acetylation of the products, and identification of the resulting substituted methyl glycosides by GLC. Periodate oxidation followed by borohydride reduction confirmed some of the linkages and demonstrated the absence of (2-8) linkages between sialic acid units. Mass spectrometry of the permethylated gangliosides gave information on sugar sequence at the nonreducing end.     

Structure of the human erythrocyte blood group P1 glycosphingolipid.

A glycosphingolipid with blood group P1 activity was extracted from an acetone powder of human erythrocyte stroma with chloroform-methanol. It was purified by chromatography on columns of silicic acid and by preparative thin-layer chromatography of the fully acetylated and deacetylated glycolipid. The purified glycolipid contained galactose, N-acetylglucosamine, and glucose in a molar ratio of 3:1:1. Treatment of the P1 glycolipid with fig alpha-galactosidase released a single galactosyl residue and destroyed the blood group activity, and the alpha-galactosidase product had the same chromatographic mobility as paragloboside. Substitution sites on the neutral sugars of the P1 glycolipid and the alpha-galactosidase product were established by identification of methylated alditol acetates, and substitution on N-acetylglucosamine was determined by identification of methyl glycoside derivatives. The terminal nonreducing disaccharide of the P1 glycolipid is Gal(alpha, 1 leads to 4)Gal. N-Acetylglucosamine was identified as the next sugar in sequence by mass spectrometric analysis of the permethylated P1 glycolipid. On the assumption that the glucose residue is linked to ceramide, we propose the following structure for the P1 glycolipid: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-NAc(beta, 1 leads to 4)Glc-Cer.     

High-resolution proton nuclear magnetic resonance characterization of seminolipid from bovine spermatozoa

The high-resolution one- and two-dimensional proton nuclear magnetic resonance (1H-NMR) characterization of seminolipid from bovine spermatozoa is presented. The 1H-NMR data was confirmed by gas-liquid chromatography-mass spectrometric analysis of the partially methylated alditol acetates of the sugar unit, mild alkaline methanolysis of the glyceryl ester, mobility on normal phase and diphasic thin-layer chromatography (HPTLC), and fast atom bombardment mass spectrometry (FAB-MS). The structure of the molecule corresponds to 1-O-hexadecyl-2-O-hexadecanoyl-3-O-beta-D-(3'-sulfo)-galactopyranosyl- sn-glycerol.     

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.