Alditols methylated

Recently we found that methylated oligosaccharide-alditol methyl esters can he resolved by reverse-phase HPLC (O Neill et al., 1986a, b, c). The technique has been applied to the separation and characterization of acidic tri-, tetra-, and pentasaccharide derivatives from a number of bacterial polysaccharides (O Neill et al., 1986a, b, c). A similar procedure can be used to establish the structural features of cell wall polysaccharides... 

This can be illustrated by our work (O Neill et al., 1986a) on the extracellular anionic polysaccharide produced by Alcaligenes (ATCC 31555) species. The polysaccharide was hydrolyzed for 1.5 h with 0.2 M trifluoroacetic acid and the acidic oligosaccharides isolated by ion-exchange chromatography. Following reduction and methylation, the methylated oligosaccharide-alditol methyl esters were separated by reverse-phase HPLC (Fig. 9). Two major and one minor components were isolated and characterized by FAB-MS, EI-MS, and H-NMR. 

The behavior of alditol methyl ethers on m.s. has been studied by using permethylated pentitols and hexitols. The mass spectra for stereoisomers are virtually identical, and the molecular ion was not observed. Primary fragments are formed by a-cleavages of the alditol chain (14), or by elimination of methanol from the molecular ion (m/ e 234). 

The mass spectra of per (trimethylsilyl )ated alditols showed that this class of compounds is fragmented in a manner closely related to that obtaining for alditol methyl ethers. Thus, primary fragments are formed by a-cleavage of the alditol chain, and secondary fragments are produced by subsequent elimination(s) of trimethylsdanol. The number of carbon atoms in the alditol chain can be determined from the (M — CH3) and (M — McsSiOH) ions. The base peak in the spectra was the trimethylsilyl ion (m/e 73). Small variations in peak intensities were found between stereoisomers, but these diflferences are probably too small to permit the identification of stereoisomers. 

In a study completed during the early development of f.a.b.-m.s., both f.d. and f.a.b. were used to characterize 101 fractions containing neutral oligosaccharides isolated from human milk. Samples were examined as their peracetylated alditols. In subsequent work, the structures of two minor acidic oligosaccharides from human milk were investigated. The per-methylated derivatives were analyzed by f.a.b.-m.s., and their compositions and sequences were defined by the f.a.b. data. Methylation analysis and partial formolysis were the other principal methods used. 

Glycosyl-linkages were determined by GC-EIMS of the partially methylated alditol acetates. RG-II samples (2 mg) were methylated using sodium methyl sulfmyl carbanion and methyl iodide in dimethyl sulfoxide [24] followed by reduction of the uronosyl groups with lithium triethylborodeuteride (Superdeuteride , Aldrich) [23,25]. Methylated and carboxyl-reduced samples were then submitted to acid hydrolysis, NaBIlt reduction and acetylation, partially methylated alditol acetates being analysed by EIMS on two fused-silica capillary columns (DB-1 and DB-225) [20]. 

Methylation analysis of PI and FIbp. The fractions were methylated according to Ciucanu and Kerek [8]. The procedure was repeated until no absorbance was detected by I.r. at 2500-3500 cm-i and the per-O-methylated polysaccharides hydrolyzed and analyzed by GLC and GLC-MS of the derived partly O-methylated alditol acetates. 

Carboxyl redution. A sample of pennethylated PI (5 mg) was carboxyl-reduced by a modification of the method described by Lindberg and Lbnngren [9], as follows. The methylated fraction was solubilized and added a mixture of LiAlH4 (25 mg) in THF (5 mL) at 20 °C for 4 h. and refluxed during 1 h. The excess of reagent was destroyed with ethyl acetate (5-6 drops) and water (10 drops) and the pH of the mixture adjusted to neutrality with acetic acid. The insoluble residue was removed by centrifugation. The reduced fi action was precipitated with EtOH. The reaction was monitored by l.r. specroscopy. Hydrolysis products were analysed by GC-MS as methyl alditol acetates... 

The removal of arabinose suggested the presence of peripheric a-L-arabinofuranosyl units. Flap was methylated by the method of Ciucanu and Kerek and products of derived O-methyl alditol acetates analysed by GLC-MS... 

Oligosaccharides were isolated from PMII by weak acid hydrolysis and separation by SEC and HPAEC-PAD. The isolated oligosaccharides were desalted, reduced and methylated. GC-MS analysis of the partially methylated alditol acetates has been used to reveal the structure of the oligosaccharides. 

Another spectrophotometric method measuring both simple and combined sugars was described in papers by Johnson and Sieburth [158] and Burney and Sieburth [159]. The basic method comprised reduction of sugars to alditols with sodium borohydride, and oxidation of the alditols to form free formaldehyde. The formaldehyde was then determined spectrophotometrically with 3-methyl-2-benzothiazolinone hydrazone hydrochloride. 

Other derivatives of aldonolactones and alditols that have found some use in g.l.c. are trifluoroacetates156 and methyl ethers.163... 

The chemical-ionization (c.i.), mass spectra of some methylated alditol acetates have been reported.51,52 The main intensities in the c.i. 

Multiple-ion monitoring is, however, of considerable value in structural studies, but only if model compounds of known structure are available for comparison. Such an approach has been used in the study of the carbohydrate structures of glycoproteins from different tissues.50 Separation of glycopeptides obtained from various tissues was performed on columns of concanavalin A-Sepharose. Structural analysis by multiple-ion monitoring of partially methylated, alditol acetates derived from the various fractions indicated that the glycopeptides were separated according to the linkage pattern of mannose (see Fig. 1). 

Fig. 1.—Mass Fragmentograms of Partially Methylated Alditol Acetates Obtained from Rat-brain Glycopeptides. [(A) Fraction A glycopeptides, (B) fraction B glycopep-tides, and (C) fraction C glycopeptides. Peak 1, 2,3,4-tri-O-methylfucitol peak 2,...

Methylation analysis Permethylation of intact oligosaccharides Hydrolysis of glycosidic linkages Reduction to monosaccharide alditols Acetylation of free hydroxyl groups previously involved in link-... 

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