Partially methylated alditol acetates

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]. 

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. 

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,...

Chromatographic analysis ages G.l.c. separation of substituent, partially methylated alditol acetates On-line e.i.- or c.i.-m.s. analysis G.l.c. analysis after sequential hydrolysis, reduction, and acetylation... 

Adapted from Kariya et al. (1997). With permission from Elsevier Publisher. a Partially methylated alditol acetate with methoxy groups at the positions shown. b Retention time on a SP2330 capillary column relative to 2,3,4-tri-O-methyl-fudtol. c The molar ratios were based on the peak area. d Not detected. 

The proposed structure is in full agreement with the results obtained by mass spectrometry, immunodiffusion and analysis of partially methylated alditol acetates (4). 

Methylation linkage analysis of the partially methylated alditol acetates gave the following derivatives ... 

Figure 9. Open tubular gas chromatogram of partially methylated alditol acetates obtained from blood-group A active tetraglycosylceramide (A) and hexaglycosyl-ceramide (B), respectively. Stationary phase was OV-1, and carrier gas was Ns. Column temperature was kept at 175°C for 14 min, then raised l°C/min. The designation above the peaks indicate actual binding positions.

E. Lau and A. Bacic, Capillary gas chromatography of partially methylated alditol acetates on a high-polarity, cross-linked, fused-silica BPX 70 column,./. Chromatogr., 637 (1993) 100-103. 

Primary fragment ions from partially methylated alditol acetates (Fig. 2) arise by -cleavage with, in general, preferred formation of ... 

Fig. 2. Primary fragment ions arising by a-cleavage in the mass spectra of partially methylated alditol acetates.

The main limitation of the use of partially methylated alditol acetates for the characterization of methylated sugars lies in the structural symmetry that may exist when the primary hydroxyl group (0-5 in pentoses and 0-6 in hexoses) is not etherified. This difficulty, however, can be overcome by introducing deuterium at C-1 by reduction of the sugar with sodium borodeuteride. 

Methylated alditol units in oligosaccharides are cleaved in a manner similar to that described previously for partially methylated alditol acetates. On the other hand, substituted cyclic carbohydrates undergo a variety of types of fragmentation, the most important of which are summarized in Fig. 4 for simple permethylated glycosides. 

Carpita NC, Shea EM (1989) Linkage structure of carbohydrates by gas chromotography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In Biermann CJ, McGinnis GD (eds) Analysis of carbohydrates by GLC and MS. CRC Press, Boca Raton, FL, p 157... 

GC-MS linkage analysis of partially methylated alditol acetates derived from the 50% (v v) aqueous acetonitrile fractions of the permethylated fV-glycans derived from the wildtype and Mgat4a knockout mouse spleen... 

Methylation is the most widely used method to determine the position of the glycosyl linkages.Permethylation of glycolipids can be done according to the methods of Hakomori (32) and Ciucanu and Kerek (33). The permethylated derivatives are hydrolyzed and the products converted into partially methylated alditol acetates and identified by gas chromatography-mass spectrometry ( CG-MS) (34). 

Developments in capillary columns for GLC have improved the resolution of the individual components with a concomitant reduction of elution times. Neeser and Schweizer (1984) have separated the 0-methyloxime acetates of several sugars using a fused-silica Carbowax 20M capillary column. Although alditol acetates can be separated by capillary GLC (Blakeney et al., 1982), this method is more suitable for separating complex mixtures of partially methylated alditol acetates derived from meth-ylation analysis. It has been reported that phthalic esters interfere with the determination of alditol acetates by GLC (Dudman and Whittle, 1976) on conventional packed columns. This problem can be overcome to some extent by the use of capillary columns with polar phases (Henry et al.,... 

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