Sugars permethylation

Fig. 11.—Sequence of a 2S-Sugar Residue Glycosphingolipid Isolated from Rabbit Erythrocyte Membranes. (Cleavage points, and the masses of fragment ions of the permethylated derivative, are shown. No fragment-ions were observed above 4000, because of the poor sensitivity at high mass.)...

PA-1 was partially hydrolyzed with O.IM TFA (40°C, 24 h) and the three fractions (PA-l-I, PA-l-II and PA-l-III) were obtained by Bio-gel P-6. Especially, about 50% of TBA-positive material (PA-l-III) was eluted in the fraction of small oligosaccharide. Permethylated oligosaccharide-alditols from PA-l-III were analyzed by GC-EIMS, and three disaccharide-alditols (IP, 2P and 3P) were detected. EI-MS and component sugar analysis suggested that the major peak, IP was Rha-(l- 5)-Kdo-ol and the minor peaks, 2P and 3P were two epimers of Araf-( l->5)-Dha. 

IV. Degradation of the Permethylated Sample, and Analysis of the Partially Methylated Sugars... 

Depolymerization of the permethylated carbohydrate is achieved by hydrolysis with acid. Under these conditions, the amino sugar residues are N-deacetylated, and the aminohexosidic linkages become resistant to hydrolysis. Stellner and coworkers29 showed that, when the acid degradation is conducted in 95% acetic acid, the amino sugar residues are also liberated, and can be analyzed by the methylation technique.29 Therefore, acetolysis followed by acid hydrolysis is now commonly used, as it allows the analysis both of hexose and hexosamine residues. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

The development of mass spectrometry (MS) procedures for the study of C-glycosylflavo-noids is particularly significant because of the resistance of the C-glycosidic bond to hydrolysis. It is important, from the structural point of view, to determine the molecular mass and to localize the sugar substituents on the aglycone moiety. This has been achieved in the past by electron impact EI-MS of permethylated derivatives. 

Permethylation of carbohydrates.1 Permethylation of carbohydrates in a solid-liquid system is not possible because of lack of solubility in benzene or methylene chloride. The difficulty can be circumvented by reaction of peracetylated sugars with methyl bromide in C6H6-aqueous NaOH with tetrabutylammonium hydrogen sulfate as catalyst. 

The peak at m/e 203 (Bi) is more intense than that at m/e 214 (As). Hence, the regularity observed for permethylated glycosides of simple hexoses (see Section III,2b,ii) does not hold for amino sugars. 

Along with permethylated derivatives, the permethylated, LiAlH4-reduced derivatives of glycolipids are also used for mass-spectrometric analysis. The amide groups of acetamido sugars and the ceramide part are reduced to alkylamino groups, and, in the case of gangliosides, the ester... 

---