A Practical Guide to the Methylation Analysis of Carbohydrates

1. Introduction

Methylation analysis is the most facile method for determining the substitution of the monosaccharide units constituting oligo- and polysaccharides or the carbohydrate moieties of glycoconjugates. The method gives details of the structural units present in the polymers, but gives no information on their sequence or the anomeric natures of their linkages. The analysis involves the conversion of all free hydroxyls in the original material into methoxy groups, followed by hydrolytic cleavage with acid of the glycosidic linkages and analysis of monomers formed. The subsitution pattern of O-methyl groups in the monomers reflects the substitution of the corresponding sugars in the native material. The hydroxyl → methoxyl substitution in the native material is usually performed by the Hakomori procedure 1 (Sections 2.1 and 2.2) and the analysis of the monomers formed on hydrolysis is carried out by gas liquid chromatography (GLC) 2 3 and mass spectrometry (MS) of the derived alditol acetates 4 5 6 7 (Sections 4 and 5). The steps involved in methylation analysis by this method are illustrated in Fig. 1.
Methylation analysis has been reviewed earlier 8 9 and therefore practical aspects will be emphasised in the following and the theoretical discussion will be kept to a minimum.
Figure 1
Hypothetical native substrate native substrate methylation →
Methylated substrate methylated substrate acid hydrolysis →
methylated sugar A methylated sugar B methylated sugar C
↓ 1) reduction 2) acetylation
Partially methylated alditol acetates (analysis by GLC and MS)