A Practical Guide to the Methylation Analysis of Carbohydrates

2.1 Methylation with Sodium Methylsulfinylmethanide - Methyl Iodide and Transformation into Alditol Acetates

The most widely used method for the permethylation of polysaccharides and glycoconjugates is that described by Hakomori 1 . In this procedure the polyalkoxide ion of the substrate in anhydrous dimethyl sulfoxide (DMSO) is first prepared by reaction with sodium methylsulfinylmethanide (dimsyl sodium) and methyl iodide is subsequently added to effect methylation. This procedure has largely replaced the Haworth (sodium hydroxide - methyl sulfate) and Purdie (silver oxide - methyl iodide) methods 10 .
The Hakomori procedure gives, with few exceptions, complete etherification in one step and if the substrate contains uronic acids (as in many bacterial and wood polysaccharides) these are simultaniously transformed into methyl esters 11 12 whilst N-acyl amino sugars are recovered as their N-acyl, N-methyl derivatives 8 13 . O-Acyl groups present in many polysaccharides and also in some glycoproteins are completely cleaved in the strongly alkaline conditions used but acetal and ketal functions, e.g. pyruvic acid ketals, present in some polysaccharides, are stable.
A prerequisite for the methylation reaction is that the substrate is soluble in DMSO. Solubilisation is often facilitated by ultrasonic treatment and/or warming to ≅ 70°C. Most "undermethylations" are due to incomplete dissolution of the substrate. In these cases a portion of the material may be completely methylated while the remaining insoluble part is unmethylated. Low solubilities of substrates can often be circumvented by acetylation using acetic anhydride - pyridine in formamide 14 (the O-acyl groups introduced will be split off during the subsequent base treatment) or by making a partial methylation using the Hakomori, Haworth or Purdie conditions. These procedures will in many cases increase the solubility of the material in DMSO, thereby making the Hakomori reaction possible. However, due care has to be exercised with polysaccharides containing uronic acids since these may be degraded by β-elimination during base treatment if the carboxyl group has first been esterified 12 .
The methylated material is recovered by dialysis (polymeric substrate) or by partiotioning between water and chloroform (oligomeric substrate). Subsequent hydrolysis is usually performed with aqueous formic acid followed by treatment with aqueous mineral acid. The conditions given the experimental part are suitable for most polysaccharides but in certain cases stronger or weaker conditions may be needed depending on particular structural features of the material. Some sugars may give rise to artifacts, e.g. formation of 1,6-anhydro sugars. After hydrolysis and neutralisation with barium carbonate, the monomers formed are transformed into alditols using sodium borohydride (deuteride) as the reductive agent. The excess of borohydride is decomposed with an acidic ion exchanger or acetic acid and the resulting boric acid is removed as its methyl ester by codistillation with methanol. Acetylation is performed with acetic anhydride - pyridine on a boiling water bath, excess reagents being removed by codistillation with toluene.