Monosaccharide permethylation products for gas chromatography - mass spectrometry: how reaction conditions can influence isomeric ratios

Abstract

Methylation analysis has been widely used for determination of carbohydrate structures by mass spectrometry. Permethylation of monosaccharides yields mixtures of anomeric pyranosides and furanosides. This paper discusses the influence of some of the permethylation reaction parameters on the proportions of isomeric products obtained. The ratios of three five- and six-membered ring products obtained from two permethylated monosaccharides, D-galactose and L-fucose, have been determined as a function of reaction parameters. The method of Ciucanu and Kerek (1) (methyl iodide in dimethyl sulfoxide (DMSO) in the presence of sodium hydroxide (NaOH)) was used as a starting point. The "conventional" method consists of mixing all of the reagents with the substrate and allowing the reaction to proceed with stirring. Both D-galactose and L-fucose under these conditions produced two main permethylated isomers, a furanoside and a pyranoside, along with two other minor isomeric components. We have investigated the effect on the proportion of products obtained of mixing DMSO, substrate, and NaOH for various times prior to the addition of methyl iodide. Results for D-galactose showed that shorter times enhanced the formation of permethylated furanoside isomers, while reducing the proportion of pyranosides. In other sets of experiments, the time and temperature of reaction, following the addition of methyl iodide, were studied. The indication is that 15 min are sufficient to produce complete methylation, with longer reaction times yielding the same results. Again for D-galactose, low reaction temperatures (ca. 10 °C) favored formation of furanoside products. Higher temperatures yielded higher pyranoside/furanoside ratios. Higher quantities of NaOH also favored formation of the main galactopyranoside product. As for L-fucose, the ratio of the main furanoside vs. pyranoside products obtained by permethylation varied in a way similar to permethylated galactoside. Thus, higher temperatures and longer reaction times favored the main fucopyranoside product. Gentler conditions (i.e., shorter reaction times and lower temperatures) significantly favored the formation of the main fucofuranoside product. These results are interesting as they show the possibility of controlling the relative abundance of permethylated isomers of fucose and galactose. They also constitute a warning to chemists who use methylation procedures in their analyses, to the effect that permethylation products may vary considerably if the reaction conditions are not carefully controlled. Keywords: glucose, galactose, fucose, TLC, GC–MS, permethylation, monosaccharides.