Upgrading Methylation Method for Structural Studies of Polysaccharides: Case Analysis of a Bioactive Polysaccharide from Acacia tortilis

Methylation has been the most widely used conventional tool in the structural characterization of polysaccharides. In the series of developments in methylation methods employing a diverse range of solvents and methylating reagents under basic conditions, the main emphasis relied upon inclusive solubilization of polysaccharides during the reaction procedure. In our laboratory, Acacia tortilis ssp. raddiana (Savi) Brenan polysaccharide (AcTP) has been reported for antidiabetic activity and hence its chemical structure has been investigated. AcTP was found to be composed of L-rhamnose, L-arabinose, D-mannose, D-galactose, D- galacturonic acid, and D-glucuronic acid in molar ratio 1:34:1:10:2:2 respectively. The polysaccharide was subjected to Hakomori methylation protocol for linkage analysis. However, the method encountered following limitations: undermethylation, partial degradation of polysaccharide, and decline in solubility of methylated product in hydrolytic and reducing reagents for further derivatization to partially methylated alditol acetates (PMAA). The limitations were circumvented by applying key modifications in standard protocol viz. in situ production of methylsulfinyl methyl sodium; purification of a product by dialysis; replacing 0.15M H₂SO₄ by 4M trifluoroacetic acid and performing the reduction by NaBH₄ in methanol/chloroform (7:3) solvent system. Permethylation of AcTP to PMAA was realized by the proposed modified method and glycosyl linkages were studied by GC-MS. The monosaccharide composition based on alditol acetate investigation and successive PMMA analysis demonstrated complete methylation without any significant degradation establishing the worthiness of the modified method. The methylation was also confirmed by FTIR results. Unlike the original protocol, the modified method is more feasible, efficient, effective and can be used for permethylation of neutral as well as acidic polysaccharides.