Esterification and glycosydation of oligogalacturonides : examination of the reaction products using MALDI-TOF MS and HPAEC

Methyl-esterified and methyl-glycosydated oligogalacturonides (oligoGalA) were produced to be used as substrates for the characterization of pectinolytic enzymes acting on the homogalacturonan backbone. The reactions were monitored with recently developed techniques like high-performance anion-exchange chromatography (HPAEC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allow sensitive monitoring of the reactions. MALDI-TOF MS reveals the degree of esterification and or glycosydation. HPAEC at neutral pH separates not only oligogalacturonides with a different degree of esterifcation but seems to separate some isomers of e.g. monomethyl- and dimethyl-triGalA as well. Breakdown products formed by hydrolysis side reactions were revealed and could even be quantified by HPAEC pH 12 analysis. Using these techniques the conditions for each of the reactions were optimized. Esterification was performed best at concentrations of maximal 0.02N sulfuric acid in anhydrous methanol at 4oC. Hardly any glycosydation occurs and the level of hydrolysis of the oligoGalA was less than 5%. Methyl-glycosydation and simultaneous esterification was performed best in 0.1N sulfuric acid in anhydrous methanol at room temperature. HPAEC revealed only a limited hydrolysis (<11%).