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

Methyl-esterified and methyl-glycosydated oligogalacturonides (oligoGalA) w ere produced to be used as substrates for the characterization of pectinoly tic enzymes acting on the homogalacturonan backbone. The reactions were mon itored with recently developed techniques like high-performance anion-excha nge chromatography (HPAEC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allow sensitive monito ring of the reactions. MALDI-TOF MS reveals the degree of esterification an d or glycosydation. HPAEC at neutral pH separates not only oligogalacturoni des with a different degree of esterifcation but seems to separate some iso mers of e.g. monomethyl- and dimethyl-triGalA as well. Breakdown products f ormed by hydrolysis side reactions were revealed and could even be quantifi ed by HPAEC pH 12 analysis. Using these techniques the conditions for each of the reactions were optimized. Esterification was performed best at conce ntrations of maximal 0.02 N sulfuric acid in anhydrous methanol at 4 degree s C. Hardly any glycosydation occurs and the level of hydrolysis of the oli goGalA was less than 5%. Methyl-glycosydation and simultaneous esterificati on was performed best in 0.1 N sulfuric acid in anhydrous methanol at room temperature. HPAEC revealed only a limited hydrolysis (< 11%). (C) 2000 Els evier Science Ltd. All rights reserved.