Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS

Amidation of methyl-esterified oligogalacturonides (oligoGalA) was studied to produce partly and fully amidated oligoGalA to be used as substrates and /or inhibitors for the characterization of pectolytic enzymes acting on the homogalacturonan backbone. The reactions were performed with varying conce ntrations of ammonia or methylamine and monitored in time using matrix-assi sted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TO F MS) that allows sensitive monitoring of the reactions. MALDI-TOF MS revea ls the degree of amidation (DAm) and extent of hydrolysis of methyl-esters. Using this technique the conditions for each of the reactions was optimize d. Amidation was performed best under anhydrous conditions at a concentrati on of 4 M ammonia or methylamine at ambient temperature. Amidation using me thylamine reached almost completeness (DAm 95) without hardly any hydrolysi s of methyl-esters while amidation with ammonia reached a DAm of 70 on aver age. After an initial fast amidation, precipitation of the partly amidated oligoGalA reduced the reaction rate enormously. The use of ammonia in aqueo us solutions instead off anhydrous ammonia resulted in 6-10% lower DAm valu es due to the hydrolysis of methyl-esters. Therefore, anhydrous conditions are preferred during amidation. Furthermore, methylamine is a better reagen t for amidation of oligoGalA and pectins then ammonia, but also results in totally different products with other properties. (C) 2001 Elsevier Science Ltd. All rights reserved.