Fluorescent labeling of pectic oligosaccharides with 2-aminobenzamide and enzyme assay for pectin

Oligogalacturonides [oligomers composed of (1-->4)-linked alpha-D-galactosyluronic acid residues] with degrees of polymerization (DP) from 1 to 10, and a tri-, penta-, and heptasaccharide generated from the backbone of rhamnogalacturonan I (RG-I) were labeled at their reducing ends using aqueous 2-aminobenzamide (2AB) in the presence of sodium cyanoborohydride in over 90% yield. These derivatives were analyzed by high-performance anion-exchange chromatography (HPAEC) and structurally characterized by electrospray-ionization mass spectrometry (ESIMS) and by H-1 and C-13 NMR spectroscopy. The 2AB-labeled oligogalacturonides and RG-I oligomers are fragmented by endo- and exo-polygalacturonase and by Driselase, respectively. 2AB-labeled oligogalacturonide is an exogenous acceptor for galacturonosyltransferase of transferring galacturonic acid from UDP-GalA. Thus, the 2AB-labeled oligogalacturonides and RG-I oligomers are useful for studying enzymes involved in pectin degradation and biosynthesis and may be of value in determining the biological functions of pectic fragments in plants. (C) 2002 Elsevier Science Ltd. All rights reserved.