An NMR solution study of the mega-oligosaccharide, rhamnogalacturonan II

Rhamnogalacturonan II (RG-II) is a structurally complex pectic mega-oligosa ccharide that is released enzymatically from the primary cell wall of highe r plants. It contains roughly 30 monosaccharide units (MW similar to 5 kDa) including very unusual residues such as Kdo, Dha, aceric acid and apiose. Previous studies have demonstrated that these monomers are arranged into fo ur structurally well-defined oligosaccharide side chains (A-D), linked to a homogalacturonan mainchain, but the specific attachment sites of these bra nches on the pectic backbone have not yet been elucidated. In the present w ork, fairly complete assignments of the 750 MHz H-1 NMR spectra and partial assignments of the C-13 NMR spectra of the sodium-borohydride-reduced RG-I I monomer were obtained for a 5 mM sample isolated from red wine. On the wh ole, these data corroborate the primary structures of the sidechains previo usly established by methylation analysis, partial hydrolysis and FAB-MS spe ctrometry but some heterogeneity has been demonstrated (partial substitutio n at B5, B6, and A5). The preferred orientations of the majority of the sid echain glycosidic linkages in the RG-II monomer have been determined from t he sequential nOe data and the solution structure is generally in good agre ement with the stable conformers previously obtained by molecular modeling (MM3) of the disaccharide and sidechain oligosaccharide building blocks. Al l of a two-residue, a three-residue, and a four-residue segment of the back bone have been tentatively identified from long range interactions between sidechain protons as well as in the mainchain. Taking into account the leng th of the 9-mer galacturonan mainchain described in prior work, these build ing blocks constitute almost the complete structure of RG-II (Scheme 2).