Synthesis and Immunological Properties of a Tetrasaccharide Portion of the B Side Chain of Rhamnogalacturonan II (RG‐II)
ABSTRACT A highly convergent strategy was used for the synthesis of a tetrasaccharide [3-aminopropyl beta-L-arabinofuranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-[alpha-L-rhamnopyranosyl-(1-->3)]-alpha-L-arabinopyranoside] portion of the B side chain of the plant cell-wall pectic polysaccharide rhamnogalacturonan II (RG-II). The terminal nonreducing beta-L-arabinofuranosyl residue of the target compound was installed by using an arabinofuranosyl donor that was protected with a 3,5-O-(di-tert-butylsilane) group to facilitate nucleophilic attack from the beta-face. The synthetic strategy also employed a chemoselective glycosylation of a trichloroacetimidate donor with a thioglycosyl acceptor; this gave a product that could be used immediately in a subsequent glycosylation. The reducing end of the tetrasaccharide contained an aminopropyl group to facilitate conjugation to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Mice that were immunized with a KLH-tetrasaccharide conjugate produced antibodies that recognized RG-II isolated from Arabidopsis thaliana cell walls, but did not recognize RG-II obtained from red wine. Our data suggest that the arabinopyranosyl residue exists in the (4)C(1) conformation in the tetrasaccharide and in A. thaliana RG-II, whereas it has the (1)C(4) conformation in wine RG-II. It is proposed that differences in the conformation of side chain B might account for the ability of antibodies to discriminate between RG-II that was isolated from Arabidopsis and wine.
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ABSTRACT: A paradigm about Rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and further employed a fast structural characterization method based on chromatography and mass spectrometry, allowing the analysis of RGII side chains from microgram amounts of cell wall. We actually found RGII structures to be much more diverse than described up to now. In chain A of wild type plants, up to 45% of the L-fucose is substituted by L-galactose, state seemingly uncorrelated with RGII dimerization capacity. This led us to a complete reinvestigation of RGII structures of the Arabidopsis thaliana fucose deficient mutant mur1, which gave insights on RGII chain A biosynthesis and suggested that chains A truncation, rather than L-fucose to L-galactose substitution, is responsible for mur1 dwarf phenotype. Mass spectrometry data of chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid giving a plausible explanation to the yet puzzling RGII antibody recognition. The β-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ specific manner. Combined with variation in side chain B length, this gives rise to a family of RGII structures instead of the unique structure described until now and paves the way to studies considering physiological roles of the modulation of RGII composition. This article is protected by copyright. All rights reserved.The Plant Journal 06/2013; DOI:10.1111/tpj.12271 · 6.82 Impact Factor
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ABSTRACT: A conformationally restricted 2-O-benzyl-3,5-O-di-tert-butylsilylene-β-d-thiogalactofuranoside donor was prepared from benzyl α-d-galactofuranoside and its donor capability was studied for stereoselective 1,2-cis α-d-galactofuranosylation. An unusual chemical behavior in benzylation and hydrogenolysis reactions was observed after the introduction of the 3,5-O-di-tert-butylsilylene protecting group into the galactofuranosyl moiety. The influence of the solvent, temperature, and activating system was evaluated. The NIS/AgOTf system, widely used in 1,2-cis β-arabinofuranosylation, was not satisfactory enough for 1,2-cis galactofuranosylation. However, moderate to high α-selectivity was obtained with all the acceptors employed when using p-NO2PhSCl/AgOTf as a promoting system, in CH2Cl2 at -78°C. The order of the addition of the reactants (premixing or preactivation) did not affect substantially the stereochemical course of the glycosylation reaction. The α-d-Galf-(1→6)-d-Man linkage was achieved with complete diastereoselectivity by preactivation of the conformationally constrained thioglycoside donor.Carbohydrate Research 10/2014; 397. DOI:10.1016/j.carres.2014.07.024 · 1.97 Impact Factor
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ABSTRACT: Extensins are plant-derived glycoproteins that are densely modified by oligo-arabinofuranosides linked to hydroxyproline residues. These glycoproteins have been implicated in many aspects of plant growth and development. Here, we describe the chemical synthesis of a tetrameric β(1-2)-linked arabinofuranoside that is capped by an α(1-3)-arabinofuranoside and a similar trisaccharide lacking the capping moiety. The challenging β(1-2)-linked arabinofuranosides were installed by using an arabinofuranosyl donor protected with 3,5-O-(di-tert-butylsilane) and a C-2 2-methylnaphthyl (Nap) ether. It was found that the cyclic silane-protecting group of the glycosyl donor greatly increased β-anomeric selectivity. It was, however, imperative to remove the silane-protecting group of an arabinosyl acceptor to achieve optimal anomeric selectivities. The anomeric linker of the synthetic compounds was modified by a biotin moiety for immobilization of the compounds to microtiter plates coated with streptavidine. The resulting microtiter plates were employed to screen for binding against a panel of antibodies elicited against plant cell wall polysaccharides.Organic & Biomolecular Chemistry 07/2013; 11(31). DOI:10.1039/c3ob40958a · 3.49 Impact Factor