Glycans are essential molecules for living beings and, in land plants, they play an important structural role as part of the plant cell wall. A major component of the cell wall of many plants is the hemicellulose xylan, which is, after cellulose, the second most abundant glycan in the plant biomass.
Xylan polysaccharides are composed of a linear 1,4-β-linked backbone of xylosyl residues and are decorated with diverse substituents such as glucuronic acid and arabinosyl residues. Only recently, the cohesive role that xylan polysaccharides play between the other major components of the secondary cell wall, cellulose and lignin, is being recognised. Developing synthetic methodologies to produce xylan oligosaccharides that can serve as substrates for enzymatic studies is important for unravelling some of the gaps in the knowledge of its biosynthesis.
Another related xylose-containing polysaccharide was recently revealed in the cell walls of barley. The newly discovered hemicellulose glucoxylan is a linear 1,4-β-linked glycan composed of alternating xylosyl and glucosyl residues. This glycan was previously identified only in the cell wall of a sea lettuce species (a type of green algae). In barley, it is synthesised by genes in the monocot-specific CslF subfamily and is believed to play a structural role in the plant cell wall.
In Chapter 2 of this thesis, a chemoenzymatic synthesis of xylan dodecasaccharides for the study of xylan-modifying enzymes is described. The synthesis of these materials was designed around two central ideas, (a) the implementation of a divergent-convergent iterative synthetic strategy, and (b) the use of a glycosynthase for the glycosylation reactions. Because of the limited solubility of long unprotected glycans in water, two parallel syntheses were performed, one to obtain xylan oligosaccharides with a methyl group at the 3-OH of the reducing-end xylosyl residue and one to obtain xylan oligosaccharides without the methylation. Besides unprotected xylosyl acceptors, the xylan glycosynthase (XynAE265G) employed in these syntheses requires unprotected α-xylosyl fluorides as donors. These donors were equipped with a THP group at the 4-OH of the non-reducing end xylosyl residue to prevent self-condensation of these molecules. The enzymatic glycosylation reactions with this donor gave exclusively the desired 1,4-β-linked glycosyl products, and no sugar-based side-products were detected.
The xylans oligosaccharides equipped with a methyl group showed superior solubility in comparison to the unprotected ones, so the methyl-substituted dodecasaccharide was used for biosynthetic studies using xylan-modifying enzymes. Treatment of this substrate with glucuronosyltransferase AtGUX3 was found to install a single GlcA substituent at the substrate. The data obtained by MS/MS analysis of the reaction products is compatible with the substituent being installed at one of the two central residues of the substrate, which is in agreement with previously reported in vivo studies.
In Chapter 3, a small library of glucoxylan oligosaccharides was produce by chemical synthesis. These molecules have the same structural features as those reported in the literature to be synthesised by the two barley glycosyltransferases HvCslF3 and HvCslF10. The synthesis of these target molecules, ranging from di- to hexasaccharides, was designed to maximize the number of convergent and divergent steps in order to minimize the number of required synthetic transformations. As a temporary protecting group for chain elongation, a TBS group was used with great success to protect the 4-OH glycosylation site. The protected glucoxylan molecules in this small library are equipped with an anomeric azido-pentyl linker, which may be used after reduction to the aminopentyl linker, for immobilization of these molecules as microarrays for biosynthetic studies and the characterization of antibodies.