Chemo‐Enzymatic Synthesis of Long‐Chain Oligosaccharides for Studying Xylan‐Modifying Enzymes

Abstract

Plant research is hampered in several aspects by a lack of pure oligosaccharide samples that closely represent structural features of cell wall glycans. An alternative to purely chemical synthesis to access these oligosaccharides is chemoenzymatic synthesis using glycosynthases. These enzymes enable the ligation of oligosaccharide donors, when activated for example as α-glycosyl fluorides, with suitable acceptor oligosaccharides. Herein, the synthesis of xylan oligosaccharides up to dodecasaccharides is reported, with glycosynthasemediated coupling reactions as key steps. The xylo-oligosaccharide donors were protected at the non-reducing end with a 4-O-tetrahydropyranyl (THP) group to prevent polymerization. Installation of an unnatural 3-O-methylether substitu-ent at the reducing end xylose of the oligosaccharides ensured good water solubility. Biochemical assays demonstrated enzymatic activity for the xylan acetyltransferase XOAT1 from Arabidopsis thaliana, xylan arabinofuranosyltransferase XAT3 enzymes from rice and switchgrass, and the xylan glucuronosyltransferase GUX3 from Arabidopsis thaliana. In case of the glucuronosyltransferase GUX3, MALDI-MS/ MS analysis of the reaction product suggested that a single glucuronosyl substituent was installed primarily at the central xylose residues of the dodecasaccharide acceptor, demonstrating the value of long-chain acceptors for assaying biosynthetic glycosyltransferases.

Phosphates as Assisting Groups in Glycan Synthesis

“…Protecting groups on a synthetic backbone can ensure site-specific enzymatic modifications. ,− Azides or N -trifluoroacetyl moieties can mask amines, and O -acetyl, O -methyl, and O -tetrahydropyranyl (THP) conceal hydroxyl groups. − ,, Enzymatic installation of fucosyl, sialyl, or C-6 oxidized galactosyl residues can help to direct further enzymatic functionalization. ,, While powerful, these blocking groups require specific enzymes, complicate chemical synthesis for installation and removal (i.e., O -acetyls), or cannot be removed (i.e., O -methyl) . Phosphorylation could offer a general and mild alternative to the site-specific enzymatic functionalization of complex glycans.…”

“… 25 , 28 − 35 Azides or N -trifluoroacetyl moieties can mask amines, and O -acetyl, O -methyl, and O -tetrahydropyranyl (THP) conceal hydroxyl groups. 28 − 31 , 33 , 36 Enzymatic installation of fucosyl, sialyl, or C-6 oxidized galactosyl residues can help to direct further enzymatic functionalization. 25 , 34 , 35 While powerful, these blocking groups require specific enzymes, 34 complicate chemical synthesis for installation and removal (i.e., O -acetyls), 33 or cannot be removed (i.e., O -methyl).…”