Oligosaccharide synthesis by coupled endo-glycosynthases of different specificity: A straightforward preparation of two mixed-linkage hexasaccharide substrates of 1,3/1,4-β-glucanases

Glycosynthases are engineered glycosidases which are hydrolytically inactive yet efficiently catalyse transglycosylation reactions of glycosyl fluoride donors, and are thus promising tools for the enzymatic synthesis of oligosaccharides. Two endo-glycosynthases, the E134A mutant of 1,3/1,4-β-glucanase from Bacillus licheniformis and the E197A mutant of cellulase Cel7B from Humicola insolens, were used in coupled reactions for the stepwise synthesis of hexasaccharide substrates of 1,3/1,4-β-glucanases. Because the two endo-glycosynthases show different specificity, towards laminaribiosyl and cellobiosyl donors, respectively, the target hexasaccharides were prepared by condensation of the corresponding disaccharide building blocks through sequential addition of the glycosynthases in a "one-pot" process. Different strategies were used to achieve the desired transglycosylation between donor and acceptor in each step, and to prevent unwanted elongation of the first condensation product and polymerization (self-condensation) of the donor: 1) selection of disaccharide donors differing in the configuration of the hydroxyl substituent normally acting as acceptor, 2) temporary protection of the polymerizable hydroxyl group of the donor, or 3) addition of an excess of acceptor to decrease the probability that the donor can act as an acceptor. The best procedure involved the condensation of α-lactosyl or 4^II-O-tetrahydropyranyl-α-cellobiosyl fluorides with α-laminaribiosyl fluoride, catalyzed by E197A Cel7B, to give tetrasaccharide fluorides, which were then the donors for in situ condensation with methyl β-cellobioside catalyzed by E134A 1,3/1,4-β-glucanase. After isolation, the final hexasaccharides Galβ4Glcβ4Glcβ3Glcβ4Glcβ4Glcβ-OMe and Glcβ4Glcβ4Glcβ3Glcβ4Glcβ4Glcβ-OMe were obtained in 70-80% overall yields.