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

Glycosynthases are engineered glycosidases which are hydrolytically inactiv e yet efficiently catalyse transglycosylation reactions of glycosyl fluorid e donors, and are thus promising tools for the enzymatic synthesis of oligo saccharides. Two endo-glycosynthases, the E134A mutant of 1,3/1,4-beta -glu canase from Bacillus licheniformis and the E197A mutant of cellulase Cel7B from Humicola insolens, were used in coupled reactions for the stepwise syn thesis of hexasaccharide substrates of 1,3/1,4-beta -glucanases. Because th e two endo-glycosynthases show different specificity, towards laminaribiosy l and cellobiosyl donors, respectively, the target hexasaccharides were pre pared by condensation of the corresponding disaccharide building blocks thr ough sequential addition of the glycosynthases in a "one-pot" process. Diff erent strategies were used to achieve the desired transglycosylation betwee n donor and acceptor in each step, and to prevent unwanted elongation of th e first condensation product and polymerization (self-condensation) of the donor: 1) selection of disaccharide donors differing in the configuration o f the hydroxyl substituent normally acting as acceptor, 2) temporary protec tion 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 a n acceptor. The best procedure involved the condensation of alactosyl or 4( 11)-O-tetrahydropyranyl-alpha -cellobiosyl fluorides with alpha -laminaribi osyl fluoride, catalyzed by E197A Cel7B, to give tetrasaccharide fluorides, which were then the donors for in situ condensation with methyl beta -cell obioside catalyzed by E134A 1,3/1,4-beta -glucanase. After isolation, the f inal hexasaccharides Gal beta 4Glc beta 4Glc beta 3Glc beta 4Glc beta 4Glc beta -OMe and Glc beta 4Glc beta 4Glc beta 3Glc beta 4Glc beta 4Glc beta -O Me were obtained in 70-80% overall yields.