Chemoenzymic synthesis of (1→3,1→4)-β-d-glucooligosaccharides for subsite mapping of (1→,3,1→4)-β-d-glucan endohydrolases

dA series of unsubstituted (1-->3,1-->4)-beta-D-glucooligosaccharides, designed for subsite mapping in which the number of glucosyl-binding subsites and the subsite-binding/transition state activation affinities at individual subsites of plant and bacterial (1-->3,1-->4)-beta-D-glucan 4-glucanohydrolases (EC 3.2.1.73) can be determined, has been synthesised through chemical and enzymic procedures. A recombinant (1-->3,1-->4)-beta-D-glucan 4-glucanohydrolase from Bacillus licheniformis has been used In organic media to catalyse the condensation of 3-O-beta-D-glucopyranosyl-beta-D-glucopyrariosyl fluoride (Glc beta 3Glc beta F, compound 1) with cellobiose (Glc beta 4Glc, 2), cellotriose (Glc beta 4Glc beta 4Glc; 3), cellotetraose (Glc beta 4Glc beta 4Glc beta 4Glc, 4) and cellopentaose (Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc; 5), to produce the (1-->3,1-->4)-beta-D-glucooligosaccharides, Glc beta 3Glc beta 4Glc beta 4Glc 6, Glc beta 3Glc beta 4Glc beta 4Glc beta 4Glc 7, Glc beta 3Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc 8, Glc beta 3Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc 9. Synthesised oligosaccharides 6-9 were isolated in yields of 15-45%, compared with compound 1. In a second series of syntheses, a cellodextrin phosphorylase (EC 2.4.1.49) from Clostridium thermocellum was used to sequentially transfer glucosyl residues from alpha-D-glucopyranosyl phosphate 10 to the 4-position of the non-reducing terminus of the trisaccharide Glc beta 3Glc beta 4Glc 11, to generate the (1-->3, 1-->4)-beta-D-glucooligosaccharides, Glc beta 4Glc beta 3Glc beta 4Glc 12, Glc beta 4Glc beta 4Glc beta 3Glc beta 4Glc 13, Glc beta 4Glc beta 4Glc beta 4Glc beta 3Glc beta 4Glc 14 in 14, 10 and 5% yield, respectively, from compound 11.