CHEMOENZYMIC SYNTHESIS OF (1-]3,1-]4)-BETA-D-GLUCOOLIGOSACCHARIDES FOR SUBSITE MAPPING OF (1-],3,1-]4)-BETA-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 affinitie s at individual subsites of plant and bacterial (1-->3,1-->4)-beta-D-g lucan 4-glucanohydrolases (EC 3.2.1.73) can be determined, has been sy nthesised 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-bet a-D-glucopyranosyl-beta-D-glucopyrariosyl fluoride (Glc beta 3Glc beta F, compound 1) with cellobiose (Glc beta 4Glc, 2), cellotriose (Glc b eta 4Glc beta 4Glc; 3), cellotetraose (Glc beta 4Glc beta 4Glc beta 4G lc, 4) and cellopentaose (Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc; 5), to produce the (1-->3,1-->4)-beta-D-glucooligosaccharides, Glc be ta 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 b eta 3Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc beta 4Glc 9. Synthesi sed oligosaccharides 6-9 were isolated in yields of 15-45%, compared w ith compound 1. In a second series of syntheses, a cellodextrin phosph orylase (EC 2.4.1.49) from Clostridium thermocellum was used to sequen tially transfer glucosyl residues from alpha-D-glucopyranosyl phosphat e 10 to the 4-position of the non-reducing terminus of the trisacchari de Glc beta 3Glc beta 4Glc 11, to generate the (1-->3, 1-->4)-beta-D-g lucooligosaccharides, Glc beta 4Glc beta 3Glc beta 4Glc 12, Glc beta 4 Glc beta 4Glc beta 3Glc beta 4Glc 13, Glc beta 4Glc beta 4Glc beta 4Gl c beta 3Glc beta 4Glc 14 in 14, 10 and 5% yield, respectively, from co mpound 11.