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

Abstract

A series of unsubstituted (1→3,1→4)-β-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)-β-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)-β-D-glucan 4-glucanohydrolase from Bacillus licheniformis has been used in organic media to catalyse the condensation of 3-O-β-D-glucopyranosyl-β-D-glucopyranosyl fluoride (Glcβ3GlcβF, compound 1) with cellobiose (Glcβ4Glc, 2), cellotriose (Glcβ4Glcβ4Glc, 3), cellotetraose (Glcβ4Glcβ4Glcβ4Glc, 4) and cellopentaose (Glcβ4Glcβ4Glcβ4Glcβ4Glc, 5), to produce the (1→3,1→4)-β-D-glucooligosaccharides, Glcβ3Glcβ4Glcβ4Glc 6, Glcβ3Glcβ4Glcβ4Glcβ4Glc 7, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glc 8, Glcβ3Glcβ4Glcβ4Glcβ4Glcβ4Glcβ4Glc 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 α-D-glucopyranosyl phosphate 10 to the 4-position of the non-reducing terminus of the trisaccharide Glcβ3Glcβ4Glc 11, to generate the (1→3,1→4)-β-D-glucooligosaccharides, Glcβ4Glcβ3Glcβ4Glc 12, Glcβ4Glcβ4Glcβ3Glcβ4Glc 13, Glcβ4Glcβ4Glcβ4Glcβ3Glcβ4Glc 14 in 14, 10 and 5% yield, respectively, from compound 11.