Regioselectivity in acylation of oligosaccharides catalyzed by the metalloprotease thermolysin

Investigation of the acylation scope of carbohydrates by metalloprotease thermolysin immobilized on Celite as biocatalyst has been carried out. The reactions were performed in DMSO, a good solvent for carbohydrates, where the enzyme has previously shown its activity in transesterifications of sucrose, maltose and maltose-containing oligosaccharides. Surprisingly, no reaction was observed for glucose or the glucose-containing disaccharides, trehalose and lactose. In contrast, laurate monoesters of several sucrose-containing tri- and tetrasaccharides were synthetized through a one step transesterification using vinyl laurate as the acylating agent. Enzyme regioselectivity was accurately determined by HPLC/MS and the structure of the main regioisomers was established by a combination of NMR experiments. The preferred position of acylation in all cases was the 2-OH of the α-d-glucopyranose moiety linked 1→2 to the β-d-fructofuranose unit. These results correlate with the regioselectivity observed in the case of the disaccharide sucrose. A general carbohydrate binding motif for catalysis by thermolysin is proposed.

Graphical Abstract