Mixed-linkage cellooligosaccharides: A new class of glycoside hydrolase inhibitors

A new class of inhibitors for beta -D-glycoside hydrolases, in which a sing le alpha-(1 -->4)-glycosidic bond is incorporated into an otherwise all-bet a-(1 -->4)-linked oligosaccharide, is described. Such mixed beta/alpha -lin kage cellooligosaccharides are not transition-state mimics, but instead are capable of utilising binding energy from numerous subsites, spanning eithe r side of the catalytic centre, without the need for substrate distortion. This binding is significant; a mixed alpha/beta -D-tetrasaccharide acts com petitively on a number of cellulases, displaying inhibition constants in th e range of 40-300 muM. Using the Bacillus agaradhaerens enzyme Cel5A as a m odel system, one such mixed beta/alpha -cellooligosaccharide, methyl 4'',4' ''-dithio-alpha -cellobiosyl-(1 -->4)-beta -cellobioside, displays a K-i va lue of 100 muM, an inhibition at least 150 times better than is observed wi th an equivalent all-beta -linked compound. the three-dimensional structure of B. agaradhaerens Cel5A in complex with methyl 4'',4'''-dithio-alpha -ce llobiosyl-(1 -->4)-beta -cellobioside has been determined at 1.8 Angstrom r esolution. This confirms the expected mode of binding in which the ligand, with all four pyranosides in the C-4(1) chair conformation, occupies the -3 , -2 and +1 subsites whilst evading the catalytic (-1) subsite. Such "by-pa ss" compounds offer great scope for the development of a new class of beta -D-glycoside hydrolase inhibitors.