Bromoketone C-glycosides, a new class of β-glucanase inactivators

Although reliable methods have been developed for the labeling of the active site nucleophiles in glycosidases, no such reliable method has been developed for the identification of the acid/base catalyst. To address this problem two novel bromoketone affinity labels based on a beta-C-glucoside (6), and a beta-C-cellobioside (9), have been synthesized via a chemoenzymatic process and tested as inactivators of the beta-glucosidase from Agrobacterium sp. and the beta-glucanases from Cellulomonas fimi. The beta-glucosidase was inactivated by 6 in a time-dependent manner according to kinetic parameters of k(i) = 0.01 min(-1) and K-I = 3.1 mM. Electrospray ionization mass spectrometric analysis revealed that multiple labeling of the enzyme had occurred. The beta-endoglucanases, CenA and CenD, were inactivated stoicheometrically by 9 according to kinetic parameters of k(i) = 0.0155 min(-1); K-I = 0.35 mM and k(i) = 0.01 min(-1); K-I = 6.0 mM, respectively. These should therefore prove to be valuable reagents for the labeling of glycosidases.