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.