The design, synthesis, and properties of the first mechanism-based inactivators for bacterial aminoglycoside 3’-phosphotransferases [APH(3’)s], enzymes which mediate resistance to kanamycins and related aminoglycoside antibiotics, are described. These molecules, whose design was based on the structures of aminoglycosides neamine and kanamycin B, serve as substrates for APH(3’)s. However, as a deviation from the turnover process subsequent to phosphorylation, enzyme inactivation ensues. A mechanism is proposed for the inactivation chemistry. The general chemical principles described for the inactivation strategy herein hold the promise for the development of novel mechanism-based inactivators for other "transferases", such as protein kinases, acetyltransferases, nucleotidyltransferases, sulfotransferases, and the like.