The design and in vitro biochemical evaluation of two novel classes of mechanism-based inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented enzyme-induced sulfonamide fragmentation cascade is described. The inhibitors incorporate in their structure either an appropriately-functionalized saccharin scaffold, or a 1,2, 5-thiadiazolidin-3-one-1,1-dioxide scaffold. The inactivation of the enzyme by these inhibitors was found to be efficient, time-dependent and to involve the active site. Biochemical, HPLC, and mass spectrometric studies show that the interaction of these inhibitors with HLE results in the initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral intermediate with the active site serine (Ser-195). Collapse of the tetrahedral intermediate with tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine which can either react with water to form a relatively stable acyl enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue (His-57). The results also demonstrate convincingly the superiority of the 1, 2, 5-thiadiazolidin-3-one-1,1-dioxide scaffold over the saccharin scaffold in the design of inhibitors of (chymo)trypsin-like serine proteases.