Peptidyl epoxides were designed as selective pseudo-mechanism-based inactivators of cysteine proteases. Both threo- and erythro-peptidyl epoxides were synthesized and tested as potential inactivators of serine proteases (chymotrypsin, subtilisin, and elastase) and of cysteine proteases (papain, cathepsin B, and clostripain). Four tripeptidyl epoxides (Cbz-Gly-Leu-Phe-epoxide, Cbz-Ala-Ala-Phe-epoxide, Cbz-Gly-Leu-Ala-epoxide, and Cbz-Ala-Ala-Ala-epoxide), bearing amino acid sequences similar to those of good substrates or known inhibitors of the serine proteases, were tested in this study. Neither the threo- nor the erythro-peptidyl epoxides exhibited any inhibitory activity toward the serine proteases, even at high concentration and long incubation time. Nor did the threo-peptidyl epoxides inhibit the cysteine proteases. On the other hand, the erythro-peptidyl epoxides were time- and concentration-dependent inactivators of the cysteine proteases. Furthermore, stereoselectivity toward the natural L-amino acid at the P-1 position was also exhibited upon inhibition of papain. In order to demonstrate selectivity within the cysteine protease family, two other erythro-peptidyl epoxides (Cbz-Phe-Ala-epoxide and Cbz-Phe-O-Bn-Tnr-epoxide) were synthesized and tested as inhibitors of the three cysteine proteases. These new peptidyl epoxides exhibited selective inactivation of cysteine proteases, with second-order rate constants (k(i)/K-i) ranging over 4 orders of magnitude (0.04-330 M(-1) s(-1)). Thus, this new family of highly selective cysteine protease inhibitors offers mechanistic implications and may have useful applications.