Ester and amide derivatives of alpha-azaglycine (carbazic acid, H2NNHCOOH), alpha-azaalanine, and alpha-azaphenylalanine (i.e., Ac-l-Phe-NHN(R)CO-X, where X = H, CH3, or CH2Ph, respectively) were synthesized and evaluated as inhibitors of the cysteine proteinases papain and cathepsin B. The ester derivatives inactivated papain and cathepsin B at rates which increased dramatically with leaving group hydrophobicity and electronegativity. For example, with 8 (R = H, X = OPh) the apparent second-order rate constant for papain inactivation was 67 600 M-1 s-1. Amide and P1-thioamide derivatives do not inactivate papain, nor are they substrates; instead they are weak competitive inhibitors (0.2 mM < Ki < 4 mM). Inactivation of papain involves carbamoylation of the enzyme, as demonstrated by electrospray mass spectrometry. Active site titration indicated a 1:1 stoichiometry for the inactivation of papain with 8, and both inactivated papain and cathepsin B are highly resistant to reactivation by dialysis (t1/2 > 24 h at 4 degrees C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400- 900-fold more slowly than their azaglycine analogues, consistent with the planar configuration at Nalpha of the P1 residue and the very substantial stereoselectivity of papain for L- vs D- residues at the P1 position of its substrates. Azaglycine derivative 9 (R = H, X = OC6H4NO2-p) inactivates papain extremely rapidly (>70 000 M-1 s-1), but it also decomposes rapidly in buffer with release of nitrophenol (kobs = 0.13 min-1); under the same conditions 8 shows <7% hydrolysis over 24 h. This nitrophenol release probably involves cyclization to an oxadiazolone since 17 (R = CH3, X = OC6H4NO2-p), which cannot form an isocyanate, releases nitrophenol almost as rapidly (kobs = 0.028 min-1). Cathepsin C, another cysteine proteinase with a rather different substrate specificity (i.e., aminopeptidase), was not inactivated by 8, indicating that the inactivation of papain and cathepsin B by azapeptide esters is a specific process. Their ease of synthesis coupled with good solution stability suggests that azapeptide esters may be useful as active site titrants of cysteine proteinases and probes of their biological function in vivo.