1344
J. Med. Chem. 1998, 41, 1344-1351
Azapeptides as Inhibitors and Active Site Titrants for Cysteine Proteinases Ruye Xing and Robert P. Hanzlik* Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506 Received November 25, 1997
Ester and amide derivatives of R-azaglycine (carbazic acid, H2NNHCOOH), R-azaalanine, and R-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 °C). Azaalanine derivatives Ac-L-Phe-NHN(CH3)CO-X inactivate papain ca. 400900-fold more slowly than their azaglycine analogues, consistent with the planar configuration at NR 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 70000a 4 1 0.03 0.05 0.05 12 110 a 0.3b
cathepsin B
54 90 730 310 7730 0 0
0.34b 18c amide > urea. Azapeptide amide 10, having a urealike carbonyl, is thus a very poor inactivator and not a substrate for papain. Even adding an electron-withdrawing p-nitrophenyl substituent to the potential leaving group nitrogen fails to make the carbonyl carbon in 11 attractive to the cysteine nucleophile of papain. Although papain hydrolyzes simple peptide amides quite well, it does not hydrolyze peptide thioamides unless they have an electron-withdrawing nitrogen substituent such as p-nitrophenyl;3 thus 12 is virtually inert to papain (Table 1). Nevertheless, compounds 1012 still interact noncovalently with papain reasonably well, as shown by the fact that they are reasonable
papain kcat/Km (M-1 s-1) 203 000 800 000 120 000 853 000 k2/Ki (M-1 s-1) 67 600 110 0.3
cathepsin B 123 000 450 000 80 000 776 000 7 730 18
