IRREVERSIBLE INHIBITION OF SERINE PROTEASES BY PEPTIDE DERIVATIVES OF (ALPHA-AMINOALKYL)PHOSPHONATE DIPHENYL ESTERS

Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates have been synthesized and are effective and specific inhibitors of serine proteases at low concentration. Z-Phe(P)(OPh)2 irreversibly reacts with chymotrypsin (k(obsd)/[I] = 1200 M-1 s-1) and does not react with two elastases. The best inhibitor for most chymotryspin-like enzymes including bovine chymotrypsin, cathepsin G, and rat mast cell protease II is the tripeptide Suc-Val-Pro-Phe(P)(OPh)2 which corresponds to the sequence of an excellent p-nitroanilide substrate for several chymases. The valine derivative Z-Val(P)(OPh)2 is specific for elastases and reacts with human leukocyte elastase (HLE, 280 M-1 s-1) but not with chymotrypsin. The tripeptide Boc-Val-Pro-Val(P)(OPh)2, which has a sequence found in a good trifluoromethyl ketone inhibitor of HLE, is the best inhibitor for HLE (K(obsd)/[I] = 27 000 M-1 s-1) and porcine pancreatic elastase (PPE, k(obsd)/[I] = 11 000 M-1 s-1). The rates of inactivation of chymotrypsin by MeO-Suc-Ala-Ala-Pro-Phe(P)(OPh)2 and PPE and HLE by MeO-Suc-Ala-Ala-Pro-Val(P)(OPh)2 were decreased 2-5-fold in the presence of the corresponding substrate, which demonstrates active site involvement. Only one of two diastereomers of Suc-Val-Pro-Phe(P)(OPh)2 reacts with chymotrypsin (146 000 M-1 s-1), and the enzyme-inhibitor complex had one broad signal at 25.98 ppm in the P-31 NMR spectrum corresponding to the Ser-195 phosphonate ester. Phosphonylated serine proteases are extremely stable since the half-time for reactivation was > 48 h for the inhibited elastases and 7.5-26 h for chymotrypsin. Peptidyl derivatives of diphenyl (alpha-aminoalkyl)phosphonates are relatively easy to synthesize, are chemically stable in buffer and in human plasma, form very stable derivatives with serine proteases, do not react with acetylcholinesterase, and thus should have considerable potential utility as therapeutic agents.