Peptidyl inverse esters of p-methoxybenzoic acid: a novel class of potent inactivator of the serine proteases

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Abstract
A series of novel synthetic peptides, containing a C-terminal β-amino alcohol linked to p-methoxybenzoic acid via an ester linkage, have been prepared and tested as inhibitors against typical members of the serine protease family. For example, the sequences Ac-Val-Pro-NH-CH-(CH2-C6H5)-CH2O-CO-C6H4-OCH3 (I) and Ac-Val-Pro-NH-CH-[CH-(CH3)2]-CH2O-CO-C6H4-OCH3 (II), which fulfil the known primary and secondary specificity requirements of chymotrypsin and elastase respectively, have been found to behave as exceptionally potent irreversible inactivators of their respective target protease. Thus I was found to inactivate chymotrypsin with an overall second-order rate constant (k2/Ki) of approx. 6.6×106 M-1·s-1, whereas II is an even more potent inactivator of human neutrophil elastase, exhibiting a second-order rate constant of inactivation of approx. 1.3×107 M-1·s-1. These values represent the largest rate constants ever reported for the inactivation of these proteases with synthetic peptide-based inactivators. On prolonged incubation in substrate-containing buffers, samples of the inactivated proteases were found to regain activity slowly. The first-order rate constants for the regeneration of enzymic activity from chymotrypsin and human neutrophil elastase inactivated by I and II respectively were determined to be approx. 5.8×10-5 s-1 and approx. 4.3×10-4 s-1. We believe that the most likely mechanism for the inactivation and regeneration of enzymic activity involves the formation and subsequent slow hydrolysis of long-lived acyl enzyme intermediates.