Synthesis of Peptidyl Ene Diones: Selective Inactivators of the Cysteine Proteinases

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Abstract

A series of synthetic peptides in which the C-terminal carboxyl grouping (-CO2H) of each has been chemically converted into a variety of ene dione derivatives (-CO-CH=CH-CO-X; X = -H, -Me, -OBut, -OEt, -OMe, -CO-OMe), have been prepared and tested as inactivators against typical members of the serine and cysteine protease families. For example, the sequences Cbz-Pro-Phe-CH=CH-CO-OEt (I) which fulfils the known primary and secondary specificity requirements of the serine protease chymotrypsin, and Cbz-Phe-Ala-CH=CH-CO-OEt (II) which represents a general recognition sequence for cysteine proteases such as cathepsins B, L and S, have been tested as putative irreversible inactivators of their respective target proteases. It was found that, whereas II, for example, functioned as a time-dependent, irreversible inactivator of each of the cysteine proteases, I behaved only as a modest competitive reversible inhibitor of chymotrypsin. Within the simple ester sequences Cbz-Phe-Ala-CH=CH-CO-R, the rank order of inhibitor effectiveness decreases in the order R = -OMe > -OEt >> -OBut. It was also found that the presence of both an unsaturated double bond and an ester (or α-keto ester) moiety were indispensable for obtaining irreversible inactivators. Of the irreversible inactivators synthesized, Cbz-Phe-Ala-CH=CH-CO-CO-OEt (which contains a highly electrophilic α-keto ester grouping) was found to be the most effective exhibiting, for example, second-order rate constants of approximately 1.7 × 106m−1min−1 and approximately 4.9 × 104m−1min−1 against recombinant human cathepsin S and human spleenic cathepsin B, respectively. This initial study thus holds out the promise that this class of inactivator may well be specific for the cysteine protease subclass.

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