Peptidyl fluoromethyl ketones as thiol protease inhibitors

The fluoromethyl ketone derivatives of peptides are now available through several synthetic approaches and can be examined with respect to their properties as protease inhibitors. It had been expected that the fluoro atom might be too inert for nucleophilic displacement and that irreversible inactivation might not be achievable by this type of derivative in contrast to chloromethyl ketones. However, with serine and cysteinyl proteases, alkylation of the enzyme does take place although the rates are not similar to those of the chloromethyl ketones. Of the two classes, thiol proteases are more readily inactivated and the fluoromethyl ketones are almost as effective as the chloromethyl ketones. Our observations confirm and extend those of Rasnick (Anal. Biochem. 149, 461-465 (1985)). The structure of the peptidyl portion of the reagent controls specificity of inhibition in the typical manner of affinity-labels for proteases. However, fluoromethyl ketones are considerably less reactive to nucleophiles such as the thiol group of glutathione, than chloromethyl ketones and, therefore, this new class of inhibitors may provoke fewer side reactions when used in biological studies.