Irreversible inhibition of the HIV-1 protease by agents that specifically alkylate its catalytic aspartate residues is a potentially useful approach for circumventing the evolution of HIV strains that are resistant to protease inhibitors. Five haloperidol- and two FMOC-based epoxides of differing reactivities have been synthesized and tested as irreversible inhibitors of the HIV-1 protease (HIV-1 PR). Of these, two trisubstituted epoxides, a cis-1,2- disubstituted epoxide, a 1,1-disubstituted epoxide, and a monosubstituted epoxide function as irreversible inhibitors, but two trans-1,2-disubstituted epoxides do not. The most effective of the epoxides (6) inactivates HIV-1 PR with K-inact = 65 mu M and V-inact = 0.009 min(-1). 1,2-Epoxy-3-(p-nitrophenoxy)propane (EPNP), a nonspecific inactivating agent for aspartyl proteases, has been used to validate a protocol for establishing the stoichiometry and site of protein alkylation. Mass spectrometric analysis of the inactivated enzyme shows that one molecule of either EPNP or the cyclic 1,2-disubstituted epoxide 6 is covalently bound per HIV-1 PR dimer. Mass spectrometric sequencing of labeled proteolytic peptides shows that both inhibitors are covalently bound to a catalytic aspartate residue. The covalent binding of three alpha,beta-unsaturated ketone derivatives of haloperidol has been similarly examined. Analysis of HIV-1 PR inactivated by these agents establishes that they bind covalently to the two cysteines and the N-terminal amino group but not detectably to the catalytic aspartate residues. The results indicate that aspartate-targeted inactivation of HIV-1 PR depends on (a) matching the reactivity of the alkylating functionality to that of the aspartates, preferably by exploiting the two-aspartate catalytic motif of the protease to activate the alkylating agent, and (b) appropriate positioning of the alkylating functionality within the active site. These requirements are readily met by a monosubstituted, 1,1-disubstituted, or cyclic cis-1,2-disubstituted epoxide but not by trans-1,2-disubstituted epoxides or alpha,beta-unsaturated ketones.