Peptidic Phosphonylating Agents as Irreversible Inhibitors of Serine Proteases and Models of the Tetrahedral Intermediates

Peptide analogues incorporating an electrophilic phosphorus moiety (2–6) have been synthesized and studied as inhibitors of a variety of serine proteases. Inhibition is irreversible and, for α-lytic protease (ALP), shown to result from covalent binding to the active site serine hydroxyl [Bone, R., Sampson, N. S., Bartlett, P. A., & Agard, D. A. (1991) Biochemistry (following paper in this issue)]. For reaction of human leukocyte elastase (HLE) with the thiophenyl esters 6s-V (Boc-AAPVΓ[P=O(SPh)O]AA-OMe), 4s-V (BocAAPVΓ[P=O(SPh)O]-Me), and 3s-V (Boc-Vψ[P=O(SPh)O]AA-OMe), evidence is presented to suggest that the S₄–S₁] subsites, but not the S₁′ and S₂′ positions, are occupied by the inhibitors during the inactivation process. The selectivity that is observed between the proteases and the hexapeptide phosphonates 60-V (Boc-AAPVΓ[P=O(OPh)O]AA-OMe) and 60-F (Boc-AAPFΓ[P=O(OPh)O]AA-OMe) parallels that between these enzymes and their substrates: ALP and HLE are selectively inactivated by the Val^P-containing analogue 60-V, while subtilisin (SUB) shows a preference for the Phe^P derivative 60-F. A detailed kinetic analysis of the enzyme–inhibitor interactions was complicated by the susceptibility of the inhibitors to enzymatic degradation. The configuration at phosphorus was found not to have a significant influence on the rate at which the inhibitors react with the peptidases. Moreover, in the case of inactivation of ALP by the hexapeptide 60-V, the same covalent adduct is formed from both stereoisomers (Bone et al., 1991), indicating that one of these diastereomers undergoes substitution with retention of configuration.