Synthesis and Kinetic Studies of Diphenyl 1-(N-Peptidylamino)alkanephosphonate Esters and Their Biotinylated Derivatives as Inhibitors of Serine Proteases and Probes for Lymphocyte Granzymes

Diphenyl 1-(N-peptidylamino)alkanephosphonate esters are highly reactive, specific, and aqueously stable irreversible inhibitors which can be used to probe the functions of many serine proteases, including the lymphocyte granzymes. We synthesized 16 peptide phosphonates with Ala, Met, Phe, or Val P1 amino acid residues, including two biotinylated derivatives for future functional and biochemical characterization of granzymes. The reactivity of the inhibitors was characterized with human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine chymotrypsin, and the granzymes of natural killer (NK) cells, which include a number of proteolytic activities (Asp-ase, Met-ase, etc.) that cleave peptide substrates with these residues in the P1 position. The reactivity and specificity of the phosphonates depended on the length and sequence of the peptidyl moiety and on the leaving group. Z-Ala-Ala-AlaP(OPh)2was a good inhibitor of HLE and PPE (kobsd/[I] = 38 and 30m−1s−1, respectively) and had little reactivity with chymotrypsin. Z-Phe-Pro-PheP(OPh)2was a good inhibitor of chymotrypsin (kobsd/[I] = 17,000m−1s−1) and had little reactivity with the elastases. The leaving group of Z-MetP(OPh-4-Cl)2made it a more effective chymotrypsin inhibitor than Z-MetP(OPh)2(kobsd/[I] values of 142 and 30m−1s−1, respectively). With granzymes, the compounds reacted with a fraction of the Met-ase, chymase, and Ser-ase activities and lacked reactivity with Asp-ase and tryptase. Z-MetP(OPh-4-Cl)2was an excellent inhibitor of Met-ase 1. Bi-Aca-Aca-Phe-Leu-PheP(OPh)2appears to react specifically with one chymase while leaving other chymases untouched. Perforin-dependent lysis mediated by cytotoxic lymphocyte granules was inactivated by Z-Ala-Ala-AlaP(OPh)2, Z-MetP(OPh-4-Cl)2, Z-Leu-PheP(OPh)2, and Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. The biochemical properties and biological efficacy of these inhibitors make them suitable for cellular and physiological studies of granzyme function.