Novel amidine-containing peptidyl phosphonates as irreversible inhibitors for blood coagulation and related serine proteases

Abstract

A series of new peptidyl (alpha-aminoalkyl)phosphonate diphenyl esters containing the 4-amidinophenyl group were synthesized and tested as irreversible inhibitors for thrombin and other trypsin-like enzymes. These phosphonates irreversibly inhibited several coagulation enzymes and trypsin. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 is the best human thrombin inhibitor in the series with a k(obs)/[I] value of 11,000 M-1 s-1, and it inhibits thrombin more than 5-fold more effectively than the other enzymes tested. Z-(4-AmPhGly)P(OPh)2 is the best inhibitor for plasma kallikrein with a k(obs)/[I] value of 18,000 M-1 s-1. Generally, the (4-AmPhGly)P(OPh)2 derivatives are better inhibitors of thrombin and trypsin than the corresponding (4-AmPhe)P(OPh)2 derivatives which contain an extra CH2 separating the amidinophenyl group from the peptide backbone. The amidino phosphonates did not inhibit acetylcholinesterase and were chemically stable in neutral buffers. In addition, the inhibited trypsin derivative did not regain any enzyme activity after removal of excess inhibitor and incubation in a pH 7.5 buffer for 1 day. Boc-D-Phe-Pro-(4-AmPhGly)P(OPh)2 and D-Phe-Pro-(4-AmPhe)P(OPh)2 prolonged the prothrombin time ca. 2-fold and prolonged the activated partial thromboplastin time ca. 3-4-fold in human plasma at concentrations of 63 and 125 microM, respectively. The novel amidine-containing peptidyl phosphonates reported here are thus effective anticoagulants in vitro, and they may have utility for use in vivo.

Constitutive Production of Catalytic Antibodies to a Staphylococcus aureus Virulence Factor and Effect of Infection

“…trypsin in Fig. 4B, lane 1 (25,46,47). Irreversible phosphonate 1-IgG complexes were evident by denaturing electrophoresis of boiled reaction mixtures (Fig.…”

Proteolytic Inhibition in Regulating the Insulin-Like Growth Factor-Binding Proteins in Prostate Cancer

“…Consequently, the selectivity and the irreversible inhib ition make these phosphonate esters useful in the study of the regulatory role of PSA in physiology and pathology. Peptidyl (α-aminoalkyl) derivatives of phosphonates diphenyl esters have been shown to be specific and potent irreversible inhibitors of elastases and various serine enzymes [51]. Due to the stability and irreversible nature of these inhibitions [52], the peptidyl diphenyl phosphonate esters based on modeling studies of the active site of PSA can be further explored as potentially specific inhibitors for PSA.…”

Toward Selective Covalent Inactivation of Pathogenic Antibodies

“…CRAs-Diphenyl N-(6-biotinamidohexanoyl)amino(4-amidinophenyl)methanephosphonate (compound 1) was prepared from diphenylamino(4-amidinophenyl)methanephosphonate (19,20) and 6-biotinamidohexanoic acid (Anaspec, San Jose, CA) by the aid of (benzotriazole-1-yl)oxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) (Novabiochem, San Diego, CA). The HPLC-purified material (retention time 20.76 min, purity 95% (220 nm); YMC ODS-AM column (4.6 ϫ 250 mm), 0.05% trifluoroacetic acid in water (A):0.05% trifluoroacetic acid in acetonitrile (B), from 90:10 to 20:80 in 45 min (1.0 ml/min)) was characterized by electrospray ionization-mass spectrometry (observed m/z 721.…”