X-Ray Crystallographic Analyses of Human α-Thrombin Complexed to Peptidyl Aminophosphonates: Evidence of a Binding Mechanism

We sought to study O,O-Diphenyl dipeptidylaminophosphonates and their interaction with the coagulation protease, human a-thrombin. The 'fibrinogen-like' recognition sequence D-Phe-Pro-Arg, is a template of high affinity for thrombin. This was modified by replacing the 'P1' Arg by a neutral side chain, and the unnatural amino acid diphenylalanine was used in place of the 'P3' Phe. The tripeptides of general formula D-Dpa-Pro-NHCHRP(OPh)(2) are highly selective for thrombin amongst other serine proteases; and, more importantly fbr the design of an efficacious anti-thrombotic agent, show particularly low activity toward other coagulation serine proteases of the cascade. However the kinetics of thrombin inhibition were seen to be dependent on the compounds' structure. To explain this we have studied compounds (1), where R is pentyl and compound (2) where ii is (3-methoxy)propyl, for which the kinetics of inhibition were analysed as competitive (2 mu M) and slow, tight-binding (final K-i 20nM) respectively. Analysis of the X-Ray crystal structure of these compounds complexed to human a-thrombin, unusually shows no covalent bond formed between the phosphorus nucleus and the serine 195 in the catalytic site of the enzyme. These observations are at variance to the proposed mechanism of action of other phosphorus based serine protease inhibitors.