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 α-thrombin. The ‘fibrinogen-like’ recognition sequence D-Phe-Pro-Arg, is a template of high affinity for thrombin. This was modified by replacing the ‘PI’ 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 for 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 R is (3-methoxy)propyl, for which the kinetics of inhibition were analysed as competitive (2 μM) and slow, tight-binding (final Ki 20nM) respectively. Analysis...