Synthesis of peptide p-nitroanilides mimicking fibrinogen- and hirudin-binding to thrombin: Design of slow reacting thrombin substrates

The design and synthesis of 20 peptide p-nitroanilides is described. The nitroanilides are used as thrombin substrates that share uncommon properties. These substrates are tested for their applicability to measure thrombin generation in activated plasma. This technique requires substrates that must slowly but selectively be hydrolyzed by thrombin. To ensure selectivity, thrombin's natural substrate and its most potent inhibitor were used as lead compounds. Eighteen peptide p-nitroanilides were synthesized using human fibrinogen A alpha [7-16] decapeptide as lead structure since this fragment constitutes the minimal sequence which binds to thrombin with high affinity. Two other chromogenic substrates were designed using 5-amino-2-nitrobenzoic acid as the chromophore. A peptide giving subsite interactions with the fibrinogen recognition exosite was coupled to the carboxylic function. As the peptide segment, the carboxyl terminal part of hirudin (residues 50-65) was chosen to ensure highly specific thrombin recognition. From the 20 nitroanilides synthesized, we indeed obtained a number of compounds which can be used as substrate in the thrombin generation assay.