Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synth etic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent, To demonstrate the strategy, we synthesized a tetrap eptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 me mbers of the library were synthesized on solid support with an alkane sulfo namide linker, and then displaced from the solid support by condensation wi th a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-l ike enzymes, plasmin and thrombin, which are involved in the blood coagulat ion pathway, The optimal P4 to P2 substrate specificity for plasmin was P4- Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological s ubstrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found i n many of the physiological substrates of thrombin, Single-substrate kineti c analysis of plasmin and thrombin was used to validate the substrate prefe rences resulting from the PS-SCL, By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identif ied potential determinants of the defined substrate specificity. This metho d is amenable to the incorporation of diverse substituents at the P1 positi on for exploring molecular recognition elements in proteolytic enzymes.