Selection of new chromogenic substrates of serine proteinases using combinatorial chemistry methods

Chemical synthesis, physicochemical characterization and kinetic investigations of a tetrapeptide library of chromogenic substrates containing the amide of 5-amino-2nitrobenzoic acid (Anb(5,2)-NH2) at their C-termini arc reported. Anb(5,2)-NH2 served as a chromophore released upon enzymatic action. The library consisting of 9567 peptides was synthesized using the portioning-mixing method and was screened against bovine alpha-chymotrypsin and human leukocyte elastase in solution applying an iterative approach. The selected chromogenic substrates were resynthesized and further modified at their N- and C-termini. Finally, two sequences, Z-Phe-Ala-Thr-Tyr-Anb(5,2)-NH2 and Z-Phe-Phe-Pro-Val-Anb(5,2)-NH2, were obtained as highly specific substrates for bovine alpha-chymotrypsin and human leukocyte elastase, respectively. The method of synthesis and selection of chromogenic substrates of serine proteinases described herein is straightforward and can be applied to design substrates for other proteases.