A simple method for selection of trypsin chromogenic substrates using combinatorial chemistry approach.

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine beta-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb(5,2)) to the C-termini. Deconvolution of the peptide library, performed in solution using an iterative method, yielded four efficient trypsin substrates. The most active one, Phe-Val-Pro-Arg-Anb(5,2)-NH(2), appeared to be 125-fold more active than Bz-D,L-Arg-pNA (BAPNA) used as a reference compound. The reported method of designing trypsin chromogenic substrate libraries is straightforward. Such p-nitroanilides may be useful for the investigation of any protease substrate specificity.