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

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Abstract

A tetrapeptide combinatorial library, considered as chromogenic substrates of bovine β-trypsin, was synthesized by the solid phase method. The peptides contain an analog of p-nitroanilide, obtained by attaching 5-amino-2-nitrobenzoic acid (Anb5,2) 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-Anb5,2-NH2, 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.

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Materials and methods

Peptide synthesis. Peptides for kinetic studies and peptide library were synthesized manually by the solid-phase method using Fmoc chemistry, as described previously [8]. TentaGel S RAM (substitution 0.23 meq/g) (RAPP Polymere, Germany) was used as a support. The amino acid derivatives used for the synthesis were as follows: Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Pro, Fmoc-Val, Fmoc-Arg(Pbf), Fmoc-Lys(Boc), Fmoc-Ser(tBu), Fmoc-Asp(OtBu), Fmoc-Glu(OtBu), and Fmoc-Tyr(tBu). 5-Amino-2-nitrobenzoic acid

Results and discussion

The deconvolution of the peptide library (consisting of 114=14641 peptides) against bovine β-trypsin is summarized in Fig. 1. The results obtained indicate that the aromatic Phe residue present in position P4 of the synthesized peptide library displays the strongest interaction with the enzyme investigated. Interestingly, the other aromatic residue (Tyr) present in the library had a significantly lower impact on the substrate activity. With the Phe residue fixed in position P4, sublibraries

Acknowledgements

This work was supported by the Polish State Committee for Scientific Research (KBN), Grant No. 1007/T09/2003/24, and by the University of Gdańsk (Grant No. BW-8000-5-0324-3).

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    The peptide library and individual peptides for kinetic studies were synthesized manually by the solid-phase method using fluorenyl-9-methoxycarbonyl (Fmoc) chemistry as described previously [12].

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