Trifunctional Resins

Figure 1: Principle structure of a trifunctional resin with 2 different cleavage sites plus 1 permanent linkage.

General

Purpose: sequential cleavage of peptides or peptide amides.

These resins contain 5 – 10% non-cleavable covalently attached ligands, 40 – 45% linker 1 for peptide acid/ peptide amide released by 5% TFA treatment and 40 – 45% linker 2 for peptide acid/ peptide amide released by 95% TFA treatment. The peptide, liberated by 5% TFA treatment must be separated from the resin and deprotected with 95% TFA in a follow up step.

Available as Polystyrene AM AC HMPA, Polystyrene AM AC RAM, TentaGel® AC HMPA and TentaGel® AC RAM.

W. Rapp^a, T. Ferain^b, M. Detheux^b

Introduction

Libraries synthesized by the split/ mix approach where each individual bead contains only one defined compound can be analyzed in different ways. On bead screening is the simplest way where the positive beads are isolated and the compounds analyzed either direct on the beads (e.g. Edman degradation) or after they are released from the beads. Another way is to add tags or codes to the resin during synthesis. Positive beads are then isolated, decoded and the chemical history can be read out by analysis of the code. This method often causes problems and restrictions during synthesis. An other way is the use of multiple cleavable resins^1-4 if the peptides are screened in a solution based assay. But many multiple release resins suffer from complicated handling or incompatibility with the assay system. We here describe a multiple release system for screening peptide and peptide amide libraries by an iterative process to identify the active compound.

Results and discussion

Figure 2: Principle of library screening and hit identification by an iterative process using a trifunctional resin with acid sensitive linkers.

Figure 3: Investigation of acid labile linkers and their compatibility during acid treatment. From the results linker 1 (AC), linker 2 (HMPA) and the amide linker 3 (RAM) were selected.

Figure 4: Synthesis of the trifunctional resin templates having two acid sensitive arms and one amino function (ß-Ala) which immobilize the peptide via an amide bond onto the resin. Template 1 is for the release of free peptides, template 2 for peptide amides.

Bradykinin was synthesized as a model peptide on resin template 1 by Fmoc chemistry. Acid treatment with 5% TFA releases the peptide from the AC handle. The cleaved peptide was consequently treated with high concentrated TFA for deprotection. The peptides linked via the HMPA handle and via the ß-Ala stay on the resin. At this stage the resin can be washed neutral and stored. A follow up treatment with 95 % TFA cleaves off the peptide from the HMPA handle and deprotect the peptides.

Neuropeptide NPFF (SQAFLFQPQRF NH2) was synthesized on template 2 as a model peptide by Fmoc chemistry. Acid treatment with 5% TFA in DCM releases the peptide from the AC linker as a C-terminal protected peptide amide. Treatment of the cleaved peptide amide with 95% TFA deprotect the peptide and forms the free peptide amide. The peptides linked via the ß-Ala and the HMPA-RAM arm stay on the resin. Again at this stage the resin can be neutralized and stored for further applications. Treatment with 95% TFA will then deprotect and cleave the peptide from the HMPA-RAM arm to form the free peptide amide. The peptide linked via the amide bond to the resin is now also deprotected but still immobilized and ready for analysis by Edman degradation.

The calculated total loading of single beads was confirmed by single bead amino acid analysis 5. To confirm the expected and calculated release from each handle arm, single beads out of the Bradykinin synthesis are treated by the described way (5% TFA, 95% TFA, washed, dried, followed by 95% TFA) and dose response curves are taken for each release. The expected results are confirmed by the dose response experiment.

Figure 5: results of Branykinin synthesis.

Figure 6: Results of Neuropeptide NPFF synthesis.

Figure 7: Functional activation of B2 receptor expressing cells (CHO-K1; Eursocreen, Aequoscreen^TM ES-090-A) by bradykinin: Dose response curves for bardykinin from commercial source (red) or released from successive acid cleavages from polystyrene beads (green and blue).

Original publication: W. Rapp^a, T. Ferain^b, M. Detheux^b

^a Rapp Polymere GmbH, Ernst Simon Str.9, 72072 Tuebingen, Germany.

^bEuroscreen S.A. Route de Lenik 802, 1070 Brussels, Belgium.