Synthetic receptors for the differentiation of phosphorylated peptides and synthesis and use of tetrahydrofuran amino acids

Abstract

In the first part of the work a combination of bis(Zn(II)-cyclen) triazine metal complex binding sites with guanidinium moieties or Zn(II)-NTA complexes leads to artificial receptors for the differentiation of phosphorylated peptides which contain either a histidine side chain or a glutamic acid side chain as a second binding site. General methods for the synthesis of such bidentate receptors consisting either of two bis(Zn(II)-cyclen) triazine complexes or of one bis(Zn(II)-cyclen) triazine complex and a guanidinium moiety were developed and several receptors varying in length were synthesized. These complexes in combination with previously prepared complexes consisting of a bis(Zn(II)-cyclen) triazine complex in combination with a Zn(II)-NTA complex were tested in a fluorescence polarization assay against the peptide-protein interaction of different peptides and their corresponding proteins STAT1, STAT3 and GST-Lck. All receptors were found to be active showing an influence on the peptide-protein binding at receptor concentrations of 200 - 600 uM. Unfortunately, it was also found that the incorporation of a second binding site into the receptors did not alter the activity of the compounds significantly. In addition, the synthesized complexes were used to determine their binding affinities towards the fluorescently labeled peptides 5/6-Carboxyfluorescein-Gly-pSer-Ala-Ala-His-Val-NH2 and 5/6-Carboxyfluorescein-Gly-pSer-Ala-Ala-Glu-Val-NH2. The right combination of binding moieties leads to nanomolar peptide binding affinities in aqueous media at physiological pH. To the best of our knowledge these are the highest affinities of phosphopeptide binding by artificial receptors reported so far. Depending on the second functional group (His or Glu/Asp) beside the phosphate ester, selectivities of up to three orders of magnitude of the binding constant are observed. Although the bidentate receptors did not show the supposed increased activity in the STAT assay, their remarkably high binding affinities and also selectivities to certain peptide sequences could make them a versatile tool for the inhibition of peptide-protein interactions. Therefore further effort should be undertaken to test the receptors in other biologically relevant systems. In the second part the unnatural Calpha-tetrasubstituted tetrahydrofuran alpha-amino acid (TAA) was used for the synthesis of 13 new peptides based on an alternated sequence of the S- or R-configured alpha-amino acid valine and the TAA. Homo- and heterochiral stereoisomers with up to eight residues in length were systematically synthesized in good yields and high purity by solution phase chemistry. X-ray crystallography, NMR- and CD-measurements showed that all homochiral peptides, even the tetrapeptides, form helical structures in the solid state and in solution. The handedness of the helix is determined by the use of S-amino acids for right-handed or R-amino acids for left-handed peptide helices. The stable and predictable secondary structure of the new peptides makes them suitable for applications as scaffolds and peptidomimetics. Additional moieties e.g. dyes, can be introduced by metal catalyzed functionalization of the brominated arene substituent. In addition the TAA was used in CuI-catalyzed N-arylation which proceed in moderate to good yields with a variety of amines allowing to introduce aliphatic aldehydes and different functional group containing amines into the side chain of the protected TAA. An intramolecular Cu(I)-catalyzed N-arylation reaction was used for the synthesis of the 17-membered macrocycle compounds, but the obtained yield was low. Pd(0)-catalyzed O-arylations gave higher yields for the macrocylization of non-natural tripeptides containing the TAA: A 15-membered macrocycle was obtained in good yield using Pd(OAc)2 and a sterically demanding ligand. The examples illustrate that copper(I)-catalyzed N-arylations and palladium(0)-catalyzed O-arylations give access to side chain modified derivatives of the unnatural amino acid TAA and macrocyclic peptidomimetics