A Constrained Tetrapeptide as a Model of Cu(I) Binding Sites Involving CuS Clusters in Proteins.

Peptide design is an efficient strategy to create relevant models of natural metal binding sites found in proteins. The two short tetrapeptides Ac-Cys-dPro-Pro-Cys-NH (CdPPC) and Ac-Cys-Pro-Gly-Cys-NH (CPGC) were synthesized and studied as mimics of Cu(I) binding sites involved in Cu homeostasis. Both sequences contain β turn inducing motifs to rigidify the peptide backbone structure and thereby preorganize the metal-binding side chains. The more constrained structure of the peptide CdPPC with respect to CPGC was evidenced by the measurements of the temperature coefficients of the amide protons by H NMR, which suggest a solvent-shielded intramolecular hydrogen bond in CdPPC, and no H-bond in CPGC. The Cu(I) complexes were studied by UV, circular dichroism (CD), and NMR spectroscopies as well as electrospray ionization mass spectrometry (ESI-MS) experiments in aqueous solution at physiological pH. The complexes formed with CPGC showed a complicated speciation with the possible formation of many polymetallic species. By contrast, the better preorganization in CdPPC leads to the formation of a unique CuL complex involving a CuS core. The formation of this specific cluster was confirmed by ESI-MS and by diffusion-ordered NMR spectroscopy in solution. The affinity of CdPPC for Cu(I) (β = 10 calculated for a CuL complex) is more than 1 order of magnitude larger than the affinity measured for the less constrained peptide CPGC. Besides, this stability constant value is very similar to those reported with proteins. Therefore, the Cu(I) complex formed with the simple tetrapeptide CdPPC in water at physiological pH represents a very good model of Cu(I)-thiolate clusters found in proteins. The extremely large selectivity (10) in favor of Cu(I) with respect to Zn(II), an abundant competitor in cells, makes it a promising candidate to be targeted to the liver cells for the localized treatment of Cu overload in Wilson's disease.