Cu(II) coordination to His-containing linear peptides and related branched ones: Equalities and diversities

The two branched peptides (AAHAWG)₄-PWT2 and (HAWG)₄-PWT2 where synthesized by mounting linear peptides on a cyclam-based scaffold (PWT2), provided with four maleimide chains, through a thio-Michael reaction. The purpose of this study was primarily to verify if the two branched ligands had a Cu(II) coordination behavior reproducing that of the single-chain peptides, namely AAHAWG-NH₂, which bears an Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) Motif, and HAWG-NH₂, which presents a His residue as the N-terminal amino acid, in a wide pH range. The study of Cu(II) binding was performed by potentiometric, spectroscopic (UV-vis absorption, CD, fluorescence) and ESI-MS techniques. ATCUN-type ligands ((AAHAWG)₄-PWT2 and AAHAWG-NH₂) were confirmed to bind one Cu(II) per peptide fragment at both pH 7.4 and pH 9.0, with a [NH₂, 2N^-, N_Im] coordination mode. On the other hand, the ligand HAWG-NH₂ forms a [CuL₂]²⁺ species at neutral pH, while, at pH 9, the formation of 1:2 Cu(II):ligand adducts is prevented by amidic nitrogen deprotonation and coordination, to give rise solely to 1:1 species. Conversely, Cu(II) binding to (HAWG)₄-PWT2 resulted in the formation of 1:2 copper:peptide chain also at pH 9: hence, through the latter branched peptide we obtained, at alkaline pH, the stabilization of a specific Cu(II) coordination mode which results unachievable using the corresponding single-chain peptide. This behavior could be explained in terms of high local peptide concentration on the basis of the speciation of the Cu(II)/single-chain peptide systems.