Mono- and Polynuclear Copper(II) Complexes of Alloferons 1 with Point Mutations (H6A) and (H12A): Stability Structure and Cytotoxicity

Mononuclear and polynuclear copper­(II) complexes of the alloferons 1 (Allo1) with point mutations (H6A) H¹GVSGA⁶GQH⁹GVH¹²G-COOH (Allo6A) and (H12A) H¹GVSGH⁶GQH⁹GVA¹²G-COOH (Allo12A) have been studied by potentiometric, UV–visible, CD, EPR spectroscopic, and mass spectrometry (MS) methods. Complete complex speciation at different metal-to-ligand ratios ranging from 1:1 to 3:1 was obtained. At physiological pH 7.4 and a 1:1 metal-to-ligand molar ratio, the Allo6A and Allo12A peptides form CuL complexes with the 4N {NH₂, N_Im-H¹,2N_Im} binding mode. The amine nitrogen donor and the imidazole nitrogen atoms (H⁹H¹² or H⁶H⁹) can be considered to be independent metal-binding sites in the species formed for the systems studied. As a consequence, di- and trinuclear complexes for the metal-to-ligand 2:1 and 3:1 molar ratios dominate in solution, respectively. The induction of apoptosis <i>in vivo</i> in <i>Tenebrio molitor</i> cells by the ligands and their copper­(II) complexes at pH 7.4 was studied. The biological results show that copper­(II) ions <i>in vivo</i> did not cause any apparent apoptotic features. The most active was the Cu­(II)–Allo12A complex formed at pH 7.4 with a {NH₂, N_Im-H¹,N_Im-H⁶,N_Im-H⁹} binding site. It exhibited 123% higher of caspase activity in hemocytes than the native peptide, Allo1