Copper(II) complexes of oligopeptides. An equilibrium and spectroscopic study on the copper(II) lys-leu-ala-his-phe-gly system

The hexapeptide L-Lys-L-Leu-L-Ala-L-His-L-Phe-Gly was prepared by solid-phase peptide synthesis. The proton and copper(II) equilibrium system and thereby the composition and solution structure of the complexes formed were investigated by potentiometric and spectrophotometric titrations, as well as by CD and EPR spectroscopy. The ligand was able to keep two equivalents of metal ion in solution in the investigated pH interval 2-10. While the protonation isomers form separately, the formation of the metal complexes of different protonation states can be characterized by overlapping processes. The detectable coordination starts at ca. pH 3-4, where, beside the possible weak carboxylate coordination, the parallel deprotonation of the imidazole nitrogen atom and the N-terminal L-lysine α-ammonium group takes place in the presence of metal ions. In this way a large chelate ring is formed. The first significant signals in the CD and EPR spectra can be attributed to the species [ML]⁺, with one deprotonated amide nitrogen in addition to the abovementioned donor groups. A species with four coordinating nitrogen atoms containing electrically neutral the MLH_-1 complex is predominant in the physiological pH region. The spectroscopic results strongly suggest that there are at least two deprotonated amide nitrogens in the copper(II) coordination sphere. The formation of the next species requires the replacement of one of the already coordinated donor groups (probably the imidazole nitrogen). The drastic changes in the absorption and CD spectra and the increase of the A₀(Cu) to 92 G support the assumed processes. The deprotonation above pH 9.5 proved to be due to the N-terminal L-lysine ε-ammonium group, which remains uncoordinated even under favorable conditions. The formation of binuclear species with well separated copper(II) centers was detected by EPR spectroscopy when an excess of metal ion was applied.