Harnessing the Flexibility of Peptidic Scaffolds to Control their Copper(II)‐Coordination Properties: A Potentiometric and Spectroscopic Study

Designing small peptides that are capable of binding Cu²⁺ ions mainly through the side‐chain functionalities is a hard task because the amide nitrogen atoms strongly compete for Cu²⁺ ion coordination. However, the design of such peptides is important for obtaining biomimetic small systems of metalloenyzmes as well as for the development of artificial systems. With this in mind, a cyclic decapeptide, C‐Asp, which contained three His residues and one Asp residue, and its linear derivative, O‐Asp, were synthesized. The C‐Asp peptide has two Pro? Gly β‐turn‐inducer units and, as a result of cyclization, and as shown by CD spectroscopy, its backbone is constrained into a more defined conformation than O‐Asp, which is linear and contains a single Pro? Gly unit. A detailed potentiometric, mass spectrometric, and spectroscopic study (UV/Vis, CD, and EPR spectroscopy) showed that at a 1:1 Cu²⁺/peptide ratio, both peptides formed a major [CuHL]²⁺ species in the pH range 5.0–7.5 (C‐Asp) and 5.5–7.0 (O‐Asp). The corrected stability constants of the protonated species (log K*_CuH(O?Asp)=9.28 and log K*_CuH(C?Asp)=10.79) indicate that the cyclic peptide binds Cu²⁺ ions with higher affinity. In addition, the calculated value of K_eff shows that this higher affinity for Cu²⁺ ions prevails at all pH values, not only for a 1:1 ratio but even for a 2:1 ratio. The spectroscopic data of both [CuHL]²⁺ species are consistent with the exclusive coordination of Cu²⁺ ions by the side‐chain functionalities of the three His residues and the Asp residue in a square‐planar or square‐pyramidal geometry. Nonetheless, although these data show that, upon metal coordination, both peptides adopt a similar fold, the larger conformational constraints that are present in the cyclic scaffold results in different behaviour for both [CuHL]²⁺ species. CD and NMR analysis revealed the formation of a more rigid structure and a slower Cu²⁺‐exchange rate for [CuH(C‐Asp)]²⁺ compared to [CuH(O‐Asp]²⁺. This detailed comparative study shows that cyclization has a remarkable effect on the Cu²⁺‐coordination properties of the C‐Asp peptide, which binds Cu²⁺ ions with higher affinity at all pH values, stabilizes the [CuHL]²⁺ species in a wider pH range, and has a slower Cu²⁺‐exchange rate compared to O‐Asp.