Incorporation of β‐Alanine in Cu(II) ATCUN Peptide Complexes Increases ROS Levels, DNA Cleavage and Antiproliferative Activity**

Abstract

Redox‐active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore leads to low ROS generation. In this work, we demonstrate that an exchange of the α‐amino acid Gly with the β‐amino acid β‐Ala at position 2 (Gly2→β‐Ala2) of the ATCUN motif reinstates ROS production (•OH and H2O2). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β‐Ala2‐containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β‐Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β‐Ala2 peptide complexes had lower IC50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP‐MS measurements.

Interactions of neurokinin B with copper(ii) ions and their potential biological consequences

“…44,45 These results also agree with the recently published DFT calculations of Cu( ii ) complexes of GGH and the related ligands. 46 The overall stability of the complexes is supported by hydrogen bonds, which stabilize the formation of helical elements in the peptide conformation. A hydrogen bond can contribute to the stability by 4–7 kcal mol −1 , 47 corresponding to ca.…”

The rates of Cu(ii)–ATCUN complex formation. Why so slow?

“…Recent DFT calculations indicate a possible axial binding of the carboxyl oxygen in a 2N type species. 12 This may affect the cis/trans equilibrium enhancing the relative abundance of the effective cis-exo isomer, which precedes the 4N complex in the mechanism proposed previously (Fig. S1 †).…”

Impact of N-heteroaromatic N-termini in Cu(ii) ATCUN metallopeptides on their biorelevant redox activity

“…This is in agreement with results for complexes of the type Cu-GGH. 19,26 Unlike Cu-AA1, Cu-AA3 and Cu-AA4 (ESI †, S4), Cu-AA2, is the only one of the type AA that does not show a d-d band in its CD spectrum. On the other hand, Cu-CA1 and Cu-CA4 (ESI †, S4) show distinctive d-d bands with maxima and minima in the range of 550 and 700 nm, but complexes Cu-CA2, Cu-CA3, and Cu-CA5 do not (ESI †, S4).…”

“…As occurred with the introduction of β-Ala, the coordination of the aromatic N-donor groups results in successive increase of the chelate ring size (5,5,6 → 5,6,6 → 6,6,6) (Chart 1), which in turn, should have a direct impact on the complex stability, 22 and consequently redox chemistry. 19,23 Chart 1 Cu(II) complexes Cu-AA1-Cu-AA4 and Cu-CA1-Cu-CA5 incorporating β-Ala and N-heteroaromatics.…”