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

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 alpha-amino acid Gly with the beta-amino acid beta-Ala at position 2 (Gly2 ->beta-Ala2) of the ATCUN motif reinstates ROS production ((OH)-O-center dot and H2O2). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these beta-Ala2-containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for beta-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 beta-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.