Expanding the DNA damaging potential of artificial metallo-nucleases with click chemistry

Recently, copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) click chemistry has emerged as a promising approach for designing new artificial metallo-nucleases (AMNs) with DNA-damaging properties. By functionalising a central organic azide with three alkyne donors, Tri-Click (TC) ligands capable of chelating three copper ions through the donor group and triazole linker can be generated. However, the versatility of this approach along with the influence of specific donors on metal binding, DNA recognition, and cellular DNA damage in an anticancer context remains poorly understood. Here, we prepare a series of Tri-Click ligands incorporating systematic cyclic and acyclic N-, O-, and S-donors and evaluate their AMN activities. Screening experiments pinpoint planar N-donor ligands as high value agents. Among these, the copper complex of Tri-Click-Pyridine (Cu₃-TC-Py) displays significant potential. We characterise its activity using single-molecule imaging, microscale thermophoresis, FRET-based binding assays, molecular dynamics, and intracellular DNA interaction studies in human and functional bacterial cells. We report the emergence of Cu₃-TC-Py as a lead AMN with high reactivity for DNA damage applications central to anticancer therapy.