Click Chemistry Derived Copper Artificial Metallo-Nucleases with Discrete Nucleic Acid Targeting Properties

Abstract

The design of copper compounds with artificial metallo-nuclease (AMN) activity is an important goal as these agents are mechanistically unique compared to established metallodrugs. This thesis reports the development of a new dinuclear copper AMN, called Cu2 -BPL-C6, which was prepared using click chemistry methodology. This compound demonstrates site-specific DNA recognition with low micromolar damaging activity. The complex was rationally designed to induce enhanced DNA damage by coordinating two redox-active copper centres coordinated to distal phenanthroline groups that are linked by an aliphatic spacer. DNA binding experiments—including circular dichroism spectroscopy, agarose gel electrophoresis, and fluorescence quenching experiments—revealed preferential binding for AT-rich DNA. The oxidative cleavage mechanism of Cu2 BPL-C6 was elucidated using in vitro molecular and biophysical assays with spin trapping antioxidants and free radical scavengers. Next, the quantification of genomic DNA damage, along with the types of oxidative lesions produced, were monitored using single molecule analysis of peripheral blood mononuclear cells exposed to Cu2 -BPL-C6. Broad spectrum anticancer screening in collaboration with the National Cancer Institute (NCI-60) revealed selectivity against several melanoma, breast, colon, and non-small cell lung cell lines. A small library of Cu2 BPL-C6 congeners were then synthesised to observe the effects of modifying the linker length and composition of this class of agent. To extend the application of targeted AMNs further, a library of bis-acridine ligand scaffolds were screened with Holliday Junction (HJ) DNA for their recognition properties. The lead compound in the screen, a C6-linked acridine derivative, was then covalently modified using click chemistry. Here, an azide-phenanthroline group was tethered to a mono-acridine derived scaffold and coordinated with copper(II). Efforts to generate a functionalised bis-acridine scaffold are established. Multiplex PAGE and microscale thermophoresis assays were established for high-throughput screening of HJ recognising compounds. Using these platforms, the mono-acridine hybrid was screened for its selective HJ DNA cleavage properties