Herein we provide an accessible account of molecular methods to probe inorganic–nucleic acid interactions. Techniques are described using copper(ii) and platinum(ii) complexes prepared in our laboratories.

Here we report the synthesis and isolation of a series of bis-chelate Cu(2+) phenanthroline-phenazine cationic complexes of [Cu(DPQ)(Phen)](2+), [Cu(DPPZ)(Phen)](2+), and [Cu(DPPN)(Phen)](2+) (where Phen = 1,10-phenanthroline, DPQ = dipyridoquinoxaline, DPPZ = dipyridophenazine, and DPPN = benzo[i]dipyridophenazine). These compounds have enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2](2+) (bis-Phen), with calf thymus DNA binding constants of DPQ and DPPZ agents (∼10(7) M(bp)(-1)) being the highest currently known for Cu(2+) phenanthrene compounds. Complex DNA binding follows DPQ ≈ DPPZ > DPPN > bis-Phen, with fluorescence quenching and thermal melting experiments on poly[d(A-T)2] and poly[d(G-C)2] supporting intercalation at both the minor and major groove. Phenazine complexes, however, show enhanced targeting and oxidative cleavage on cytosine-phosphate-guanine-rich DNA and have comparable in vitro cytotoxicity toward the cisplatin-resistant ovarian cancer line, SKOV3, as the clinical oxidative DNA-damaging drug doxorubicin (Adriamycin). In this study we also describe how a novel "on-chip" method devised for the Bioanalyser 2100 was employed to quantify double-stranded DNA damage, with high precision, by the complex series on pUC19 DNA (49% A-T, 51% G-C). Both DPQ and bis-Phen complexes are highly efficient oxidizers of pUC19, with DPQ being the most active of the overall series. It is apparent, therefore, that oxidative chemical nuclease activity on homogeneous canonical DNA is not entirely dependent on dynamic nucleotide binding affinity or intercalation, and this observation is corroborated through catalytic interactions with the superoxide anion radical and Fenton breakdown of hydrogen peroxide.

The building of robust and versatile inorganic scaffolds with artificial metallo-nuclease (AMN) activity is an important goal for bioinorganic, biotechnology, and metallodrug research fields. Here, a new type of AMN combining a tris-(2-pyridylmethyl)amine (TPMA) scaffold with the copper(II) N,N'-phenanthrene chemical nuclease core is reported. In designing these complexes, the stabilization and flexibility of TPMA together with the prominent chemical nuclease activity of copper 1,10-phenanthroline (Phen) were targeted. A second aspect was the opportunity to introduce designer phenazine DNA intercalators (e.g., dipyridophenazine; DPPZ) for improved DNA recognition. Five compounds of formula [Cu(TPMA)(N,N')] (where N,N' is 2,2-bipyridine (Bipy), Phen, 1,10-phenanthroline-5,6-dione (PD), dipyridoquinoxaline (DPQ), or dipyridophenazine (DPPZ)) were developed and characterized by X-ray crystallography. Solution stabilities were studied by continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE), and Davies electron-nuclear double resonance (ENDOR) spectroscopies, which demonstrated preferred geometries in which phenanthrene ligands were coordinated to the copper(II) TPMA core. Complexes with Phen, DPQ, and DPPZ ligands possessed enhanced DNA binding activity, with DPQ and DPPZ compounds showing excellent intercalative effects. These complexes are effective AMNs and analysis with spin-trapping scavengers of reactive oxygen species and DNA repair enzymes with glycosylase/endonuclease activity demonstrated a distinctive DNA oxidation activity compared to classical Sigman- and Fenton-type reagents.

Free radical generation is an inevitable consequence of aerobic existence and is implicated in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing and neurodegenerative disorder. Free radicals can, however, be used to our advantage since their production is catalysed by synthetic inorganic molecules—termed artificial metallonucleases—that cut DNA strands by oxidative cleavage reactions. Here, we report the rational design and DNA binding interactions of a novel di-Cu2+ artificial metallonuclease [Cu2(tetra-(2-pyridyl)-NMe-naphthalene)Cl4] (Cu2TPNap). Cu2TPNap is a high-affinity binder of duplex DNA with an apparent binding constant (Kapp) of 107 M(bp)−1. The agent binds non-intercalatively in the major groove causing condensation and G-C specific destabilization. Artificial metallonuclease activity occurs in the absence of exogenous reductant, is dependent on superoxide and hydrogen peroxide, and gives rise to single strand DNA breaks. Pre-associative molecular docking studies with the 8-mer d(GGGGCCCC)2, a model for poly[d(G-C)2], identified selective major groove incorporation of the complex with ancillary Cu2+-phosphate backbone binding. Molecular mechanics methods then showed the d(GGGGCCCC)2 adduct to relax about the complex and this interaction is supported by UV melting experiments where poly[d(G-C)2] is selectively destabilized.

The deleterious effects of metal-catalyzed reactive oxygen species (ROS) in biological systems can be seen in a wide variety of pathological conditions including cancer, cardiovascular disease, aging, and neurodegenerative disorder. On the other hand however, targeted ROS production in the vicinity of nucleic acids—as demonstrated by metal-activated bleomycin—has paved the way for ROS-active chemotherapeutic drug development. Herein we report mechanistic investigations into the oxidative nuclease activity and redox properties of copper(II) developmental therapeutics [Cu(DPQ)(phen)]2+ (Cu-DPQ-Phen), [Cu(DPPZ)(phen)]2+ (Cu-DPPZ-Phen), and [{Cu(phen)2}2(μ-terph)](terph) (Cu-Terph), with results being compared directly to Sigman's reagent [Cu(phen)2]2+ throughout (phen = 1,10-phenanthroline; DPQ = dipyridoquinoxaline; DPPZ = dipyridophenazine; Terph = terephthalate). Oxidative DNA damage was identified at the minor groove through use of surface bound recognition elements of methyl green, netropsin, and [Co(NH3)6]Cl3 that functioned to control complex accessibility at selected regions. ROS-specific scavengers and stabilizers were employed to identify the cleavage process, the results of which infer hydrogen peroxide produced metal-hydroxo or free hydroxyl radicals (•OH) as the predominant species. The extent of DNA damage owing to these radicals was then quantified through 8-oxo-2′-deoxyguanosine (8-oxo-dG) lesion detection under ELISA protocol with the overall trend following Cu-DPQ-Phen > Cu-Terph > Cu-Phen > Cu-DPPZ. Finally, the effects of oxidative damage on DNA replication processes were investigated using the polymerase chain reaction (PCR) where amplification of 120 base pair DNA sequences of varying base content were inhibited—particularly along A-T rich chains—through oxidative damage of template strands.

In order to expand the current repertoire of cancer treatments and to help circumvent limitations associated with resistance, the identification of new metallodrugs with high potency and novel mechanisms of action is of significant importance. Here we present a class of di-copper(II) complex based on the synthetic chemical nuclease [Cu(Phen)2]+ (where Phen = 1,10-phenanthroline) that is selective against solid epithelial cancer cells from the National Cancer Institute’s 60 human cell line panel (NCI-60). Two metallodrug leads are studied and in each case two [Cu(Phen)2]+ units are bridged by a dicarboxylate linker but the length and rigidity of the linkers differ distinctly. Both agents catalyze intracellular superoxide (O2•−) and singlet oxygen (1O2) formation with radical species mediating oxidative damage within nuclear DNA in the form of double strand breaks and to the mitochondria in terms of membrane depolarization. The complexes are effective DNA binders and can discriminate AT/AT from TA/TA steps of duplex DNA through induction of distinctive Z-like DNA or by intercalative interactions.

The in cellulo catalytic production of reactive oxygen species (ROS) by copper(II) and iron(II) complexes is now recognized as a major mechanistic model in the design of effective cytotoxins of human cancer. The developmental complex, [Cu(o-phthalate)(1,10-phenanthroline)] (Cu-Ph), was recently reported as an intracellular ROS-active cytotoxic agent that induces double strand breaks in the genome of human cancer cells. In this work, we report the broad-spectrum action of Cu-Ph within the National Cancer Institute's (NCI) Developmental Therapeutics Program (DTP), 60 human cancer cell line screen. The activity profile is compared to established clinical agents-via the COMPARE algorithm-and reveals a novel mode of action to existing metal-based therapeutics. In this study, we identify the mechanistic activity of Cu-Ph through a series of molecular biological studies that are compared directly to the clinical DNA intercalator and topoisomerase II poison doxorubicin. The presence of ROS-specific scavengers was employed for in vitro and intracellular evaluation of prevailing radical species responsible for DNA oxidation with superoxide identified as playing a critical role in this mechanism. The ROS targeting properties of Cu-Ph on mitochondrial membrane potential were investigated, which showed that it had comparable activity to the uncoupling ionophore, carbonyl cyanide m-chlorophenyl hydrazine. The induction and origins of apoptotic activation were probed through detection of Annexin V and the activation of initiator (8,9) and executioner caspases (3/7) and were structurally visualized using confocal microscopy. Results here confirm a unique radical-induced mechanistic profile with intracellular hallmarks of damage to both genomic DNA and mitochondria.

Nucleic acid click chemistry was used to prepare a family of chemically modified triplex forming oligonucleotides (TFOs) for application as a new gene‐targeted technology. Azide‐bearing phenanthrene ligands—designed to promote triplex stability and copper binding—were ‘clicked’ to alkyne‐modified parallel TFOs. Using this approach, a library of TFO hybrids was prepared and shown to effectively target purine‐rich genetic elements in vitro. Several of the hybrids provide significant stabilisation toward melting in parallel triplexes (>20 °C) and DNA damage can be triggered upon copper binding in the presence of added reductant. Therefore, the TFO and ‘clicked’ ligands work synergistically to provide sequence‐selectivity to the copper cutting unit which, in turn, confers high stabilisation to the DNA triplex. To extend the boundaries of this hybrid system further, a click chemistry‐based di‐copper binding ligand was developed to accommodate designer ancillary ligands such as DPQ and DPPZ. When this ligand was inserted into a TFO, a dramatic improvement in targeted oxidative cleavage is afforded.