A study of Pt(II)-phenanthroline complex interactions with double-stranded and G-quadruplex DNA by ESI-MS, circular dichroism, and computational docking.

The binding interactions of a series of square-planar platinum(II)-phenanthroline complexes of the type [Pt(PL)(AL)]2+ [where PL = variously methyl-substituted 1,10-phenanthroline (phen) and AL = ethane-1,2-diamine (en)] were assessed with a G-quadruplex DNA (5'-TTG GGG GT-3', G4DNA) and a double-stranded DNA (5'-CGC GAA TTC GCG-3', dsDNA) sequence by ESI-MS. The results indicate a strong correlation between G4DNA affinity and increasing phenanthroline methyl substitution. Circular dichroism (CD) spectroscopy and molecular docking studies also support the finding that increased substitution of the phenanthroline ligand increased selectivity for G4DNA. ESI-MS was used to probe the interaction of a range of square-planar Pt(II)-phenanthroline complexes with double-stranded and G-quadruplex DNA.

Effect of substituents on the ability of nickel Schiff base complexes with four pendant groups to bind to G-quadruplexes.

The synthesis of eleven new nickel Schiff base complexes each bearing four pendant groups is reported. The structures of the complexes differ in the identity of the pendant groups and/or diamine moiety. All complexes were characterised by microanalysis, Nuclear Magnetic Resonance (NMR) spectroscopy and Electrospray Ionisation Mass Spectrometry (ESI-MS), while the solid-state structures of two of the molecules were also determined using X-ray crystallographic methods. The DNA binding properties of the nickel complexes with double stranded DNA and a range of G-quadruplex DNA structures was explored using different spectroscopic methods as well as computational techniques. Results from ESI-MS experiments and Fluorescent Indicator Displacement (FID) assays were consistent with each other and indicated that varying the diamine moiety had less influence on DNA affinity than changing the pendant groups. These conclusions were also generally supported by results obtained from UV melting experiments and Fluorescence Resonance Energy Transfer (FRET) assays. The cytotoxicity of selected examples of the new complexes, and close analogues reported recently, towards V79 Chinese hamster lung cancer cells and THP-1 human leukemia cells was measured. All were found to display modest cytotoxicity, with flow cytometry experiments suggesting an apoptotic pathway was the most likely mechanism of cell death.

G-quadruplex DNA binding properties of novel nickel Schiff base complexes with four pendant groups.

Three new nickel Schiff base complexes were prepared using a two-step procedure that involves initial selective dialkylation of 2,4,6-trihydroxybenzaldehyde, followed by reaction with 1,2-phenylenediamine and nickel(II) acetate. Each of the complexes possessed the same Schiff base core but differed in the identity of the four pendant groups. All complexes were characterised by microanalysis, NMR spectroscopy and ESI mass spectrometry. In addition, two of the complexes were also characterised in the solid state using X-ray crystallography, which confirmed the presence of a square planar geometry around the metal ion. The DNA binding properties of the three nickel complexes with double stranded DNA and a range of G-quadruplex DNA structures were explored using ESI mass spectrometry, CD spectroscopy, UV melting curves, Fluorescence Resonance Energy Transfer (FRET) assays, Fluorescent Indicator Displacement (FID) assays and molecular docking studies. These techniques confirmed the ability of the three nickel complexes to bind to most of the DNA molecules examined, as well as stabilise the latter in several instances. In addition, the results of these investigations provided evidence that pendant groups with morpholine rings generally reduced DNA binding behaviour, whilst pendants featuring piperidine ring systems attached to the Schiff base core by three and not two methylene linkers often showed the greatest extent of binding or DNA stabilisation.

Understanding G-Quadruplex Biology and Stability Using Single-Molecule Techniques.

The link between the chemical stability of G-quadruplex (qDNA) structures and their roles in eukaryotic genomic maintenance processes has been an area of interest now for several decades. This Review seeks to demonstrate how single-molecule force-based techniques can provide insight into the mechanical stabilities of a variety of qDNA structures as well as their ability to interconvert between different conformations under conditions of stress. Atomic force microscopy (AFM) and magnetic and optical tweezers have been the primary tools used in these investigations and have been used to examine both free and ligand-stabilized G-quadruplex structures. These studies have shown that the degree of stabilization of G-quadruplex structures has a significant effect on the ability of nuclear machinery to bypass these roadblocks on DNA strands. This Review will illustrate how various cellular components including replication protein A (RPA), Bloom syndrome protein (BLM), and Pif1 helicases are capable of unfolding qDNA. Techniques such as single-molecule fluorescence resonance energy transfer (smFRET), often in conjunction with the aforementioned force-based techniques, have proven extremely effective at elucidating the factors underpinning the mechanisms by which these proteins unwind qDNA structures. We will provide insight into how single-molecule tools have facilitated the direct visualization of qDNA roadblocks and also showcase results obtained from experiments designed to examine the ability of G-quadruplexes to limit the access of specific cellular proteins normally associated with telomeres.

An unusually flexible expanded hexaamine cage and its Cu(II) complexes: variable coordination modes and incomplete encapsulation.

The bicyclic hexaamine "cage" ligand Me(8)tricosaneN(6) (1,5,5,9,13,13,20,20-octamethyl-3,7,11,15,18,22-hexaazabicyclo[7.7.7]tricosane) is capable of encapsulating octahedral metal ions, yet its expanded cavity allows the complexed metal to adopt a variety of geometries comprising either hexadentate or pentadentate coordination of the ligand. When complexed to Cu(II) the lability of the metal results in a dynamic equilibrium in solution between hexadentate- and pentadentate-coordinated complexes of Me(8)tricosaneN(6). Both [Cu(Me(8)tricosaneN(6))](ClO(4))(2) (6-coordinate) and [Cu(Me(8)tricosaneN(6))](S(2)O(6)) (5-coordinate) have been characterized structurally. In weak acid (pH 1) a singly protonated complex [Cu(HMe(8)tricosaneN(6))](3+) has been isolated that finds the ligand binding as a pentadentate with the uncoordinated amine being protonated. vis-NIR and electron paramagnetic resonance (EPR) spectroscopy show that the predominant solution structure of [Cu(Me(8)tricosaneN(6))](2+) at neutral pH comprises a five-coordinate, square pyramidal complex. Cyclic voltammetry of the square pyramidal [Cu(Me(8)tricosaneN(6))](2+) complex reveals a reversible Cu(II/I) couple. All of these structural, spectroscopic, and electrochemical features contrast with the smaller cavity and well studied "sarcophagine" (sar, 3,6,10,13,16,19-hexaazabicyclo[6.6.6]eicosane) Cu(II) complexes which are invariably hexadentate coordinated in neutral solution and cannot stabilize a Cu(I) form.

The effect of isomerism and other structural variations on the G-quadruplex DNA-binding properties of some nickel Schiff base complexes.

A series of novel isomeric nickel Schiff base complexes, as well as nickel complexes of related ligands having asymmetric structures have been prepared and characterised using microanalysis, 1H and 13C NMR spectroscopy and ESI-MS. The Schiff base ligands were prepared by condensation reactions involving ethylenediamine and different derivatives of benzophenone. The solid-state structures of eight of the complexes were also determined and revealed that each possessed a regular square planar coordination geometry around the metal ion. Many of the new complexes featured at least one, and in many instances two, protonatable pendant groups that enhance aqueous solubility. This enabled the DNA binding properties of the latter complexes to be explored using a variety of instrumental approaches, including ESI-MS, circular dichroism (CD) spectroscopy, FRET melting assays and FID assays, as well as molecular docking studies. The results of experiments performed using ESI-MS suggested that none of the nickel complexes exhibit a high affinity towards either a double stranded DNA (dsDNA) molecule D2, or the parallel unimolecular quadruplex DNA (qDNA) molecule Q1. In contrast, complexes (8) and (12) both gave spectra which reflected a significant level of binding to the parallel tetramolecular qDNA Q4. The results of binding experiments performed using CD spectroscopy suggested that (12) exhibits a significant level of affinity towards most types of DNA, while (4) shows a preference for interacting with parallel, unimolecular qDNA molecules. Complex (4) produced the lowest values of DC50 in FID assays performed using parallel Q1 or Q4, confirming its affinity for these qDNA molecules. The results of FRET melting experiments provided further evidence that (12), along with (8), can interact extensively with anti-parallel unimolecular qDNA. Experiments which monitored the effect of the nickel complexes on the melting temperature of D2 showed that none had a stabilising effect on this dsDNA molecule.

A new class of quadruplex DNA-binding nickel Schiff base complexes.

We have prepared six new nickel Schiff base complexes via reactions of substituted benzophenones with different diamines in the presence of nickel(ii). These new complexes were then reacted with 1-(2-choroethyl)piperidine to afford a further six novel nickel(ii) Schiff base complexes bearing pendant ethylpiperidine groups. The complexes bearing the ethylpiperidine moieties had greater solubility in water, and were therefore suitable for use in DNA binding experiments. ESI mass spectra of solutions containing 4 and the parallel, tetramolecular quadruplex Q4, contained ions attributable to formation of non-covalent complexes. In contrast, no ions from non-covalent complexes were observed when the experiments were repeated using 4 and either a double stranded DNA (dsDNA) molecule (D2), or parallel Q1, a unimolecular quadruplex DNA (qDNA). The ESI-MS binding study also revealed that 14 has a significant ability to form non-covalent complexes with qDNA, but does not interact to the same extent with D2. This is supported by the large changes to the ellipticity of bands observed in the circular dichroism spectra of two different unimolecular qDNA molecules (c-kit1 and Q1), including the latter annealed under conditions designed to induce formation of alternative topologies (antiparallel and hybrid). In Fluorescent Indicator Displacement (FID) assays conducted using the new nickel complexes, 14 gave the lowest values of DC50 for experiments conducted with Q1 and Q4. Furthermore, 14 showed greater stabilisation of an antiparallel qDNA molecule in FRET assays than when the other new complexes were examined. These results highlight the potential of 14 as a lead complex for future structure/DNA binding investigations. This is reinforced by the results obtained from cytotoxicity studies performed using four of the nickel complexes, including 14, and Chinese hamster lung cancer (V79) cells, which gave IC50 values between 4 and 12 µM. These complexes were also shown to have the ability to induce apoptosis in the same cancer cell line.

Comparison of mass spectrometry and other techniques for probing interactions between metal complexes and DNA.

Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding interactions of two series of ruthenium complexes, [Ru(phen) 2L] (2+) and [RuL' 2(dpqC)] (2+), to a double stranded DNA hexadecamer, and derive orders of relative binding affinity. These were shown to be in good agreement with orders of relative binding affinity derived from absorption and circular dichroism (CD) spectroscopic examination of the same systems and from DNA melting curves. However, the extent of luminescence enhancement caused by the addition of DNA to solutions of the ruthenium complexes showed little correlation with orders of binding affinity derived from ESI-MS or any of the other techniques. Overall the results provide support for the validity of using ESI-MS to investigate non-covalent interactions between metal complexes and DNA.

Effect of structure variations on the quadruplex DNA binding ability of nickel Schiff base complexes.

Two different series of nickel Schiff base complexes were prepared as part of a study aimed at discovering new compounds with high affinity and selectivity for quadruplex DNA (qDNA). The new complexes were prepared by modification of a literature method for synthesising N,N'-bis-(4-((1-(2-ethyl)piperidine)-oxy)salicylidene)phenylenediaminenickel(ii) (complex (1)). For Series 1 complexes, the phenylenediamine head group of the literature complex was replaced with ethylenediamine, phenanthrenediamine, R,R- and S,S-diaminocyclohexane. These complexes, as well as an asymmetric molecule featuring a naphthalene moiety on one side and a single ethyl piperidinyl salicylidene group on the other, were prepared in order to examine the effect of varying the number and position of aromatic groups on DNA binding. Series 2 complexes were isomers of those in Series 1, in which pendant ethyl piperidine groups were located at different positions. All new complexes were characterised by 1D and 2D NMR spectroscopic methods alongside microanalysis and ESI-MS. In addition, the solid state structures of eight new complexes were determined using single crystal X-ray diffraction methods. N,N'-Bis-(4-((1-(2-ethyl)piperidine)oxy)-salicylidine)diaminophenanthrenenickel(ii) (9), was shown by ESI-MS, CD spectroscopy and UV melting studies to exhibit a greater affinity towards, and ability to stabilise, dsDNA than all other complexes in the first series. ESI-MS revealed (9) to have a strong tendency to form a 1 : 1 complex with the tetramolecular, parallel qDNA molecule Q4, however it exhibited low affinity towards the parallel unimolecular qDNA molecule Q1. The enantiomeric complexes (5) and (7), featuring R,R- and S,S-diaminocyclohexane moieties, respectively, showed similar binding profiles towards all DNA molecules investigated, whereas the asymmetric complex (11), exhibited very low DNA affinity in all cases. Series 2 complexes showed very similar DNA affinity and selectivity to their isomeric counterparts in Series 1. For example, (14) and (15), both of which contain a phenylenediamine head group, showed higher affinity towards D2, Q1 and Q4, than any of the other Series 2 complexes. In addition, complex (21), which contains a meso-1,2-diphenylethylenediamine moiety, interacted strongly with Q4, but not D2 or Q1. This observation was very similar to that made previously for the isomeric complex (3).

Carbon Nanotube Membranes: Synthesis, Properties, and Future Filtration Applications.

Over the course of the past decade, there has been growing interest in the development of different types of membranes composed of carbon nanotubes (CNTs), including buckypapers and composite materials, for an ever-widening range of filtration applications. This article provides an overview of how different types of CNT membranes are prepared and the results obtained from investigations into their suitability for different applications. The latter involve the removal of small particles from air samples, the filtration of aqueous solutions containing organic compounds and/or bacteria, and the separation of individual liquids present in mixtures. A growing number of reports have demonstrated that the incorporation of CNTs into composite membranes confers an improved resistance to fouling caused by biomacromolecules and bacteria. These results are discussed, along with evidence that demonstrates it is possible to further reduce fouling by taking advantage of the inherent conductivity of composite membranes containing CNTs, as well as by using different types of electrochemical stimuli.

Trigonal prismatic metal complexes: a not so rare coordination geometry?

The solid state structures of two metal complexes of a hexaamine macrobicyclic ligand, in which the metal ion has an exact trigonal prismatic geometry, have been determined. Theoretical calculations showed this is the most stable geometry for d(0), d(10) and high spin d(5) metal complexes of the ligand with M-N bond distances >∼2.35 Å.

Identification of bifunctional GA and AG intrastrand crosslinks formed between cisplatin and DNA.

A combination of enzymatic digestion and electrospray ionisation mass spectrometry (ESI-MS) was used to characterise bifunctional adducts in which cisplatin is bound to GA base sequences in 8mer and 16mer oligonucleotides that do not contain other, higher affinity binding sites. The extent of formation of bifunctional adducts with GA base sequences was significant, but less than that seen with similar oligonucleotides containing either AG or GG sequences.

Frontiers in Nucleic Acid Nanotechnology.

This Special Issue of Nanomaterials highlights innovative work from around the world focused on harnessing the physical, chemical and topological properties of nucleic acids. [...].

Synthesis and characterisation of nickel Schiff base complexes containing the meso-1,2-diphenylethylenediamine moiety: selective interactions with a tetramolecular DNA quadruplex.

As part of a program of preparing metal complexes which exhibit unique affinities towards different DNA structures, we have synthesised the novel Schiff base complex N,N'-bis-4-(hydroxysalicylidine)meso-diphenylethylenediaminenickel(ii) (), via the reaction of meso-1,2-diphenylethylenediamine and 2,4-dihydroxybenzaldehyde. This compound was subsequently reacted with 1-(2-chloroethyl)piperidine or 1-(2-chloropropyl)piperidine, to afford the alkylated complexes N,N'-bis-(4-((1-(2-ethyl)piperidine)oxy)salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) () and N,N'-bis-(4-((1-(3-propyl)piperidine)oxy)-salicylidine)meso-1,2-diphenylethylenediaminenickel(ii) (), respectively. These complexes were characterised by microanalysis and X-ray crystallography in the solid state, and in solution by (1)H and (13)C NMR spectroscopy. Electrospray ionisation mass spectrometry (ESI-MS) was used to confirm the identity of () and (). The affinities of () and () towards a discrete 16 mer duplex DNA molecule, and examples of both tetramolecular and unimolecular DNA quadruplexes, was explored using a variety of techniques. In addition, the affinity of two other complexes () and (), towards the same DNA molecules was examined. Complexes () and () were prepared by methods analogous to those which afforded () and (), however 1,2-phenylenediamine was used instead of meso-1,2-diphenylethylenediamine in the initial step of the synthetic procedure. The results of ESI-MS and DNA melting temperature measurements suggest that () and () exhibit a lower affinity than () and () towards the 16 mer duplex DNA molecule, while circular dichroism (CD) spectroscopy suggested that none of the four complexes had a major effect on the conformation of the nucleic acid. In contrast, ESI-MS and CD spectroscopy suggested that both () and () show significant binding to a tetramolecular DNA quadruplex. The results of ESI-MS and Fluorescence Resonance Energy Transfer (FRET) assays indicated that () and () did not bind as tightly to a unimolecular DNA quadruplex, although both complexes had a major effect on the CD spectrum of the latter. These results highlight that the presence of the meso-1,2-diphenylethylenediamine moiety in metal complexes of this type may provide a general method for instilling selectivity for some DNA quadruplexes over dsDNA.

An investigation into the interactions of gold nanoparticles and anti-arthritic drugs with macrophages, and their reactivity towards thioredoxin reductase.

Gold(I) complexes are an important tool in the arsenal of established approaches for treating rheumatoid arthritis (RA), while some recent studies have suggested that gold nanoparticles (Au NPs) may also be therapeutically efficacious. These observations prompted the current biological studies involving gold(I) anti-RA agents and Au NPs, which are aimed towards improving our knowledge of how they work. The cytotoxicity of auranofin, aurothiomalate, aurothiosulfate and Au NPs towards RAW264.7 macrophages was evaluated using the MTT assay, with the former compound proving to be the most toxic. The extent of cellular uptake of the various gold agents was determined using graphite furnace atomic absorption spectrometry, while their distribution within macrophages was examined using microprobe synchrotron radiation X-ray fluorescence spectroscopy. The latter technique showed accumulation of gold in discrete regions of the cell, and co-localisation with sulfur in the case of cells treated with aurothiomalate or auranofin. Electrospray ionization mass spectrometry was used to characterize thioredoxin reductase (TrxR) in which the penultimate selenocysteine residue was replaced by cysteine. Mass spectra of solutions of TrxR and aurothiomalate, aurothiosulfate or auranofin showed complexes containing bare gold atoms bound to the protein, or protein adducts containing gold atoms retaining some of their initial ligands. These results support TrxR being an important target of gold(I) drugs used to treat RA, while the finding that Au NPs are incorporated into macrophages, but elicit little toxicity, indicates further exploration of their potential for treatment of RA is warranted.

Comparison of the binding stoichiometries of positively charged DNA-binding drugs using positive and negative ion electrospray ionization mass spectrometry.

Positive and negative ion electrospray ionization (ESI) mass spectra of complexes of positively charged small molecules (distamycin, Hoechst 33258, [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2) have been compared. [Ru(phen)2dpq]Cl2 and [Ru(phen)2dpqC]Cl2 bind to DNA by intercalation. Negative ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA showed ions from DNA-ligand complexes consistent with solution studies. In contrast, only ions from free DNA were present in positive ion ESI mass spectra of mixtures of [Ru(phen)2dpq]Cl2 or [Ru(phen)2dpqC]Cl2 with DNA, highlighting the need for obtaining ESI mass spectra of non-covalent complexes under a range of experimental conditions. Negative ion spectra of mixtures of the minor groove binder Hoechst 33258 with DNA containing a known minor groove binding sequence were dominated by ions from a 1:1 complex. In contrast, in positive ion spectra there were also ions present from a 2:1 (Hoechst 33258: DNA) complex, suggesting an alternative binding mode was possible either in solution or in the gas phase. When Hoechst 33258 was mixed with a DNA sequence lacking a high affinity minor groove binding site, the negative ion ESI mass spectra showed that 1:1 and 2:1 complexes were formed, consistent with existence of binding modes other than minor groove binding. The data presented suggest that comparison of positive and negative ion ESI-MS spectra might provide an insight into various binding modes in both solution and the gas phase.

Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry.

ESI mass spectra show that up to five ruthenium molecules can bind non-covalently to double stranded 16mer DNA, and provide information on the relative affinity and DNA sequence selectivity of different ruthenium complexes.

Does cytotoxicity of metallointercalators correlate with cellular uptake or DNA affinity?

The cytotoxicity of the metallointercalators, [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1R,2R-diaminocyclohexane)](2+) ([56MERR]) and [Pt(5,6-dimethyl-1,10-phenanthroline)(trans-1S,2S-diaminocyclohexane)](2+) ([56MESS]), towards A549 human lung cancer cells was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The IC(50) value obtained following exposure of A549 cells to [56MESS] for 4 h was approximately three times smaller than that obtained when [56MERR] was administered under the same conditions, indicating that the former complex displayed greater cytotoxicity. Both IC(50) values were greater than that obtained after exposure of A549 cells to cisplatin, demonstrating that the latter compound was the most cytotoxic of the three examined. Microprobe synchrotron radiation X-ray fluorescence (SR-XRF) analyses of metallointercalator-treated A549 cells showed that platinum became localised in DNA-rich regions of the nucleus. In contrast, when the same cells were treated with cisplatin the metal became distributed throughout the cell. Determination of the maximum concentration of platinum present inside the cells using graphite furnace atomic absorption spectrophotometry (GFAAS) of platinum-treated cells suggested that there was greater uptake of [56MERR] compared to [56MESS] by the A549 cells, and that platinum uptake did not account for the greater toxicity of [56MESS], as assessed by the MTT assay. Electrospray ionization mass spectrometric (ESI-MS) and circular dichroism (CD) spectroscopic studies of solutions containing either [56MERR] or [56MESS], and a duplex hexadecamer molecule, showed the two metallointercalators displayed very similar affinity towards the nucleic acid. Overall these results indicate that the difference in cytotoxicity of the two platinum metallointercalators is probably the result of variations in their interactions with other cellular components.

Quadruplex DNA: a promising drug target for the medicinal inorganic chemist.

Compounds that can bind to and stabilize quadruplex DNA structures in telomeres, or induce formation of such structures from ssDNA, represent an attractive general approach to the treatment of cancer. Until recently most effort in this area has been directed towards the synthesis of organic compounds for this purpose. More recently there has been growing recognition that metal complexes offer a number of potential advantages for the preparation of lead complexes that bind with high affinity and selectivity for quadruplex DNA. This review seeks to discuss the work that has been reported in this area to date. While most early studies focused on metal complexes of porphyrin ligands, during the past 4 years there has been a dramatic increase in the number of papers in the literature examining the potential of mononuclear complexes of a variety of other ligands, particularly Schiff base ligands and those based on phenanthroline, as quadruplex DNA binders and telomerase inhibitors. In addition, there has been growing interest in exploiting supramolecular chemistry to prepare novel multinuclear complexes that bind to this new drug target.

A novel enzymatic bioelectrode system combining a redox hydrogel with a carbon NanoWeb.

A novel bioelectrode system has been prepared in which an enzyme and a conducting polymer hydrogel are combined in a nanostructured scaffold. The latter consists of fibres of carbon NanoWeb, grown by chemical vapour deposition onto reticulated vitreous carbon (RVC). The catalytic currents produced by this new bioelectrode system are significantly larger than those obtained using conventional electrodes.