Structural basis and effect of copper(II) complexes with 4-oxo-thiazolidine ligands on DNA binding and nuclease activity.

Seven novel Copper(II) complexes, namely [Cu(Am4DHotaz)(H2O)2](ClO4) (1), [Cu(Am4DHotaz)(NO3)(MeOH)]·H2O (2), [Cu(Am4Motaz)2(H2O)](ClO4)2·0.83H2O (3), [Cu(Am4Motaz)2(NO3)]NO3·MeOH (4), [Cu(Am4Eotaz)2(NO3)]3(NO3)3·2H2O (5), [Cu(Am4Eotaz)2(ClO4)](ClO4) (6) and [Cu(Am4Eotaz)(ClO4)(H2O)](ClO4) (6a) (HAm4DHotaz = N'-(4-oxothiazolidin-2-ylidene)pyridine-2-carbohydrazonamide, Am4Motaz = N'-(3-methyl-4-oxothiazolidin-2-ylidene)pyridine-2-carbohydrazonamide and Am4Eotaz = N'-(3-ethyl-4-oxothiazolidin-2-ylidene)pyridine-2-carbohydrazonamide), have been successfully synthesized and characterized by several physicochemical techniques and, for 1-6 complexes, single crystal X-ray diffraction. Having the structural data as a base, complexes 1, 2 and 3 exhibited square pyramidal to square pyramidal slightly distorted geometry, whereas 4, 5 and 6 an intermediate between square pyramidal and trigonal bipyramidal. The ability of complexes 1-6 to cleave DNA was assayed with the aid of gel electrophoresis on supercoiled pUC18-DNA. Except for [Cu(Am4Motaz)2(H2O)](ClO4)2·0.83H2O (3), the compounds were not able to perform DNA cleavage (data not shown). Since 3 has been shown to behave as a nuclease, its interaction with DNA was studied by means of thermal denaturation and viscosimetry measurements.

Half Sandwich Rhodium(III) and Iridium(III) Complexes as Cytotoxic and Metallonuclease Agents.

Half sandwich complexes of the type [(η5-C5Me5)M(L1-3)Cl]Cl.2H2O were synthesized using [{(η5-C5Me5)M(µ-Cl)Cl}2], where M = Rh(III)/Ir(III) and L1-3 = pyrimidine-based ligands. The complexes were characterized by spectral analysis. DNA interaction studies by absorption titration and hydrodynamic measurement and suggest intercalative mode of binding of complexes with CT-DNA. The molecular docking study also supports intercalation of the complexes between the stacks of nucleotide base pairs. The gel electrophoresis assay demonstrated the ability of the complexes to interact and cleave plasmid DNA. Minimum inhibitory concentrations (MIC) of the complexes were investigated by the microdilution broth method. The cytotoxic properties of the metal complexes were evaluated using brine shrimp lethality bioassay.

Sulfonamide-containing copper(II) metallonucleases: Correlations with in vitro antimycobacterial and antiproliferative activities.

The bis-(1,10-phenanthroline)copper(I) complex, [Cu(I)(phen)2]+, was the first copper-based artificial nuclease reported in the literature. The biological and ligand-like properties of sulfonamides make them good candidates for fine-tuning the reactivity of the [Cu(phen)2] motif with biomolecules. In this context, we developed three novel copper(II) complexes containing the sulfonamides sulfameter (smtrH) and sulfadimethoxine (sdmxH) and (N^N)-bidentate ligands (2,2'-biyridine or 1,10-phenantroline). The compounds were characterized by chemical and spectroscopic techniques and single-crystal X-ray crystallography. When targeting plasmid DNA, the phen-containing compounds [Cu(smtr-)2(phen)] (1) and [Cu(sdmx-)2(phen)] (2) demonstrated nuclease activity even in the absence of reducing agents. Addition of ascorbic acid resulted in a complete cleavage of DNA by 1 and 2 at concentrations higher than 10 µM. Experiments designed to evaluate the copper intermediates involved in the nuclease effect after reaction with ascorbic acid identified at least the [Cu(I)(N^N)2]+, [Cu(I)(sulfa)(N^N)]+ and [Cu(I)(sulfa)2]+ species. The compounds interact with DNA via groove binding and intercalation as verified by fluorescence spectroscopy, circular dichroism (CD) and molecular docking. The magnitude and preferred mode of binding are dependent on the nature of both N^N ligand and the sulfonamide. The potent nuclease activity of compounds 1 and 2 are well correlated with their antiproliferative and anti-M. tuberculosis profiles. The results presented here demonstrated the potential for further development of copper(II)-sulfonamide-(N^N) complexes as multipurpose metallodrugs.

A new thio-Schiff base fluorophore with copper ion sensing, DNA binding and nuclease activity.

Copper ion recognition and DNA interaction of a newly synthesized fluorescent Schiff base (HPyETSC) were investigated using UV-vis and fluorescent spectroscopy. Examination using these two techniques revealed that the detection of copper by HPyETSC is highly sensitive and selective, with a detection limit of 0.39 µm and the mode of interaction between HPyETSC and DNA is electrostatic, with a binding constant of 8.97×10(4) M(-1). Furthermore, gel electrophoresis studies showed that HPyETSC exhibited nuclease activity through oxidative pathway.

Highly efficient synthetic iron-dependent nucleases activate both intrinsic and extrinsic apoptotic death pathways in leukemia cancer cells.

The nuclease activity and the cytotoxicity toward human leukemia cancer cells of iron complexes, [Fe(HPClNOL)Cl2]NO3 (1), [Cl(HPClNOL)Fe(µ-O)Fe(HPClNOL)Cl]Cl2·2H2O (2), and [(SO4)(HPClNOL)Fe(µ-O)Fe(HPClNOL)(SO4)]·6H2O (3) (HPClNOL=1-(bis-pyridin-2-ylmethyl-amino)-3-chloropropan-2-ol), were investigated. Each complex was able to promote plasmid DNA cleavage and change the supercoiled form of the plasmid to circular and linear ones. Kinetic data revealed that (1), (2) and (3) increase the rate of DNA hydrolysis about 278, 192 and 339 million-fold, respectively. The activity of the complexes was inhibited by distamycin, indicating that they interact with the minor groove of the DNA. The cytotoxic activity of the complexes toward U937, HL-60, Jukart and THP-1 leukemia cancer cells was studied employing 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), fluorescence and electronic transmission microscopies, flow cytometry and a cytochrome C release assay. Compound (2) has the highest activity toward cancer cells and is the least toxic for normal ones (i.e. peripheral blood mononuclear cells (PBMCs)). In contrast, compound (1) is the least active toward cancer cells but displays the highest toxicity toward normal cells. Transmission electronic microscopy indicates that cell death shows features typical of apoptotic cells, which was confirmed using the annexin V-FITC/PI (fluorescein isothiocyanate/propidium iodide) assay. Furthermore, our data demonstrate that at an early stage during the treatment with complex (2) mitochondria lose their transmembrane potential, resulting in cytochrome C release. A quantification of caspases 3, 9 (intrinsic apoptosis pathway) and caspase 8 (extrinsic apoptosis pathway) indicated that both the intrinsic (via mitochondria) and extrinsic (via death receptors) pathways are involved in the apoptotic stimuli.

Copper(II) complexes with 2NO and 3N donor ligands: synthesis, structures and chemical nuclease and anticancer activities.

A series of water soluble copper(II) complexes of the types [Cu(L)Cl] 1-2, where LH is 2-(2-(1H-benzimidazol-2-yl)ethyliminomethyl)phenol (H(L1)), and 2-(2-(1H-benzimidazol-2-yl)-ethyliminomethyl)-4-methylphenol (H(L2)), and [Cu(L)Cl2] 3-6, where L is (2-pyridin-2-yl-ethyl)pyridin-2-ylmethyleneamine (L3), 2-(1H-benzimidazol-2-yl)ethylpyridin-2-yl-methyleneamine (L4), 2-(1H-benzimidazol-2-yl)ethyl(1H-imidazol-2-ylmethylene)amine (L5), and 2-(1H-benzimidazol-2-yl)ethyl-(4,4a-dihydroquinolin-2-ylmethylene)amine (L6), have been isolated and characterized by elemental analysis, electronic absorption, ESI-MS and EPR spectral techniques and the electrochemical method. The single crystal X-ray structures of [Cu(L1)Cl] 1 and [Cu(L2)Cl] 2 possess a distorted square-based coordination geometry while [Cu(L4)Cl2] 4 and [Cu(L6)Cl2] 6 possess a distorted trigonal bipyramidal coordination geometry. Both absorption spectral titration and an EthBr displacement assay reveal that all the complexes bind with calf thymus (CT) DNA through covalent mode of DNA interaction involving the replacement of an easily removable chloride ion with DNA nucleobases. All the complexes exhibit oxidative cleavage of supercoiled (SC) plasmid DNA in the presence of hydrogen peroxide as an activator. It is remarkable that at 50 µM concentration 5 and 6 completely degrade SC DNA into undetectable minor fragments and thus they act as efficient chemical nucleases. All the complexes are remarkable in displaying cytotoxicity against the HBL-100 human breast cancer cell line with potency more than that of the widely used drug cisplatin and hence they have the potential to act as promising anticancer drugs. Interestingly, they are non-toxic to normal cell lymphocytes isolated from human blood samples, revealing that they are selective in killing only the cancer cells.

Mn(II) complexes with sulfonamides as ligands. DNA interaction studies and nuclease activity.

Sulfonamides derived from 8-aminoquinoline react with Mn(II) and Mn(III) salts to form Mn(II) complexes; the Mn(III) species are reduced to the divalent state in the presence of 1,10 phenanthroline and bipyridine. Their molecular structure, determined by single crystal X-ray diffraction, show that all the complexes present a distorted octahedral geometry, in which the deprotonated sulfonamide acts as a bidentate ligand. UV-visible spectroscopy and changes in the melting temperature (Tm) of calf thymus DNA show a strong interaction of these complexes with DNA. The significant hypochromicity of the charge transfer transition at 370 nm without an appreciable change in wavelength and the minor changes in the relative viscosity of calf thymus DNA have been attributed to an interaction between the surface of DNA and the complexes. [Mn(qbsa)(2)(MeOH)(2)], [Mn(qbsa)(2)(phen)], [Mn(qtsa)(2)(H(2)O)(2)] and [Mn(qtsa)(2)(phen)] (where qbsa=N-quinolin-8-yl-bencenesulfonamide, qtsa=N-quinolin-8-yl-p-toluenesulfonamide and qnsa=N-quinolin-8-yl-naftalenesulfonamide) exhibit a prominent nuclease activity and the mechanism of DNA cleavage is investigated.

Cu(II) and Mn(II) complexes containing macroacyclic ligand: synthesis, DNA binding, and cleavage studies.

The two new metal complexes of Cu(II) and Mn(II) containing macroacyclic ligand of the type [Cu(hpn)](PF(6))(2) (1) and [Mn(hpn)](PF(6))(2) (2) [where hpn = [1-{[2-{[2-hydroxynaphthalen-1-yl)methylidine]amino}phenyl)imino]methyl}naphthalene-2-ol]] have been synthesized and characterized by employing analytical and spectral methods. The DNA binding properties of the complexes with calf thymus-DNA were studied by using absorption spectra and viscosity measurements, as well as thermal denaturation experiments. The absorption spectra indicated that the complexes intercalate tightly between the base pairs of the DNA with intrinsic DNA binding constants of 1.8 × 10(4) M(-1) for (1) and 3.7 × 10(4) M(-1) for (2) in 5 mM Tris-HCl/50 mM NaCl buffer at pH 7.2, respectively. The enhancement in the relative viscosity of DNA on binding to the ligand supports the proposed DNA binding modes. The oxidative cleavage activity of complexes (1) and (2) were carried out on double-stranded pUC19 circular plasmid DNA using gel electrophoresis. The complexes show significant nuclease activity.

Carbohydrate-conjugate heterobimetallic complexes: synthesis, DNA binding studies, artificial nuclease activity and in vitro cytotoxicity.

New carbohydrate-conjugated heterobimetallic complexes [C(32)H(62)N(10)O(8)NiSn(2)Cl(4)]Cl(2)(1) and [C(32)H(62)N(10)O(8)CuSn(2)Cl(4)]Cl(2) (2) were synthesized and characterized by spectroscopic (IR, (1)H, (13)C, and (119)Sn NMR, EPR, UV-vis, ESI-MS) and analytical methods. The interaction studies of 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 toward CT DNA. The extent of interaction was further confirmed by the interaction of 2 with the nucleotides viz.; 5'-AMP, 5'-CMP, 5'-GMP, and 5'-TMP, by absorption titration. (1)H, (31)P, (119)Sn NMR spectroscopy further validated the interaction mode of 2 with 5'-GMP. The electrophoresis pattern observed for 2 with supercoiled pBR322 DNA, exhibited significantly good nuclease activity following oxidative pathway. The preferential selectivity of 2 toward the major groove was observed on interaction of 2 with pBR322 DNA, in the presence of standard groove binders viz.; DAPI and methyl green. Additionally, in vitro antitumor activity of 2 was evaluated on a panel of human cancer cell lines, exhibiting remarkable cytotoxicity activity against Colo205 (colon) and MCF7 (breast) cell lines with GI(50) values <10 µg/mL.

Synthesis, structure, DNA binding and cleavage properties of ternary amino acid Schiff base-phen/bipy Cu(II) complexes.

Ternary Cu(II) complexes [Cu(II)(saltrp)(B)] (1,2), (saltrp=salicylidene tryptophan, B=1,10 phenathroline (1) or 2,2' bipyridine (2)) were synthesized and characterized. Complex 2 was structurally characterized by single crystal X-ray crystallography. The molecular structure shows a distorted square pyramidal coordination geometry (CuN(3)O(2)) in which the ONO donor Schiff base is bonded to the Cu(II) in the basal plane. The N,N donor heterocyclic base displays an axial-equatorial binding mode. CT-DNA binding studies revealed that the complexes show good binding propensity (Intrinsic binding constant, K(b)=3.32×10(5) M(-1) for 1 and K(b)=3.10×10(5) M(-1) for 2). The catalytic role of these complexes in the oxidative and hydrolytic cleavage of DNA was studied in detail. Complex 1 binds and cleaves DNA more efficiently as compared to 2. From the kinetic experiments, rate constants for the hydrolysis of phosphodiester bond of DNA backbone were determined as 1.94 h(-1) and 1.05 h(-1) for 1 and 2 respectively. It amounts to (2.93-5.41)×10(7) fold rate enhancement compared to uncatalyzed double stranded DNA, which is impressive as compared to related Cu(II) Schiff base complexes.

Structural analysis of zinc-finger (TTK) + [Cu(BPA)]2+ /[Cu(IDB)]2+ + DNA complexes: an investigation by molecular dynamics simulation.

In the present study, the molecular dynamics simulation technique is employed to investigate the hydrogen abstraction possibility from sugar of DNA in two designed complexes of copper-based chemical nuclease [Cu(BPA)](2+) bis(2-pyridylmethyl) amine (BPA) or [Cu(IDB)](2+) N,N-bis(2-benzimidazolylmethyl) amine (IDB) bound to the zinc finger protein Tramtrack (TTK). The simulated results show that each of the designed complexes can form a stable conformation within 30 ns of simulation time with the substrate OOH(-) and an 18-base pair (bp) DNA segment and is located in the major groove of the DNA segment. The active terminal O atom of the OOH(-) substrate is found in close proximity to the target C2'H, C3'H, C4'H or C5'H proton of the DNA in TTK + [Cu(BPA)OOH](+) + DNA or TTK + [Cu(IDB)OOH](+) + DNA complex, which is crucial to propose the hydrogen abstraction possibility that is responsible for the DNA cleavage. The positions of copper-based chemical nucleases bound to TTK may substantially influence the hydrogen abstraction possibility. The structures and sizes of ligands in copper-based nucleases are also found to have influence on the order of difficulty of the hydrogen abstraction from the sugars of DNA.

Nucleic acids--genes, drugs, molecular lego and more.

Chemically modified nucleic acids find widespread use as tools in research, as diagnostic reagents and even as pharmaceutical compounds. On the background of antisense research and development, the synthesis and evaluation of modified oligonucleotides was intensively pursued in the early to mid nineties in corporate research of former Ciba. Most of these efforts concentrated on the development of sugar and/or backbone-modified derivatives for pharmaceutical applications. Additionally, oligonucleotide metal conjugates were investigated with the goal to develop artificial ribonucleases. Since the turn of the millennium also the potential of non-nucleosidic and non-hydrogen bonding building blocks has increasingly been recognized. Such derivatives possess unique properties that may have an impact in the fields of materials and genetic research. In this brief account, we take a personal look back on some past as well as some recent results.

DNA photocleavage activity of cobalt(III) polypyridyl complexes containing dpq ligand.

Four cobalt(III) polypyridyl complexes, [Co(phen)(3-)(n)(dpq)(n)](3+) (phen=1,10-phenanthroline, dpq=dipyrido[3,2-f:2',3'-h]-quinoxaline) (n=0, 1, 2, and 3) were synthesized and the influences of the dpq ligand on the photophysical properties, electrochemical properties, DNA binding affinities, as well as photonuclease activities of the complexes, were examined in detail. The presence of dpq ligand increases the DNA binding affinities of the corresponding complexes remarkably with respect to [Co(phen)(3)](3+). With the sequential substitution of phen ligand by dpq ligand, the (1)O(2) quantum yields of the corresponding complexes are enhanced greatly. As a result, the photonuclease activities follow the order of [Co(dpq)(3)](3+)>[Co(phen)(dpq)(2)](3+)>[Co(phen)(2)(dpq)](3+)>>[Co(phen)(3)](3+). It was found all the examined complexes can generate ()OH upon UV irradiation, and ()OH is also involved in DNA photocleavage as reactive oxygen species.

Synthesis and copper-mediated nuclease activity of a tetracationic tris(2,2'-bipyridine) ligand.

We report herein the synthesis of a novel tetracationic tris(2,2'-bipyridine) ligand 4. We show that this ligand metalated with copper(II), and in the presence of ascorbate as a reducing agent, strongly damages pUC18 plasmid DNA. Copper complex formation was demonstrated by ESI-MS (electrospray ionization-mass spectrum) at a 1:3 ligand to metal ratio. Binding of both 4 and its copper(II) complex to CT-DNA (calf thymus DNA) was characterized by viscosimetry, thermal denaturation and fluorescence-based competition assays. The viscosimetric data indicated that 4 and its copper(II) complex bind DNA through partial intercalation and thermal denaturation studies revealed a significant increase of duplex DNA stability in the presence of these species (DeltaT(m)=16.4 and 18.3 degrees C, respectively). Moreover, 4 and its copper(II) complex were found to effectively compete with ethidium bromide for the intercalative binding sites of DNA. Overall, the copper(II)-4 complex constitutes a very efficient DNA cleaving agent in the presence of ascorbate. Experiments with scavengers further suggest that the generation of Cu(I), hydrogen peroxide, superoxide, hydroxyl radical and singlet oxygen-like species contributes to the DNA breakage induced by the Cu(II) complex of 4.

Metal-free artificial nucleases based on simple oxime and hydroxylamine scaffolds.

Hydrolysis of DNA is of increasing importance in biotechnology and medicine. In this Letter, we present the DNA-cleavage potential of metal-free hydroxylamines and oximes as new members of nucleic acid cleavage agents.

Synthesis, structure, and nuclease properties of several binary and ternary complexes of copper(II) with norfloxacin and 1,10 phenantroline.

Three new binary Cu(II) complexes of norfloxacin have been synthesized and characterized. We also report the synthesis, characterization and X-ray crystallographic structures of a new binary compound, [Cu(HNor)(2)]Cl(2).2H(2)O (2) and two new ternary complexes norfloxacin-copper(II)-phen, [Cu(Nor)(phen)(H(2)O)](NO(3)).3H(2)O (4), and [Cu(HNor)(phen)(NO(3))](NO(3)).3H(2)O (5). The structure of 2 consists of two crystallographically independent cationic monomeric units of [Cu(HNor)(2)](2+), chloride anions, and uncoordinated water molecules. The Cu(II) ion is placed at a center of symmetry and is coordinated to two norfloxacin ligands which are related through the inversion center. The structures of 4 and 5 consist of cationic units ([Cu(Nor)(phen)(H(2)O)](+) for 4 and [Cu(HNor)(phen)(NO(3))](+) for 5), nitrate counteranions, and lattice water molecules that provide crystalline stability through a network of hydrogen-bond interactions. The complexes exhibit a five coordinated motif in a square pyramidal environment around the metal center. The ability of compounds 4 and 5 to cleave DNA has also been studied. Mechanistic studies with different inhibiting reagents reveal that hydroxyl radicals, singlet oxygen, and superoxide radicals are all involved in the DNA scission process mediated by these compounds.

Hydrolysis of plasmid DNA and RNA by amino alkyl naphthalimide as metal-free artificial nuclease.

A strategy of dimethylamino alkyldiimide conjugated with an intercalator of naphthalimide for hydrolysis of DNA was suggested and evaluated. 4 can hydrolyze 4 kb plasmid DNA into 2 kb fragments with GC and GG selectivity, which represents a novel example of sequence- or site-selective metal-free DNA artificial nuclease. Results also show it could hydrolyze RNA efficiently.

Design, construction and in vitro testing of zinc finger nucleases.

Zinc finger nucleases (ZFNs) are hybrid proteins that have been developed as targetable cleavage reagents for double-stranded DNA, both in vitro and in vivo. This protocol describes the design and construction of new DNA-binding domains comprised of zinc fingers (ZFs) directed at selected DNA sequences. Because the ZFNs must dimerize to cut DNA, they are designed in pairs for any new site. The first step is choosing a DNA segment of interest and searching it for sequences that can be recognized by combinations of existing ZFs. The second step is the construction of coding sequences for the selected ZF sets. Third, these coding sequences are linked to that of the nonspecific cleavage domain from the FokI restriction endonuclease in a cloning vector of choice. Finally, the ZFNs are expressed in Escherichia coli, partially purified, and tested in vitro for cleavage of the target sequences to which they were designed. If all goes smoothly, design, construction and cloning can be completed in about two weeks, with expression and testing completed in one additional week.

Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly.

Engineered zinc finger nucleases can stimulate gene targeting at specific genomic loci in insect, plant and human cells. Although several platforms for constructing artificial zinc finger arrays using "modular assembly" have been described, standardized reagents and protocols that permit rapid, cross-platform "mixing-and-matching" of the various zinc finger modules are not available. Here we describe a comprehensive, publicly available archive of plasmids encoding more than 140 well-characterized zinc finger modules together with complementary web-based software (termed ZiFiT) for identifying potential zinc finger target sites in a gene of interest. Our reagents have been standardized on a single platform, enabling facile mixing-and-matching of modules and transfer of assembled arrays to expression vectors without the need for specialized knowledge of zinc finger sequences or complicated oligonucleotide design. We also describe a bacterial cell-based reporter assay for rapidly screening the DNA-binding activities of assembled multi-finger arrays. This protocol can be completed in approximately 24-26 d.

Artificial metallonucleases.

The development of synthetic agents able to hydrolytically cleave DNA with high efficiency and selectivity is a fascinating challenge that will show the way to obtaining artificial nucleases able to compete with the natural enzymes. This Feature Article highlights the progress reported toward the realization of synthetic nucleases with particular attention to the strategies that can be pursued to improve efficiency and sequence selectivity.