Two Distinct Thermal Stabilities of DNA and Enzymatic Activities of DNase I in a Multistep Assembly with Carbazole Ligands: Different Binding Characteristics for Duplex and Quadruplex DNA

A partially hydrophobic carbazole ligand ((Im⁺)₂Cz: 2,2′‐(9‐ethyl‐9 H‐carbazole‐3,6‐diyl)bis(ethyne‐2,1‐diyl)bis(1,3‐dimethyl‐1 H‐imidazol‐3‐ium)) adopts two different binding states (binding states I and II) in its interactions with calf‐thymus (ct‐) DNA. Two distinct binding states were identified by biphasic UV/Vis and circular dichroism (CD) spectral changes during the titration of DNA into the carbazole ligand. At low concentrations of ct‐DNA, (Im⁺)₂Cz binds to nearly every part of ct‐DNA (binding state I). By contrast, an increased concentration of ct‐DNA results in a switch in the DNA‐binding state, so that the ligands are bound per five DNA base pairs. Similarly, a monocationic carbazole ligand (Im⁺Cz: 2‐((6‐bromo‐9‐ethyl‐9 H‐carbazol‐3‐yl)ethynyl)‐1,3‐dimethyl‐1 H‐imidazol‐3‐ium) also shows biphasic UV/Vis spectral changes during the titration of ct‐DNA into Im⁺Cz, which suggests two different binding states of the Im⁺Cz ligand with ct‐DNA. The stepwise equilibrium of the ligand–DNA‐complex formation is capable of switching the thermal stability of ct‐DNA, as well as the enzymatic activity of deoxyribonuclease (DNase I). In binding state I, the (Im⁺)₂Cz ligands interact with nearly every base pair in ct‐DNA and stabilize the double‐helix structure, which results in a larger increase in the melting temperature of the ct‐DNA than that observed with binding state II. On the other hand, the (Im⁺)₂Cz ligand significantly reduces the enzymatic activity of DNase I in binding state I, although the enzymatic activity is recovered once the binding state of the ligand–DNA complex is changed to binding state II. The (Im⁺)₂Cz ligand was also employed as a binder for G‐quadruplex DNA. In contrast to the stepwise complex formation between (Im⁺)₂Cz and ct‐DNA, (Im⁺)₂Cz shows a monotonous UV/Vis spectral response during the titration of G‐quadruplex DNA into (Im⁺)₂Cz, which suggests a single binding state for (Im⁺)₂Cz with G‐quadruplex DNA.     

The cis‐Diammineplatinum(II) Complex of Curcumin: A Dual Action DNA Crosslinking and Photochemotherapeutic Agent

[Pt(cur)(NH₃)₂](NO₃) ( 1 ), a curcumin‐bound cis‐diammineplatinum(II) complex, nicknamed Platicur, as a novel photoactivated chemotherapeutic agent releases photoactive curcumin and an active platinum(II) species upon irradiation with visible light. The hydrolytic instability of free curcumin reduces upon binding to platinum(II). Interactions of 1 with 5′‐GMP and ct‐DNA indicated formation of platinum‐bound DNA adducts upon exposure to visible light (λ=400–700 nm). It showed apoptotic photocytotoxicity in cancer cells (IC₅₀≈15 μM ), thus forming ^?OH, while remaining passive in the darkness (IC₅₀>200 μM ). A comet assay and platinum estimation suggest Pt–DNA crosslink formation. The fluorescence microscopic images showed cytosolic localization of curcumin, thus implying possibility of dual action as a chemo‐ and phototherapeutic agent.     

Synthesis,Characterization, Crystal Structure,and Biological Activities of Transition Metal Complexes with 1‐Phenyl‐3‐methyl‐5‐hydroxypyrazole‐4‐methylene‐8′‐quinolineimine

The Schiff base ligand, 1‐phenyl‐3‐methyl‐5‐hydroxypyrazole‐4‐methylene‐8′‐quinolineimine, and its Cu^II, Zn^II, and Ni^II complexes were synthesized and characterized. The crystal structure of the Zn^II complex was determined by single‐crystal X‐ray diffraction, indicating that the metal ions and Schiff base ligand can form mononuclear six‐coordination complexes with 1:1 metal‐to‐ligand stoichiometry at the metal ions as centers. The binding mechanism and affinity of the ligand and its metal complexes to calf thymus DNA (CT DNA) were investigated by UV/Vis spectroscopy, fluorescence titration spectroscopy, EB displacement experiments, and viscosity measurements, indicating that the free ligand and its metal complexes can bind to DNA via an intercalation mode with the binding constants at the order of magnitude of 105–106 M ^–1, and the metal complexes can bind to DNA more strongly than the free ligand alone. In addition, antioxidant activities of the ligand and its metal complexes were investigated through scavenging effects for hydroxyl radical in vitro, indicating that the compounds show stronger antioxidant activities than some standard antioxidants, such as mannitol. The ligand and its metal complexes were subjected to cytotoxic tests, and experimental results indicated that the metal complexes show significant cytotoxic activity against lung cancer A 549 cells.     

Mitochondria‐Targeting Oxidovanadium(IV) Complex as a Near‐IR Light Photocytotoxic Agent

Oxidovanadium(IV) complexes [VO(L¹)(phen)] ? Cl ( 1 ) and [VO(L²)(L³)] ? Cl ( 2 ), in which HL¹ is 2‐{[(benzimidazol‐2‐yl)methylimino]‐methyl}phenol (sal‐ambmz), HL² is 2‐[({1‐[(anthracen‐9‐yl)methyl]‐benzimidazol‐2‐yl}methylimino)‐methyl]phenol (sal‐an‐ambmz), phen is 1,10‐phenanthroline and L³ is dipyrido[3,2‐a:2′,3′‐c]phenazine (dppz) conjugated to a Gly‐Gly‐OMe dipeptide moiety, were prepared, characterized, and their DNA binding, photoinduced DNA‐cleavage, and photocytotoxic properties were studied. Fluorescence microscopy studies were performed by using complex 2 in HeLa and HaCaT cells. Complex 1 , structurally characterized by X‐ray crystallography, has a vanadyl group in VO₂N₄ core with the VO²⁺ moiety bonded to N,N‐donor phen and a N,N,O‐donor Schiff base. Complex 2 , having an anthracenyl fluorophore, showed fluorescence emission bands at 397, 419, and 443 nm. The complexes are redox‐active exhibiting the V(IV)/V(III) redox couple near ?0.85 V versus SCE in DMF 0.1 M tetrabutylammonium perchlorate (TBAP). Complex 2 , having a dipeptide moiety, showed specific binding towards poly(dAdT)₂ sequence. The dppz‐Gly‐Gly‐OMe complex showed significant DNA photocleavage activity in red light of 705 nm through a hydroxyl radical (^.OH) pathway. Complex 2 showed photocytotoxicity in HaCaT and HeLa cells in visible light (400–700 nm) and red light (620–700 nm), however, the complex was less toxic in the dark. Fluorescence microscopy revealed the localization of complex 2 primarily in mitochondria. Apoptosis was found to occur inside mitochondria (intrinsic pathway) caused by ROS generation.     

Synthesis,characterization and biological evaluation of a cobalt(II) complex with 5‐chloro‐8‐hydroxyquinoline as anticancer agent

A new cobalt(II) complex ( 1 ) of 5‐chloro‐8‐hydroxyquinoline was prepared and structurally characterized using infrared spectroscopy, electrospray ionization mass spectrometry, elemental analysis, single‐crystal X‐ray diffraction as well as powder X‐ray diffraction. Its biological activities including DNA binding and anticancer activity were investigated. The DNA binding study of complex 1 suggested that it interacted with calf thymus DNA mainly via an intercalative binding mode. The in vitro anticancer activity of complex 1 was screened against a series of tumor cell lines as well as the normal liver cell line HL‐7702 using MTT assay. complex 1 showed much higher cytotoxicity than corresponding metal salt and ligand towards the five tested tumor cell lines, in which T‐24 was the most sensitive tumor cell line towards 1, with IC₅₀ value of 7.04 ± 0.06 μM. complex 1 was found to greatly induce cell cycle arrest in T‐24 cells at S phase, and consequently to induce cell apoptosis in a dose‐dependent mode suggested by cell apoptosis analysis via Hoechst 33258 and acridine orange/ethidium bromide staining assays. The cell apoptosis mechanism of 1 was studied targeting the mitochondrion‐mediated pathway, since the apoptotic mechanism in the T‐24 cells treated by 1 was investigated by reactive oxygen species (ROS) detection, intracellular calcium concentration measurement and caspase‐9/3 activity assay. The results suggested that the cell apoptosis induced by 1 was closely related to the loss of mitochondrial membrane potential, ROS production and enhancement of intracellular [Ca²⁺], which would trigger the caspase‐9/3 activation via a mitochondrial dysfunction pathway. Copyright © 2016 John Wiley & Sons, Ltd.     

New anthracene based Schiff base ligands appended Cu(II) complexes: Theoretical study,DNA binding and cleavage activities

New anthracene based Schiff base ligands L ¹ and H( L ² ), their Cu(II) complexes [Cu( L ¹ )Cl₂] ( 1 ) and [Cu( L ² )Cl] ( 2 ) , (where L ¹  = N¹,N²‐bis(anthracene‐9‐methylene)benzene‐1,2‐diamine, L ²  = (2Z,4E)‐4‐(2‐(anthracen‐9‐ylmethyleneamino)phenylimino)pent‐2‐en‐2‐ol) have been prepared and characterized by elemental analysis, NMR, FAB‐mass, EPR, FT‐IR, UV–Vis and cyclic voltammetry. The electronic structures and geometrical parameters of complexes 1 and 2 were analyzed by the theoretical B3LYP/DFT method. The interaction of these complexes 1 and 2 with CT‐DNA has been explored by using absorption, cyclic voltammetric and CD spectral studies. From the electronic absorption spectral studies, it was found that the DNA binding constants of complexes 1 and 2 are 8.7 × 10³ and 7.0 × 10⁴ M^?1, respectively. From electrochemical studies, the ratio of DNA binding constants K₊/K₂₊ for 2 has been estimated to be >1. The high binding constant values, K₊/K₂₊ ratios more than unity and positive shift of voltammetric E_1/2 value on titration with DNA for complex 2 suggest that they bind more avidly with DNA than complex 1 . The inability to affect the conformational changes of DNA in the CD spectrum is the definite evidences of electrostatic binding by the complex 1 . It can be assumed that it is the bulky anthracene unit which sterically inhibits these complexes 1 and 2 from intercalation and thereby remains in the groove or electrostatic. The complex 2 hardly cleaves supercoiled pUC18 plasmid DNA in the presence of hydrogen peroxide. The results suggest that complex 2 bind to DNA through minor groove binding.     

Serum Albumin Binding Inhibits Nuclear Uptake of Luminescent Metal‐Complex‐Based DNA Imaging Probes

The DNA binding and cellular localization properties of a new luminescent heterobimetallic Ir^IIIRu^II tetrapyridophenazine complex are reported. Surprisingly, in standard cell media, in which its tetracationic, isostructural Ru^IIRu^II analogue is localized in the nucleus, the new tricationic complex is poorly taken up by live cells and demonstrates no nuclear staining. Consequent cell‐free studies reveal that the Ir^IIIRu^II complex binds bovine serum albumin, BSA, in Sudlow’s Site I with a similar increase in emission and binding affinity to that observed with DNA. Contrastingly, in serum‐free conditions the complex is rapidly internalized by live cells, where it localizes in cell nuclei and functions as a DNA imaging agent. The absence of serum proteins also greatly alters the cytotoxicity of the complex, where high levels of oncosis/necrosis are observed due to this enhanced uptake. This suggests that simply increasing the lipophilicity of a DNA imaging probe to enhance cellular uptake can be counterproductive as, due to increased binding to serum albumin protein, this strategy can actually disrupt nuclear targeting.     

UVA and UVB‐Induced 8‐Methoxypsoralen Photoadducts and a Novel Method for their Detection by Surface‐Enhanced Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (SELDI‐TOF MS)

UVA‐activated psoralens are used to treat hyperproliferative skin conditions due to their ability to form DNA photoadducts, which impair cellular processes and may lead to cell death. Although UVA (320–400 nm) is more commonly used clinically, studies have shown that UVB (280–320 nm) activation of psoralen can also be effective. However, there has been no characterization of UVB‐induced adduct formation in DNA alone. As psoralen derivatives have a greater extinction coefficient in the UVB region (11 800 cm^?1 M^?1 at 300 nm) compared with the UVA region (2016 cm^?1 M^?1 at 365 nm), a greater extent of adduct formation is expected. SELDI‐TOF, a proteomic technique that combines chromatography with mass spectrometry, was used to detect photoadduct formation in an alternating A–T oligonucleotide. 8‐Methoxypsoralen (8‐MOP) and DNA solutions were irradiated with either UVA or UVB. An adduct peak was obtained with SELDI‐TOF. For UVB‐activated 8‐MOP, the extent of adducts was three times greater than for UVA. HPLC ESI‐MS analysis showed that UVB irradiation yielded high levels of 3,4‐monoadducts (78% of total adducts). UVA was more effective than UVB at conversion of 4′,5′‐monoadducts to crosslinks (17% vs 4%, respectively). This report presents a method for comparing DNA binding efficiencies of interstrand crosslink inducing agents.     

Cyclometalated iridium(III) complex of 6‐hydroxydipyrido[3,2‐a:2′,3′‐c]phenazine: synthesis,and acid–base and avid DNA binding properties

A new cyclometalated Ir(III) complex [Ir(ppy)₂(hdppz)]PF₆ (Hppy = 2‐phenylpyridine and hdppz = 6‐hydroxydipyrido[3,2‐a:2′,3′‐c]phenazine) was synthesized and characterized. The pH effects on the UV–vis absorption spectra were studied and ground‐state acid ionization constant pK_a values of the complex were derived. The calf thymus DNA (ct‐DNA) binding properties of the complex were investigated with UV‐vis absorption spectrophotometric titrations, DNA competitive binding with ethidium bromide, DNA melting experiments, viscosity measurements and density functional theory (DFT) calculations. The complex was demonstrated to act as a ct‐DNA intercalator with a large DNA binding constant value of (6.06 ± 0.32) × 10⁶ M ⁻¹ in 50 mM NaCl. The avid DNA binding affinity observed was rationalized by the DFT calculations. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and Fluorescence Quenching Study of the Novel Cationic Probe Derived from Luminarosine

A simple and efficient transformation of the zwitterionic luminarosine into a brightly fluorescent cationic analogue, namely 1‐amino‐9‐methoxy‐2,4,10‐triaza‐4b‐azoniaphenanthrene ( 3 ), is reported. The fluorescence quenching of 3 by common nucleotides, calf‐thymus (CT) DNA, and halide ions was investigated by means of spectrophotometric and spectrofluorometric methods. Intermolecular static and dynamic fluorescence‐quenching constants for quenching of 3 by nucleotides and halide ions were determined in aqueous solution. Evidence for formation of nonfluorescent ground‐state complexes of 3 with nucleotides and CT‐DNA is presented. Scatchard analysis of the CT‐DNA quenching data resulted in a binding constant of 2.8×10⁴ M ⁻¹ and a number of binding sites per base pair of 0.049.     

Effect of glucocorticoids and their complexes with apolipoprotein A‐I on the secondary structure of eukaryotic DNA

The tetrahydrocortisol–apolipoprotein A‐I complex specifically interacts with eukaryotic DNA isolated from rat liver. This interaction is highly cooperative and of a saturating nature. One DNA molecule binds about 54 molecules of the complex. Small‐angle X‐ray scattering has shown that hydrogen bonds between nitrous bases are destroyed and that single‐stranded structures are formed at the interaction of the tetrahydrocortisol–apolipoprotein A‐I complex with eukaryotic DNA. The most probable site of binding the tetrahydrocortisol–apolipoprotein A‐I complex with DNA is the sequence of the CC(GCC)_n type entering the structure of many genes, among them the structure of the human apolipoprotein A‐I gene. Oligonucleotide of this type has been synthesized. The association constant (K_ass) of its complexation was shown to be 1.66 · 10⁶ M^?1. Substitution of tetrahydrocortisol for cortisol in the complex results in a considerable decrease of K_ass. IR‐spectroscopy study has shown that the interaction of tetrahydrocortisol with oligonucleotide CC(GCC)_3–5 is accompanied by the formation of hydrogen bonds via the CO‐NH, PO₂, and OH groups of desoxycytidinephosphate. The tetrahydrocortisol–apolipoprotein A‐I complex alters the DNA secondary structure formed at the interaction with the hormone, causing the structural transition “order → tangle.” It is assumed that in the GC‐pairs of the given DNA sequence, tetrahydrocortisol initiates the rupture of hydrogen bonds, while the hydrophobic interactions between nitrous bases and apoA‐I intensify this process. © 2004 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Ga(III) complex with morin for kidney cancer cell labelling

The formation of a complex between Ga(III) and morin (3,5,7,2′,4′‐pentahydroxyflavone) was studied. UV–visible, infrared and mass spectroscopies were used to characterize the complex. The stoichiometric ratio for the reaction between metal ion and flavonoid was determined using the methods of Yoe–Jones and Job, which confirmed that a 1:1 Ga–morin complex was formed (estimated binding constant = 2.31 × 10⁴ l mol⁻¹). It was found that the coordination to Ga(III) occurs through the carbonyl oxygen atom and the 3‐OH group of the morin molecule. According to developed conditions, complexation reaction with ⁶⁸Ga was performed and the complex was used to label kidney cancer cells (CAKI‐1, CAKI‐2, ACHN and 786‐O). The knowledge gained from this study should be useful for the development of new radiopharmaceuticals for diagnostic purposes containing ⁶⁸Ga.     

Structure‐Based Design of Platinum(II) Complexes as c‐myc Oncogene Down‐Regulators and Luminescent Probes for G‐Quadruplex DNA

A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G‐quadruplex DNA within the c‐myc gene promoter were evaluated. Complex 1 , which has a flat planar 2,6‐bis(benzimidazol‐2‐yl)pyridine (bzimpy) scaffold, was found to stabilize the c‐myc G‐quadruplex structure in a cell‐free system. An in silico G‐quadruplex DNA model has been constructed for structure‐based virtual screening to develop new Pt^II‐based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit‐to‐lead optimization, bzimpy and related 2,6‐bis(pyrazol‐3‐yl)pyridine (dPzPy) scaffolds containing amine side‐chains emerge as the top candidates. Six of the top‐scoring complexes were synthesized and their interactions with c‐myc G‐quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c‐myc G‐quadruplex. Complex 3 a ([Pt^II L2R ] ⁺ ; L2 =2,6‐bis[1‐(3‐piperidinepropyl)‐1H‐enzo[d]imidazol‐2‐yl]pyridine, R =Cl) displayed the strongest inhibition in a cell‐free system (IC₅₀=2.2 μM ) and was 3.3‐fold more potent than that of 1 . Complexes 3 a and 4 a ([Pt^II L3R ]⁺; L3 =2,6‐bis[1‐(3‐morpholinopropyl)‐1H‐pyrazol‐3‐yl]pyridine, R =Cl) were found to effectively inhibit c‐myc gene expression in human hepatocarcinoma cells with IC₅₀ values of ≈17 μM , whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM . Complexes 3 a and 4 a have a strong preference for G‐quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G‐quadruplex DNA with binding constants (K) of approximately 10⁶–10⁷ dm³ mol^?1, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3′‐terminal face of the G‐quadruplex. Intriguingly, binding of c‐myc G‐quadruplex DNA by 3 b is accompanied by an increase of up to 38‐fold in photoluminescence intensity at λ_max=622 nm.     

RNA Interference Controlled by Light of Variable Wavelength

Known molecular, “caged” siRNAs are activated by UV light. Since the light of this type is toxic to cells, the uncaging can cause undesired side effects. A modular, molecular system for designing siRNAs is reported, which can be activated by non‐toxic light in live cells. For example, siRNAs responsive to green and red light are described. The uncaging is mediated by ¹O₂ photogenerated on a photosensitizer, which is attached to the 3′‐terminus of the lagging strand. The 5′‐terminus of the guide strand is alkylated (“caged”) with a 9‐anthracenyl residue. The latter fragment reacts with the ¹O₂ with formation of the free (uncaged) 5′‐OH terminus. Simultaneously with the uncaging the photosensitizer is bleached and no more ¹O₂ is generated after this process is completed. The photoactivation of the siRNAs described here is not toxic to cells.     

Nucleotide Binding Preference of the Monofunctional Platinum Anticancer‐Agent Phenanthriplatin

The monofunctional platinum anticancer agent phenanthriplatin generates covalent adducts with the purine bases guanine and adenine. Preferential nucleotide binding was investigated by using a polymerase stop assay and linear DNA amplification with a 163‐base pair DNA double helix. Similarly to cisplatin, phenanthriplatin forms the majority of adducts at guanosine residues, but significant differences in both the number and position of platination sites emerge when comparing results for the two complexes. Notably, the monofunctional complex generates a greater number of polymerase‐halting lesions at adenosine residues than does cisplatin. Studies with 9‐methyladenine reveal that, under abiological conditions, phenanthriplatin binds to the N¹ or N⁷ position of 9‐methyladenine in approximately equimolar amounts. By contrast, comparable reactions with 9‐methylguanine afforded only the N⁷‐bound species. Both of the 9‐methyladenine linkage isomers (N¹ and N⁷) exist as two diastereomeric species, arising from hindered rotation of the aromatic ligands about their respective platinum–nitrogen bonds. Eyring analysis of rate constants extracted from variable‐temperature NMR spectroscopic data revealed that the activation energies for ligand rotation in the N¹‐bound platinum complex and the N⁷‐linkage isomers are comparable. Finally, a kinetic analysis indicated that phenanthriplatin reacts more rapidly, by a factor of eight, with 9‐methylguanine than with 9‐methyladenine, suggesting that the distribution of lesions formed on double‐stranded DNA is kinetically controlled. In addition, implications for the potent anticancer activity of phenanthriplatin are discussed herein.     

DNA-binding studies of a new Cu(II) complex containing reverse transcriptase inhibitor and anti-HIV drug zalcitabine

Abstract

In this study, a new copper(II) complex with zalcitabine (ddC) drug was synthesized and characterized by Fourier-transform infrared spectroscopy (FT-IR), ultraviolet–visible spectroscopy (UV–vis), mass spectroscopy, thermal gravimetric analysis and density functional theory. Then, its effect on calf-thymus DNA (CT-DNA) was investigated using absorption and fluorescence spectroscopy and viscometry technique. On the basis of FT-IR and computational studies, zalcitabine chelates with copper using its C(2)=O and N(3) group in the [Cu(zalcitabine)Cl₂] ([CuCl₂(ddC)]) complex. On the basis of the electrospray ionization mass spectroscopy of the Cu–ddC complex, monomeric copper complex [C₉H₁₃N₃O₃CuCl₂] was formed. The results of fluorescence studies indicated increasing to around 2.5 times in emission intensity of fluorescence signal of the complex. The enhancement of emission intensity and also the positive ΔH and positive ΔS values suggested that the hydrophobic interaction plays a major role in the binding with overall binding constant of 1(±0.25)×10⁵ M^?1. The ΔG value implied that the interaction occurred between DNA and the complex formation was spontaneous. Finally, changes in the relative viscosity showed that groove binding must be the predominant form of binding. Evidences are provided that [Cu(ddC)Cl₂] could interact with DNA via minor groove interaction mode.     

Stabilization of G‐Quadruplex DNA with Platinum(II) Schiff Base Complexes: Luminescent Probe and Down‐Regulation of c‐myc Oncogene Expression

The interactions of a series of platinum(II) Schiff base complexes with c‐myc G‐quadruplex DNA were studied. Complex [PtL ^1a ] ( 1 a ; H₂L ^1a =N,N′‐bis(salicylidene)‐4,5‐methoxy‐1,2‐phenylenediamine) can moderately inhibit c‐myc gene promoter activity in a cell‐free system through stabilizing the G‐quadruplex structure and can inhibit c‐myc oncogene expression in cultured cells. The interaction between 1 a and G‐quadruplex DNA has been examined by ¹H NMR spectroscopy. By using computer‐aided structure‐based drug design for hit‐to‐lead optimization, an in silico G‐quadruplex DNA model has been constructed for docking‐based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL ³ ] ( 3 ; H₂L ³ = N,N′‐bis{4‐[1‐(2‐propylpiperidine)oxy]salicylidene}‐4,5‐methoxy‐1,2‐phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c‐myc G‐quadruplex. The inhibitory activity of 3 (IC₅₀=4.4 μM ) is tenfold more than that of 1 a . The interaction between 1 a or 3 with c‐myc G‐quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1 a and 3 bind c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3’ terminal face of the G‐quadruplex. Such binding of G‐quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at λ_max=652 nm. Complex 3 also effectively down‐regulated the expression of c‐myc in human hepatocarcinoma cells.     

A Tri‐copper(II) Complex Displaying DNA‐Cleaving Properties and Antiproliferative Activity against Cancer Cells

A new disubstituted terpyridine ligand and the corresponding tri‐copper(II) complex have been prepared and characterised. The binding affinity and binding mode of this tri‐copper complex (as well as the previously reported mono‐ and di‐copper analogues) towards duplex DNA were determined by using UV/Vis spectroscopic titrations and fluorescent indicator displacement (FID) assays. These studies showed the three complexes to bind moderately (in the order of 10⁴ M ^?1) to duplex DNA (ct‐DNA and a 26‐mer sequence). Furthermore, the number of copper centres and the nature of the substituents were found to play a significant role in defining the binding mode (intercalative or groove binding). The nuclease potential of the three complexes was investigated by using circular plasmid DNA as a substrate and analysing the products by agarose‐gel electrophoresis. The cleaving activity was found to be dependent on the number of copper centres present (cleaving potency was in the order: tri‐copper>di‐copper>mono‐copper). Interestingly, the tri‐copper complex was able to cleave DNA without the need of external co‐reductants. As this complex displayed the most promising nuclease properties, cell‐based studies were carried out to establish if there was a direct link between DNA cleavage and cellular toxicity. The tri‐copper complex displayed high cytotoxicity against four cancer cell lines. Of particular interest was that it displayed high cytotoxicity against the cisplatin‐resistant MOLT‐4 leukaemia cell line. Cellular uptake studies showed that the tri‐copper complex was able to enter the cell and more importantly localise in the nucleus. Immunoblotting analysis (used to monitor changes in protein levels related to the DNA damage response pathway) and DNA‐flow cytometric studies suggested that this tri‐copper(II) complex is able to induce cellular DNA damage.     

Effective Homogeneous Hydrolysis of Phosphodiester and DNA Cleavage by Chitosan‐copper Complex

We aimed to explore the role of chitosan‐based metal complexes in catalyzing the hydrolysis of phosphodiesters. To this end, we performed detailed studies on the kinetics of the chitosan copper complex (CSCu)‐catalyzed hydrolysis of bis(4‐nitrophenol) phosphate (BNPP) in Tris‐H⁺ buffer and in an organic solvent. A significant enhancement in the rate of reaction (up to 3×10⁵‐fold acceleration) was observed at pH 8.0 (25°C). The pH dependence of BNPP hydrolysis at pH 5.5–9.5 and the UV spectra revealed that the copper‐bounded water molecules underwent deprotonation to form the active catalytic species CSCu‐OH. The kinetic behavior of BNPP catalytic hydrolysis in the Tris‐H⁺ buffer was consistent with that predicted by the Michaelis‐Menten kinetics model. An intramolecular nucleophilic attack by the copper‐bonded hydroxide group on the same activated phosphodiester substrate was proposed as the catalytic mechanism for CSCu‐catalyzed reaction system. The results of DNA binding and cleavage experiments indicated electrostatic binding mode of CSCu to DNA as well as the strong capability of CSCu to disturb the supercoiled strand of DNA and cleave it to nicked circular form.     

Probing the Coordination Environment of the Human Copper Chaperone HAH1: Characterization of HgII‐Bridged Homodimeric Species in Solution

Although metal ion homeostasis in cells is often mediated through metallochaperones, there are opportunities for toxic metals to be sequestered through the existing transport apparatus. Proper trafficking of Cu^I in human cells is partially achieved through complexation by HAH1, the human metallochaperone responsible for copper delivery to the Wilson and Menkes ATPase located in the trans‐Golgi apparatus. In addition to binding copper, HAH1 strongly complexes Hg^II, with the X‐ray structure of this complex previously described. It is important to clarify the solution behavior of these systems and, therefore, the binding of Hg^II to HAH1 was probed over the pH range 7.5 to 9.4 using ¹⁹⁹Hg NMR, ^199mHg PAC and UV–visible spectroscopies. The metal‐dependent protein association over this pH range was examined using analytical gel‐filtration. It can be concluded that at pH 7.5, Hg^II is bound to a monomeric HAH1 as a two coordinate, linear complex (HgS₂), like the Hg^II–Atx1 X‐ray structure (PDB ID: 1CC8). At pH 9.4, Hg^II promotes HAH1 association, leading to formation of HgS₃ and HgS₄ complexes, which are in exchange on the μs–ns time scale. Thus, structures that may represent central intermediates in the process of metal ion transfer, as well as their exchange kinetics have been characterized.