Experimental and theoretical investigations of novel oxidovanadium(IV) juglone complex: DNA/HSA interaction and cytotoxic activity

Abstract

A new oxidovanadium(IV) complex VO(L)(Jug) (HL = 5-methoxy-1,3-bis (1-methyl-1H-benzo[d]imidazol-2-yl)benzene, Jug?=?juglone) was synthesized and characterized. Interactions of the V(IV) complex with calf thymus DNA (CT DNA) and human serum albumin were studied using different techniques such as UV–vis and fluorescence emission spectroscopy. The experimental results were confirmed by the molecular docking study. The oxidovanadium(IV) complex can efficiently cleave pUC19 DNA in the presence of Hydrogen peroxide. Also, the in vitro cytotoxicity properties of the oxidovanadium(IV) complex was evaluated against MCF-7, HPG-2 and HT-29 cancer cell lines and HEK293 non-malignant fibroblasts were evaluated and compared with free ligands, VOSO₄ and cisplatin as reference drugs.

Communicated by Ramaswamy H. Sarma     

First syntheses and X-ray structures of a meso-alkyl-substituted corrole and its Ga(III) complex

The solvent-free condensation of heptafluorobutanal and pyrrole leads to the corresponding meso-alkyl-substituted corrole, which was metallated by gallium chloride to provide the first first-row non-transition-metal corrole. Both the ligand and the complex were characterized by X-ray crystallography.     

Synthesis, crystal structure and interaction with DNA and HSA of (N,N'-dibenzylethane-1,2-diamine) transition metal complexes

A new ligand, N,N′-dibenzylethane-1,2-diamine (L) and its four transition metal(II) complexes, ML₂(OAc)₂ · 2H₂O (M = Cu, Ni, Zn, Co), have been synthesized and characterized by elemental analysis, mass spectra, molar conductivity, NMR and IR. Moreover, the crystals structure of Cu(II) and Ni(II) complexes characterized by single crystal X-ray diffraction showed that the complexes have a similar molecular structure. Ni(II) has an regular octahedral coordination environment complexes, but typical Jahn Teller effect influenced Cu(II) in an elongated octahedral environment. The interaction between complexes and calf thymus DNA were studied by UV and fluorescence spectra measure, which showed that the binding mode of complexes with DNA is intercalation. Under physiological pH condition, the effects of Cu(OAc)₂L₂ · 2H₂O and Ni(OAc)₂L₂ · 2H₂O on human serum albumin were examined by fluorescence. The results of spectroscopic measurements suggested that the hydrophobic interaction is the predominant intermolecular force. The enthalpy change ΔH⁰ and the entropy change ΔS⁰ of Cu(OAc)₂L₂ · 2H₂O and Ni(OAc)₂L₂ · 2H₂O were calculated to be −11.533 kJ mol⁻¹ and 46.339 J mol⁻¹ K⁻¹, −11.026 kJ mol⁻¹ and 46.396 J mol⁻¹ K⁻¹, respectively, according to the Scatchard’s equation. The quenching mechanism and the number of binding site (n ≈ 1) were also obtained from fluorescence titration data.     

Phosphorus(V) corrole: DNA binding,photonuclease activity and cytotoxicity toward tumor cells

A new phosphorus(V) corrole, 10-(4-hydroxylphenyl)-5,15-bis(pentafluorophenyl)corrolato (trans-dihydroxo)phosphorus(V) 1-P, was synthesized and characterized. The interaction of 1-P with calf thymus DNA (CT-DNA) was studied by multi-spectroscopic methods. The photonuclease activity of this complex was examined by agarose gel electrophoresis. 1-P may bind to CT-DNA via an outside binding mode and display good photonuclease activity. 1-P displayed low dark toxicity but high photocytotoxic activity against H460 and A549 tumor cell lines.     

First syntheses and X-ray structures of a meso-alkyl-substituted corrole and its Ga(III) complex

The solvent-free condensation of heptafluorobutanal and pyrrole leads to the corresponding meso-alkyl-substituted corrole, which was metallated by gallium chloride to provide the first first-row non-transition-metal corrole. Both the ligand and the complex were characterized by X-ray crystallography.     

Palladium(II) complexes of biorelevant ligands. Synthesis,structures, cytotoxicity and rich DNA/HSA interaction studies

In this work, a pair of new palladium(II) complexes, [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)], (where Gly is glycine, Phe is phenylalanine, and Tyr is tyrosine) were synthesized and characterized by UV–Vis, FT-IR, elemental analysis, ¹H-NMR, and conductivity measurements. The detailed ¹H NMR and infrared spectral studies of these Pd(II) complexes ascertain the mode of binding of amino acids to palladium through nitrogen of -NH₂ and oxygen of -COO^? groups as bidentate chelates. The Pd(II) complexes have been tested for in vitro cytotoxicity activities against cancer cell line of K₅₆₂. Interactions of these Pd(II) complexes with CT-DNA and human serum albumin were identified through absorption/emission titrations and gel electrophoresis which indicated significant binding proficiency. The binding distance (r) between these synthesized complexes and HSA based on Forster?s theory of non-radiation energy transfer were calculated. Alterations of HSA secondary structure induced by complexes were confirmed by FT-IR measurements. The results of emission quenching at three temperatures have revealed that the quenching mechanism of these Pd(II) complexes with CT-DNA and HSA were the static and dynamic quenching mechanism, respectively. Binding constants (K_b), binding site number (n), and the corresponding thermodynamic parameters were calculated and revealed that the hydrogen binding and hydrophobic forces played a major role when Pd(II) complexes interacted with DNA and HSA, respectively. We bid that [Pd(Gly)(Phe)] and [Pd(Gly)(Tyr)] complexes exhibit the groove binding with CT-DNA and interact with the main binding pocket of HSA. The complexes follow the binding affinity order of [Pd(Gly)(Tyr)] > [Pd(Gly)(Phe)] with CT-DNA- and HSA-binding.     

Determination of DNA polymerase and nuclease activities of DNA-dependent polymerases using fluorescence detection under real-time conditions

A new method is proposed for estimation of polymerase activities using fluorescence detection during isothermal reaction. The method allows simultaneous determination of DNA-dependent DNA polymerase and 5'-3'-exonuclease activities using amplifiers supplied with an optical module for fluorescence detection under real-time conditions. Different primer-template combinations used as polymerase substrates were compared. Primer elongation (polymerase reaction) is detected by changes in SYBR Green I fluorescence upon binding to dsDNA during reaction; nuclease activities are detected by changes in fluorescence due to cleavage of the probe, containing the reporter fluorophore and fluorescence quencher, and hybridized in advance to the template single-stranded region. It was also shown that the method can be used for determination of relative activities of DNA polymerase preparations, estimation of temperature-time dissociation parameters of polymerase complexes with specific antibodies to its active center, and analysis of effects of inhibitors and activators of different nature on reaction rates of dsDNA polymerization and 5'-3'-exonuclease cleavage by polymerase. The method can be also used for estimation of endonuclease activities of DNA polymerases.     

DNA‐binding affinity and nuclease activity of two cytotoxic copper terpyridine complexes

Two copper(II) terpyridine complexes, [Cu(atpy)(NO₃)(H₂O)](NO₃) ? 3H₂O ( 1 ) and [Cu(ttpy)(NO₃)₂] ( 2 ) (atpy = 4′‐p‐N9‐adeninylmethyl‐phenyl‐2,2′:6,2″‐terpyridine; ttpy = 4′‐p‐tolyl‐2,2′:6,2″‐terpyridine) exhibited high cytotoxicity, with average ten times more potency than cisplatin against the human cervix carcinoma cell line (HeLa), the human liver carcinoma cell line (HepG2), the human galactophore carcinoma cell line (MCF7), and the human prostate carcinoma cell line (PC‐3). The cytotoxicity of the complex 1 was lower than that of the complex 2 . Both complexes showed more efficient oxidative DNA cleavage activity under irradiation with UV light at 260 nm than in the presence of ascorbic acid. Especially, complex 1 exhibited evident photoinduced double‐stranded DNA cleavage activity. The preliminary mechanism experiments revealed that hydrogen peroxide was involved in the oxidative DNA damage induced by both complexes. From the absorption titration data, the DNA‐binding affinity of the complexes with surpersoiled plasmid pUC19 DNA, polydAdT, and polydGdC was calculated and complex 2 showed higher binding affinity than complex 1 with all these substrates. The DNA cleavage ability and DNA‐binding affinity of both complexes depended on the substituent group on the terpyrdine ligands. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:295–302, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20292     

嗜热古菌Archaeoglobus fulgidus RecJ核酸酶的表达纯化及酶学特征

[目的]克隆表达嗜热古菌Archaeoglobus fulgidus(A.fulgidus)来源的RecJ核酸酶基因(ORF编号AF_0699,NCBI数据库基因登陆号为AF_RS03550),对该重组蛋白的核酸酶活性及酶学特征进行鉴定和分析.[方法]将A.fulgidus RecJ(AfuRecJ)核酸酶在大肠杆菌中...     

Surface trapping and AFM detection of DNA topological intermediates generated from an oxidative chemical nuclease

Direct observation of DNA topological intermediates generated from a 'chemical nuclease' treatment has been made by atomic force microscopy (AFM). The intermediates were trapped at the mica-water interface and imaging was carried out in the dynamic force mode. Complete conversion from supercoiled circular state to relaxed circular/linear state has been observed over a time scale of 8 min. Implication of such studies in complementing gel electrophoresis data has been predicted.     

Investigation on the interaction of newly designed potential antibacterial Zn(II) complexes with CT-DNA and HSA

Two Zn(II) complexes of formula [Zn(bpy)(Gly)]NO₃ (I) and [Zn(phen)(Gly)]NO₃ (II) (where bpy = 2,2′-bipyridine, phen = 1,10-phenanthroline and Gly = glycine) were synthesized and characterized by elemental analysis, molar conductance measurements, UV–vis, FT-IR, and ¹H NMR spectra. The interaction ability of these complexes with calf thymus DNA was monitored using spectroscopic methods, including UV–vis absorption spectroscopy, ethidium bromide displacement, Fourier transform infrared, and electrophoretic mobility assay. Further, the human serum albumin interactions of complexes I and II were investigated using UV–vis absorption spectroscopy, fluorescence quenching, circular dichroism, and Fourier transform infrared. The results obtained from these analyses indicated that both complexes interact effectively with CT-DNA and HSA. The binding constant (K_b), the Stern–Volmer constant (K_sv), and the number of binding sites (n) at different temperatures were determined for CT-DNA and HSA. Also, the negative ΔH° and ΔS° values showed that both hydrogen bonds and van der Waals forces played major roles in the association of CT-DNA-Zn(II) and HSA-Zn(II) complex formation. The displacement experiments suggested that Zn(II)-complexes primarily bound to Sudlow’s site II of HSA. The distance between the donor (HSA) and the acceptor (Zn(II) complexes) was estimated on the basis of the Forster resonance energy transfer (FRET) and the alteration of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. The complexes follow the binding affinity order of I > II with DNA and II > I with HSA. Finally, Antibacterial activity of complexes I and II have been screened against gram positive and gram negative bacteria.     

A new Co(III)-hypocrellin B complex with enhanced photonuclease activity

Hypocrellin B (HB), a naturally occurring photosensitizer, has been extensively and intensively studied as a promising photodynamic therapy (PDT) agent. In this work, a new Co(III) complex [Co₂(HB)(tmp)₄]⁴⁺ (tmp = 3,4,7,8-tetramethyl-1,10-phenanthroline) was designed and synthesized with HB as bridging ligand and tmp as terminal ligand. [Co₂HB(tmp)₄]⁴⁺ exhibits improved water solubility, enhanced absorptivity in the phototherapeutic window, increased binding affinity and DNA photocleavage capability toward dsDNA with respect to HB. The photodynamic activity of [Co₂(HB)(tmp)₄]⁴⁺ stems from its ¹O₂ photosensitization ability, in sharp contrast to [Cu₂(HB)(tmp)₂]²⁺ which relies on superoxide anion radical (O₂^-) and hydroxyl radical (·OH) to photocleave DNA, though the both complexes possess similar electrochemical properties. The remarkable difference between the photodynamic mechanisms of [Co₂(HB)(tmp)₄]⁴⁺ and [Cu₂(HB)(tmp)₂]²⁺ was discussed in detail.     

Mammalian DNA2 helicase/nuclease cleaves G‐quadruplex DNA and is required for telomere integrity

Efficient and faithful replication of telomeric DNA is critical for maintaining genome integrity. The G‐quadruplex (G4) structure arising in the repetitive TTAGGG sequence is thought to stall replication forks, impairing efficient telomere replication and leading to telomere instabilities. However, pathways modulating telomeric G4 are poorly understood, and it is unclear whether defects in these pathways contribute to genome instabilities in vivo. Here, we report that mammalian DNA2 helicase/nuclease recognizes and cleaves telomeric G4 in vitro. Consistent with DNA2's role in removing G4, DNA2 deficiency in mouse cells leads to telomere replication defects, elevating the levels of fragile telomeres (FTs) and sister telomere associations (STAs). Such telomere defects are enhanced by stabilizers of G4. Moreover, DNA2 deficiency induces telomere DNA damage and chromosome segregation errors, resulting in tetraploidy and aneuploidy. Consequently, DNA2‐deficient mice develop aneuploidy‐associated cancers containing dysfunctional telomeres. Collectively, our genetic, cytological, and biochemical results suggest that mammalian DNA2 reduces replication stress at telomeres, thereby preserving genome stability and suppressing cancer development, and that this may involve, at least in part, nucleolytic processing of telomeric G4.     

DNA核酸酶与猪链球菌9型毒力的关系

【目的】除了猪链球菌2型外,猪链球菌9型(SS9)也是目前流行血清型,同时也是人畜共患病原菌。前期研究发现,DNA核酸酶(Ssn A)存在于SS9毒力株中,在SS9无毒株中不存在。为明确Ssn A对SS9毒力的影响,本研究构建ssn A缺失株Δssn A,并研究其生物学功能。【方法】用穿梭质粒p SET-4s构建Δssn A,并通过斑马鱼毒力试验、HEp-2细胞黏附、猪全血存活和酶活检测等试验,评价Ssn A对SS9毒力的影响。【结果】斑马鱼毒力试验显示,Δssn A对斑马鱼毒力显著降低,半数致死量是野生株的11.2倍;Δssn A对HEp-2细胞的黏附率为野生株的60.61%;Δssn A在猪全血中的存活率为野生株的71.88%;酶活试验表明,Ssn A可降解线性和环状DNA。【结论】本研究表明SS9 Ssn A具有降解线性和环状DNA能力,该基因缺失后细菌对斑马鱼毒力、黏附HEp-2细胞能力、在猪全血中存活及分解DNA能力都显著降低,证实Ssn A是SS9的一个毒力因子。     

Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis,characterization, antitumor activities and rich DNA/HSA interaction studies

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of –NH₂ and the oxygen of –COO^– groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K₅₆₂) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide–DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA.

Communicated by Ramaswamy H. Sarma     

DNA binding and nuclease activity of copper(II) complexes of tridentate ligands

Two copper(II) complexes, 1 and 2 with L₁ and L₂ [L₁ = 2-hydroxybenzyl(2-(pyridin-2-yl)ethylamine); L₂ = 2-hydroxybenzyl(2-(pyridin-2-yl)methylamine)] ligands, respectively, have been synthesized and characterized. The interaction of both the complexes with DNA has been studied to explore their potential biological activity. The DNA binding properties of the complexes with calf thymus (CT) DNA were studied by spectroscopic titration. The complexes show binding affinity to CT DNA with binding constant (K_b) values in the order of 10⁵ M⁻¹. Thermal denaturation and circular dichroism studies suggest groove binding of the complexes to CT DNA. Complexes also exhibit strong DNA cleavage activity in presence of reducing agents like 3-mercaptopropionic acid and β-mercaptoethanol. Mechanistic studies reveal the involvement of reactive hydroxyl radicals for their DNA cleavage activity.     

DNA interaction and photodynamic antitumor activity of transition metal mono-hydroxyl corrole

A series of iron(III), manganese(III) and copper(III) mono-hydroxyl corrole complexes had been prepared and well characterized by UV–vis, ¹H NMR, ¹⁹F NMR and HR-MS. These metallocorroles may bind to CT-DNA through external binding mode. Metallocorrole Fe-2c exhibited significant phototoxicity and low toxicity toward A549 tumor cells. While manganese (III) and copper (III) corroles showed hypotoxicity to A549, MCF-7 and HepG-2 tumor cells, whether under dark or illumination conditions. All tested metallocorroles exhibited non-toxicity to human normal cells (GES-1) with or without irradiation at 625 nm. Cell cycle analysis indicated that metallocorrole Fe-2c arrested the cell cycle at G2/M phase and increased the Sub-G1 phase in A549 cell lines. It was mainly localized at mitochondria and could significantly reduce mitochondrial membrane potential after photodynamic treatment, which would further induce tumor cell apoptosis.     

DNA binding property, nuclease activity and cytotoxicity of Zn(II) complexes of terpyridine derivatives

Two zinc(II) terpyridine complexes Zn(atpy)₂(PF₆)₂ (1) (atpy = 4′-p-N9′-adeninylmethylphenyl-2,2′:6,2′′-terpyridine) and Zn(ttpy)₂(PF₆)₂ (2) (ttpy = 4′-p-tolyl-2,2′:6,2′′-terpyridine) have been synthesized and characterized by elemental analysis, ¹H NMR and electrospray mass spectroscopy. The structure of complex 2 was also determined by X-ray crystallography, which revealed a ZnN₆ coordination in an octahedral geometry with two terpyridine acting as equatorial ligands. The circular dichroism data showed that complex 1 exhibited an ICD signal at around 300 nm and induced more evident disturbances on DNA base stacking than complex 2, reflecting the impact of the adenine moiety on DNA binding modes. Complex 1 exhibited higher cleavage activity to supercoiled pUC 19 DNA than complex 2 under aerobic conditions, suggesting a promotional effect of adenine moiety in DNA nuclease ability. Interestingly, both complexes demonstrated potent in vitro cytotoxicity against a series human tumor cell lines such as human cervix carcinoma cell line (HeLa), human liver carcinoma cell line (HepG2), human galactophore carcinoma cell line (MCF-7) and human prostate carcinoma cell line (pc-3). The cytotoxicity is averagely 10 times more active than the anticancer drug cisplatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

核酸酶P1催化水解热变性DNA的研究

用桔青霉菌株IM0 2 (PenicilliumcitrinumIM0 2 )发酵制得的核酸酶P1催化水解热变性DNA ,在研究温度、底物浓度、pH、酶加入量等因素对水解结果的影响的实验基础上,通过正交实验获得最佳的工艺条件:温度5 8℃,pH6 5 ,DNA质量浓度4 0g/L ,酶加入量4 % ,脱氧核苷酸的得率92 %。在此基础上推导出其催化米氏常数Km=5 818×10 -2 g/L。     

DNA binding and nuclease activity of a one-dimensional heterometallic nitrosyl complex

The interaction of a structurally characterized Sr–Fe nitrosyl complex with DNA has been studied by UV–vis and fluorescence spectroscopy, viscometric, and gel electrophoresis techniques. From the absorption titration studies the intrinsic binding constant of the complex with DNA was calculated to be 1.6 × 10⁴ M⁻¹. Fluorimetric studies indicate that the complex compete with EB in binding to DNA. The complex shows nuclease activity on pUC19 supercoiled DNA in presence of H₂O₂.