人工核酸酶的设计和与核酸的相互作用

人工核酸酶在基因药物设计和分子生物学研究领域具有重要的科学意义,因此人工核酸酶的设计和酶模拟方面的研究引起了广泛关注并取得了重要进展．本文介绍近年来国内外学者和作者研究组在“双功能协同催化”、“双核协同催化”、“无金属催化”的人工核酸酶方面的研究进展,尤其是氮杂冠醚衍生物作为人工核酸酶与DNA相互作用的研究进展．     

DNA Binding and Cleavage Activity of Zinc(II) Complex of N,N′‐Bis(2‐guanidinoethyl)‐2,6‐pyridinedicarboxamide

The interaction of zinc(II) complex of N,N′‐bis(guanidinoethyl)‐2,6‐pyridinedicarboxamide (Gua) with DNA was studied by CD spectroscopy and agarose gel electrophoresis analysis. The results indicate that the DNA binding affinity of Zn²⁺‐Gua is stronger than that of Gua and the Zn²⁺‐Gua can promote the cleavage of phosphodiester bond of supercoiled DNA under a physiological condition, which is ～10⁶ times higher than DNA natural degradation. The hydrolysis pathway was proposed as the possible mechanism for DNA cleavage promoted by the Zn²⁺‐ Gua. The acceleration is due to cooperative catalysis of the zinc cation center and the functional groups (bisguanidinium groups).     

Synthesis and DNA Cleavage Properties of Triazacrown Derivatives

New metal‐free DNA cleaving reagent 1 , 1,4,7‐triazacrown (TACN) both with aminoethyl, hydroxyethyl side arms and a planar anthraquinone linked by an alkyl (1,6‐hexamethylene) spacer has been synthesized and characterized by NMR and MS spectrometry. For comparison, the corresponding aminoethyl, hydroxyethyl triazacrown derivative 2 without the anthraquinone has also been synthesized. DNA‐binding properties via fluorescence and CD spectroscopy indicate that the binding affinity of 1 with DNA is much stronger than that of 2 . Agarose gel electrophoresis was used to assess plasmid pUC19 DNA cleavage. Kinetic data of DNA cleavage promoted by 1 , 2 and parent triazacrown (TACN) 3 under physiological condition give the 15‐fold and 234‐fold rate acceleration of compound 1 over 2 and parent triazacrown 3 . Radical scavenger inhibition study suggests that DNA cleavage promoted by 1 may be a non‐oxidative pathway through the transphosphorylation and then hydrolysis. The dramatic rate acceleration is due not only to the anthraquinone moiety of compound 1 intercalating into DNA base pairs via stacking interaction, but also the cooperative catalysis of the nucleophilic hydroxyl and the electrophilic ammonium group for the cleavage of phosphodiester of DNA.     

Ferrocene‐bridging dinuclear cyclen copper(II) complexes as high efficient artificial nucleases: design,synthesis and interaction with DNA

Two novel cyclen copper(II) complexes bridged by ferrocene were designed and synthesized. Both of these complexes exhibited excellent cleavage ability towards plasmid DNA via an oxidative pathway without the presence of any additives. Cyclic voltammetry was used to investigate the electrochemistry characters of the interaction between the complexes and DNA. Agarose gel electrophoresis was carried out to study the DNA restriction ability of these complexes, and the results indicated that the complexes showed higher cleavage efficiency via an oxidative pathway without the presence of any additives. The mechanism of DNA cleavage catalyzed by these complexes was examined by the addition of various scavengers, and the results showed that singlet oxygen and hydroxyl radical might be responsible for the cleavage process. Copyright © 2008 John Wiley & Sons, Ltd.     

Synthesis,crystal structure,DNA binding,and DNA cleavage of a zinc complex containing N,N-bis(2-pyridylmethyl)amine

A water-soluble zinc complex, [Zn(bpea)Cl₂] (1) (bpea?=?N,N-bis(2-pyridylmethyl)ethylamine), was prepared to serve as a nuclease mimic. The complex was characterized by X-ray, infrared, and UV spectroscopy. Interactions of the complex with calf thymus-DNA (ct-DNA) have been investigated by UV absorption and fluorescence spectroscopies; the mode of ct-DNA binding for 1 has been proposed. DNA cleavage activities by 1 were performed in the absence of external agents. The influences of different complex concentrations or reaction times on DNA cleavage were studied.     

Synthesis, spectral characterization of Schiff base transition metal complexes: DNA cleavage and antimicrobial activity studies

A new series of transition metal complexes of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), VO(IV), Hg(II) and Cd(II) have been synthesized from the Schiff base (L) derived from 4-aminoantipyrine, 3-hydroxy-4-nitrobenzaldehyde and o-phenylenediamine. Structural features were obtained from their elemental analyses, magnetic susceptibility, molar conductance, mass, IR, UV-Vis, ¹H NMR and ESR spectral studies. The data show that these complexes have composition of ML type. The UV-Vis, magnetic susceptibility and ESR spectral data of the complexes suggest a square-planar geometry around the central metal ion except VO(IV) complex which has square-pyramidal geometry. The redox behaviour of copper and vanadyl complexes was studied by cyclic voltammetry. Antimicrobial screening tests gave good results in the presence of metal ion in the ligand system. The nuclease activity of the above metal complexes shows that Cu, Ni and Co complexes cleave DNA through redox chemistry whereas other complexes are not effective.     

DNA Cleavage Promoted by Cu^2＋ Complex of N,N＇-Bis（2-aminoethyl）-2,6-pyridinedicarboxamide

The interaction of Cu^2＋ complex of N,N＇-bis（2-aminoethyl）-2,6-pyridinedicarboxamide （BAP） with DNA was studied by agarose gel electrophoresis analysis. The results indicate that the BAP-Cu^2＋ complex can promote the cleavage of phosphodiester bond of supercoiled DNA at physiological condition, which is 3.2 × 10^6 times higher than DNA natural degradation. A hydrolytic cleaving mechanism through the cooperation of copper ions and functional amino groups was proposed.     

Ternary copper(II) complex of 1,10-phenanthroline and L-glycine: crystal structure and interaction with DNA

A ternary copper(II) complex, [Cu(phen)(L-Gly)(H₂O)] ·?NO₃ ·?1.5H₂O (phen =?1,10-phenanthroline, L-Gly =?L-glycine), has been synthesized and structurally characterized. The complex crystallized in a monoclinic system with space group C2/c, a =?20.572(3) Å, b =?6.9987(10) Å, c =?23.561(3) Å, β?= 98.776(5)°. The five-coordinate copper(II) center is a distorted square pyramid. Absorption spectra, fluorescence spectra and viscosity measurements showed interaction between the copper complex and DNA through an intercalative mode. The complex exhibited efficient DNA cleavage activity at micromolar concentration in the presence of ascorbate with hydroxyl radicals as the active species.     

水溶性不对称卟啉及其金属配合物合成及与DNA的 作用

合成并表征了三(4-N-甲基吡啶基)卟啉为母体,带有乙氧羰基甲氧基或羧基甲氧基侧链的水溶正电性卟啉及其金属配合物(M=Cu,Ni),并研究了它们与DNA的相互作用。卟啉对EB－DNA荧光猝灭研究表明：卟啉与DNA作用存在单一结合模式。EB荧光竞争法测得它们与DNA作用的结合常数介于3×10^5～3×10^6L·mol^-1。电子吸收研究谱表明：DNA引起卟啉Soret带的不同程度的红移(≤9nm)和减色(≤35%)。卟啉及其金属配合物都引起DNA的熔点升高(≤2.5℃)和DNA粘度的略微下降。所有这些研究表明：这些带较长侧链的水溶性卟啉及其金属配合物与DNA作用不存在插入作用模式,只是在DNA的外部结合。     

Synthesis,structure, DNA binding studies and nuclease activities of two luminescent neodymium complexes

Two neodymium(III) complexes, [Nd(Phen)(NO₃)₃(DMF)₂] (1) and [Nd(Phen)₂(NO₃)₃] (2) (phen = 1,10-phenanthroline; DMF = dimethylformamide), have been synthesized with a view to design artificial luminescent nucleases and nuclease mimics. The complexes were characterized by spectroscopic, powder, and single crystal XRD studies. The complexes, as expected, have luminescent properties. The DNA binding studies of both complexes have been carried out by spectroscopic studies e.g. electronic absorption (UV–Vis), fluorescence emission as well as viscosity measurements. The nuclease activity of the complexes has been established by gel electrophoresis using pUC19 circular plasmid DNA. The results of DNA binding as well as DNA cleavage activity and the model studies of interaction with pNPP indicate that both neodymium complexes demonstrate nuclease activity through phosphoester bond cleavage.     

Multispectroscopic DNA interaction and Docking studies

Two new water soluble oxovanadium(IV) complexes with formulae Na[VO(his)(met)SO₄] (1) and Na[VO(gly)(met)SO₄] (2), (gly=glycine his=histidine, and met=metformin) were synthesized and characterized by LCMS, UV‐Visible absorption, infrared spectra, magnetic moment, elemental analysis, thermal analysis and electronic spectral studies. The metal center was found in an octahedral geometry. DNA binding interaction of these complexes with CT DNA has been explored by UV‐Visible absorption, fluorescence, viscosity measurements and cleavage studies. Finally the binding of the complexes with CT‐DNA could be surface binding, mainly in the groove binding. The complexes were docked in to B‐DNA sequence, 5’(D*AP*CP*CP*GP*AP*CP*GP*TP*CP*GP*GP*T)‐3’ retrieved from protein data bank (PDB ID: 423D), using Discovery Studio 2.1 software.     

Electrochemical studies of the interaction of Basic Brown G with DNA and determination of DNA

A new method of electrochemical probe has been proposed for the determination of Herring Sperm DNA (DNA) based on its interaction with Basic Brown G (BBG). The electrochemical behavior of interaction of BBG with DNA was investigated on Hg electrode. In 0.1 mol L⁻¹ NH₃-NH₄Cl buffer solution (pH 8.0), BBG can be reduced on Hg electrode with a well-defined voltammetric peak at −0.67 V (versus SCE). In the presence of DNA, the reduction peak current of BBG decreases and the peak potential shifts to a more positive potential without the appearance of new peak. The study shows that a new BBG-DNA complex is formed by linear sweep voltammetry (LSV) and spectrophotometry. The decrease of the second order derivative of reductive peak current (Δi^″p) of BBG is proportional to the concentration of DNA in the range of 0.10-36 μg mL⁻¹. Limit of detection of DNA is 0.04 μg mL⁻¹. DNA of Hepatitis B Virus in serum samples was determined satisfactorily. Additionally, the binding mechanism was preliminarily discussed. The mode of interaction between BBG and DNA was found to be intercalation binding.     

Voltammetric studies of the interaction of quinacrine with DNA

The binding interaction of the antimalarial drug quinacrine with herring sperm deoxyribonucleic acid (DNA) has been studied by square wave voltammetry. The binding parameters, the binding constant K and the binding site size s, were obtained simultaneously by the analysis of bound and free quinacrine concentration corresponding to the limits of slow and fast binding kinetics compared to the experimental timescale. The binding constant and the binding site size for the interaction of quinacrine with DNA were K=1.59 (±0.18)×10⁵ M^–1 and s=7.1 (±0.15) base pairs and K=7.35 (±0.83)×10⁵ M^–1, s=6.2 (±0.02) base pairs for the limiting conditions of static and mobile binding equilibrium respectively. The standard Gibbs free energy change (G⁰=–RT ln K) is approximately –29.67 kJ/mol at 25 °C, which highlights the spontaneity of the binding of quinacrine with DNA. The binding of quinacrine to herring sperm DNA results in peak potential shifts in voltammetric and a red shift in UV-absorption measurements. The ionic strength dependence of the binding constant is not large. Furthermore, the relative viscosity of DNA increases in the presence of quinacrine. These characteristics strongly support the intercalation of quinacrine into DNA. The results also show that the intercalation of quinacrine into DNA may occur at approximately every seventh base pair.     

Investigation of DNA cleavage activities of new oxime-type ligand complexes and molecular modeling of complex-DNA interactions

Nucleolytic activities of some new oxime-type ligand complexes were investigated by neutral agarose gel electrophoresis. Analysis of the cleavage products in agarose gel indicated that all complexes used converted supercoiled pUC18 plasmid DNA to its nicked or linear form. It was found that nucleolytic activities of the complexes depend on the complex concentration, reaction time and the presence of a cooxidant (magnesium monoperoxyphthalate, MMPP) in the reaction mixture. However, the complexes cleaved pUC18 plasmid DNA at all investigated pH values. Nucleolytic activities of complexes were investigated for different complex concentrations (0.1–100 μmol L⁻¹), pH values (6.0–10.0) and reaction times (0–60 min). Molecular modeling studies performed by the Hyperchem Software together with DNA-binding studies showed that planar sites of the complexes intercalated into double stranded DNA. It can be concluded that all oxime-type ligand complexes used can be evaluated as nuclease mimics.     

Copper(II) complexes of acylhydrazones: synthesis,characterization and DNA interaction

Two new acylhydrazone copper(II) complexes of 4‐hydroxy‐N′‐[(1E)‐1‐(4‐methylphenyl)ethylidene]benzohydrazide (HL¹) and 4 ethyl [4‐({(2E)‐2‐[1‐(4‐methylphenyl)ethylidene]hydrazinyl}carbonyl)phenoxy]acetate (HL²) have been synthesized and characterized. The structures of both acylhydrazone and copper(II) complexes were identified by elemental analysis, infrared spectra, UV–visible electronic absorption spectra, magnetic susceptibility measurements, TGA and powder X‐ray diffraction. DNA binding and DNA cleavage activities of the synthesized copper complexes were examined by using UV‐visible titration and agarose gel electrophoresis, respectively. The effect of complex concentration on the DNA cleavage reactions in the absence and presence of H₂O₂ was also investigated. The results indicate that all the complexes bind slightly to calf thymus DNA and cleavage pBR322 DNA. The mechanistic studies demonstrate that a hydrogen peroxide‐derived species and singlet oxygen (¹O₂) are the active oxidative species for DNA cleavage. Copyright © 2013 John Wiley & Sons, Ltd.     

Spectroscopic Study of DNA Hydrolysis,DNA Intercalative,and Electrostatic Interaction Activity Exerted by Drug Based Coordination Compounds

Our work emphasized on synthesizing and characterizing neutral mononuclear copper(II) complexes with second generation fluoroquinolone drug ciprofloxacin (CFL) and some bipyridine derivatives (Aⁿ) of type [Cu(CFL)(Aⁿ)Cl] · 2H₂O. The DNA binding free energies were evaluated by studying the effect of salt concentrations on DNA binding. DNA interactions were investigated by using DNA melting temperature studies, viscosity measurements, absorption titration, and gel electrophoresis experiments. Also superoxide dismutase (SOD)‐like activity (IC₅₀ values) and antibacterial activity of metal complexes were studied. To validate the proper mechanistic pathway for plasmid DNA cleavage, gel electrophoresis experiments were carried out in presence of radical scavenging agents. The bactericidal activity of metal complexes was evaluated in terms of colony forming unit.     

Preparation of silybin 23-esters and evaluation of their inhibitory ability against LPO and DNA protective properties

Twelve 23-esterified silybin derivatives with different patterns of substituents such as aromatic and aliphatic groups(1-12) were designed and synthesized.The antioxidative properties of these compounds were evaluated.The modified silybin analogues exhibited improved inhibitory effects against rat liver homogenate lipid peroxidation compared to silybin,with exception of the trimethoxylated ester(5) and the aliphatic one(9).Compounds 3,5,7,8 and 11 displayed their protective properties on DNA cleavage in ...     

Interaction of zinc and cobalt with dipeptides and their DNA binding studies

Interactions of zinc and cobalt with peptides cysteinylglycine and histidylglycine have been studied. The binding modes were identified and geometry assigned. Stabilities of these complexes and their ability to bind DNA have been investigated. It is demonstrated that only zinc complexes bind DNA as compared to cobalt complexes