菲咯啉铜配合物与核酸结合特性的研究

用电化学方法研究了菲咯啉铜配合物分别与ＣｔＤＮＡ、热变性ＣｔＤＮＡ、ｐｏｌｙ（ＡＵ）作用的循环伏安行为,得到了（ｐｈｅｎ）２Ｃｕ＋与它们的表观结合常数Ｋ及结合位点数ｎ,结果表明Ｂ型ＤＮＡ是（ｐｈｅｎ）２Ｃｕ＋的最适结合底物     

维生素C·Cu·菲咯啉系统对DNA的定位损伤

维生素Ｃ·Ｃｕ·菲咯啉系统对ＤＮＡ的定位损伤柯德森,王爱国,罗广华（中国科学院华南植物研究所,广州５１０６５０）关键词活性氧;ＤＮＡ;定位损伤活性氧对ＤＮＡ的损伤已经被很多工作所证实”””,这种伤害是由０Ｂ”直接作用于ＤＮＡ所引起的”’“。但是ＯＨ’...     

吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响分析

【背景】金黄色葡萄球菌是目前食品和临床引起感染的重要病原菌,迫切需要开发新型抗菌药物。【目的】分析吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的转录组影响和主要代谢信号通路。【方法】采用液体稀释法测定P-FAH-Cu-phen作用金黄色葡萄球菌的最低抑菌浓度(minimum inhibitory concentration, MIC)和最低杀菌浓度(minimum bactericidal concentration, MBC)。将终浓度2 μg/mL的配合物分别作用于对数生长期的金黄色葡萄球菌30 min和2 h,进行转录组测序及分析。【结果】 P-FAH-Cu-phen作用金黄色葡萄球菌的MIC和MBC分别为2 μg/mL和4 μg/mL。与空白对照相比,配合物处理细菌30 min后,其差异基因共有356个,其中上调表达180个、下调表达176个;配合物处理细菌2 h后,其差异基因共有23个,其中上调表达3个、下调表达20个。差异基因功能主要富集于膜的组成部分、细胞质、质膜、ATP结合、发病机制、金属离子结合、组氨酸生物合成过程、DNA结合、水解酶活性、跨膜转运蛋白活性、硝酸盐同化、硝酸盐代谢过程、硝酸还原酶复合物、硝酸还原酶活性等。差异基因涉及的信号通路主要有双组分系统、群体感应、氮代谢、三羧酸循环、氨基酸代谢等。【结论】影响细菌质膜组成、毒素生成、生物膜形成、细胞壁合成、能量代谢等可能是吡唑啉酮铜配合物P-FAH-Cu-phen对金黄色葡萄球菌的主要抑菌作用。研究为揭示吡唑啉酮铜配合物抑制金黄色葡萄球菌分子机制提供了理论依据。     

铜离子-半胱氨酸配合物的合成及表征

研究了铜离子-半胱氨酸配合物的合成方法,探讨了合成工艺的主要影响因素,确定了原料配比,pH值,最佳反应时间及温度,采用X射线衍射光谱对配合物进行表征,结果表明铜离子能与半胱氨酸形成配合物。     

若干铜配合物歧化超氧离子的活性

合成了若干含氮的铜配合物,用核黄素-蛋氨酸-光照法测定其歧化超氧离子的活性,与超氧化物歧化酶作了对照,发现双核的铜配合物比单核的铜配合物有较高的活性,铜的双核咪唑桥配合物的活性高于结构相近的单核配合物。     

抗心律失常新药常咯啉对心肌电活动的作用

本文报告了抗心律失常新药常咯啉对心肌电活动的作用。适当浓度的常咯啉使心肌细胞的动作电位0相的振幅和最大去极化速率减小,2相的复极化速率增大,持续期间缩短,而有效不应期在绝大多数情况下反有延长。心肌活动频率较高时,药物效应更为显著,发展更为迅速。常咯啉能减少无明显外因作用时出现的和由哇巴因引起的心肌自发活动,能对抗乌头碱引起的自发活动。从以上结果来看,常咯啉的作用大概是使控制钠沟道启闭的 m 和 h 两参数的变化时程减慢,使控制动作电位2相的离子外流沟道的 X_1参数加大。文中还讨论了常咯啉对抗心肌自发活动的机理,常咯啉作为抗心律失常药物的分类何题,以及常咯啉对心肌电活动的效应与抗心律失常作用之间的关系问题。     

常咯啉与其他抗心律失常药合并应用对电刺激家兔左心室致颤阈的影响

本实验研究常咯啉与其他抗心律失常药合并应用对电刺激家兔左心室致颤阙的影响。单用常咯啉在剂量范围1—8毫克/公斤内可提高家兔左心室电致颤阈,且剂量与阈值提高呈线性关系。实验又观察了5种抗心律失常药:利多卡因(10毫克/公斤),普鲁卡因胺(40毫克/公斤)、苯妥英钠(10毫克/公斤)、奎尼丁(10毫克/公斤)和心得宁(0.05,0.1,0.2毫克/公斤),其中除心得宁在所试剂量时对致颤阈无影响外,其他4种抗心律失常药皆能显著提高致颤阈。将常略啉的高、低两个剂量分别与一个抗心律失常药的固定剂量合并以观察阈值提高,以及由用合并药后的常咯啉剂量反应回归线与单用常咯啉的剂量反应回归线比较来估价合并效应。实验分析常咯啉2.0毫克/公斤与利多卡因5毫克/公斤合并对致颤阈的提高有相加作用;常咯啉1.3毫克/公斤与普鲁卡因胺20毫克/公斤合并也有相加作用。常咯啉20毫克/公斤与普鲁卡因胺20毫克/公斤合并有增强作用。     

常咯啉与奎尼丁治疗实验性心律失常的比较

本文在三种室性和一种房性心律失常的动物模型中,比较了常咯啉与奎尼丁的防治作用。又在大鼠坐骨神经上,比较了二药的局麻强度。静脉注射奎尼丁2.4—9.6mg/kg 对大鼠静脉注射乌头碱20μg/kg 引起室性心律失常的时间没有推迟作用,而常咯啉5mg/kg 能明显推迟。在大鼠静脉注射 BaCl_2 4mg/kg 引起的室性心律失常中,静注常咯啉10mg/kg 维持正常窦性律的时间是1.8±1.6分,而静注奎尼丁4.8mg/kg 维持窦性律时间是0.2±0.6分。常咯啉抗BaCl_2心律失常的效果明显优于奎尼丁(P<0.01)。静注奎尼丁4.8mg/kg 不能提高豚鼠哇巴因中毒引起的室速和室颤阈值,仅提高室早阈值,而常略啉10mg/kg 能提高所有三个室性律阈值。在 Ach 诱发狗的房颤实验中,静注奎尼丁和常咯啉各6mg/kg 都能对抗 Ach 房颤,而且常咯啉比奎尼丁作用更强。在等毒性剂量比较下,奎尼丁是一个强的局麻药,而常咯啉则否。     

Cu~(2+)-ASP配合物的合成及分析

研究了金属铜离子与天冬氨酸 (ASP)配合物的合成方法[1,2 ] ,采用X射线光谱对配合物进行了分析鉴定 ,结果表明铜离子与天冬氨酸能形成配合物。     

N—水杨醛氨基葡萄糖希夫碱配合物对O2和DNA的效应

采用比色法、荧光和吸收光谱法研究了Ｎ－水杨醛氨基葡萄糖希夫碱的铜Ⅱ）、锌（ｋⅡ）、钴９Ⅲ）配合物对超氧阴离子自由基和ＤＮＡ的作用。结果表明：ＣｕＳＧ和ＺｎＳＧ能显著地抑制超氧阴离子自由基的生成,抑制能力ＣｕＳＧ＞ＺｎＳＧ。     

Determination of the intracellular targets and in vitro activity of a new class of chemical nuclease complexes derived from antibiotics of the quinolone family

 Alteration of bacterial DNA structure and/or associated functions in vivo by [Cu(phen)(nal)]⁺, a metal complex of the type [Cu(phen)(antib)]⁺ (where antib is a quinolone or a fluoroquinolone), was demonstrated by the induction of a recA-lacZ fusion integrated at the amyE locus of a recombinant Bacillus subtilis strain. Using the same approach, nalidixic acid alone was shown to induce 14% of the β-galactosidase levels induced by [Cu(phen)(nal)]⁺; on the other hand none of the other components, i.e. copper, phenanthroline or the complex [Cu(phen)₂]²⁺ activated significantly the recA-directed β-galactosidase activity, suggesting that the intact structure of the complex is required to reach maximum levels of induction. Results of in vitro experiments demonstrated that under reductive conditions [Cu(phen)(nal)]⁺ behaves as a powerful nuclease capable of degrading plasmid DNA; this activity was stronger than that of the chemical nuclease copper phenanthroline [Cu(phen)₂]²⁺. The nuclease activity putatively occurred by a mechanism involving hydroxyl radicals since the reaction was partially inhibited by catalase. These results support the hypothesis that the mechanism of action of quinolones could be mediated by a transition metal ion such as copper. Received: 30 September 1997 / Accepted: 30 January 1998     

Probing the cell death signaling pathway of HepG2 cell line induced by copper-1,10-phenanthroline complex

Copper-1,10-phenanthroline (phen) complex [Cu(phen)₂] has been typically known as DNA-cleaving agent. And now it becomes more important for developing multifunctional drugs with its improved cytotoxic properties. In our study, we probed the cytophysiological mechanism of Cu(phen)₂. HepG2 cells were more sensitive to Cu(phen)₂ with an IC50 of 4.03 μM than other three kinds of cell lines. After treated by Cu(phen)₂, HepG2 cells had some typical morphological changes which happened to its nucleus. DNA ladder’s occurence and Annexin V-positive increased cells indicated that Cu(phen)₂ induced HepG2 cells into apoptosis. Further studies showed that Cu(phen)₂ treatment resulted in significant G₂/M phase arrest and collapse of mitochondrial membrane potential. Several cell cycle-related factors were down-regulated, including Cyclin A, Cyclin B1 and Cdc2. But p21 and p53 were up-regulated. DNA damage, microtubule disorganization and mitotic arrest through spindle assembly checkpoint activation were observed in Cu(phen)₂-treated cells. The activation of caspase-3, 8 & 9 were checked out. The increased-expression ratio of Bax/Bcl-2 was detected. The expression levels of Bcl-x_L and Bid were found to decrease. These meant that a mitochondrial-related apoptosis pathway was activated in treated HepG2 cells. Furthermore, some ER stress-associated signaling factors were found to be up-regulated, such as Grp78, XBP-1and CHOP. Ca²⁺ was also found to be released from the ER lumen. Collectively, our findings demonstrate that Cu(phen)₂ induces apoptosis in HepG2 cells via mitotic arrest and mitochondrial- and ER-stress-related signaling pathways.     

Synchronous fluorescence, UV-visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.     

DNA binding of iron(II) mixed-ligand complexes containing 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline

Absorption spectroscopy and circular dichroism (CD) have been used to characterize the DNA binding of [Fe(phen)3]2+, [Fe(phen)2(DIP)]2+ and [Fe(phen)(DIP)2]2+ where phen and DIP stand for 1,10-phenanthroline and 4,7-diphenyl-1,10-phenanthroline, respectively. Both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ bind weakly to calf thymus DNA (CT-DNA) in an electrostatic mode, while [Fe(phen)(DIP)2]2+ binds more strongly to CT-DNA, possibly in an intercalation mode. The hypochromicity, red shift and Kb increase in the order [Fe(phen)3]2+ < [Fe(phen)2(DIP)]2+ < [Fe(phen)(DIP)2]2+ in accordance with the increase in size and hydrophobicity of the iron(II) complexes. The thermodynamic parameters obtained suggest that the DNA binding of both [Fe(phen)3]2+ and [Fe(phen)2(DIP)]2+ is entropically driven, while that of [Fe(phen)(DIP)2]2+ is enthalpically driven. A strong CD spectrum in the UV and visible region develops upon addition of CT-DNA into the racemate solution of each iron(II) complex (Pfeiffer effect). This has revealed that a shift in diastereomeric inversion equilibrium takes place in the solution to yield an excess of one of the DNA-complex diastereomers. The striking resemblance of the CD spectral profiles to those of the pure delta-enantiomer indicates that the delta-enantiomer of the iron(II) complexes is preferentially bound to CT-DNA. The mechanism of the development of Pfeiffer CD is proposed on the basis of kinetic studies on the DNA binding of the racemic iron(II) complexes.     

[Cu(phen)2] acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)₂]²⁺, where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)₂]²⁺ acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)₂]²⁺ were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10⁻¹² to 1 × 10⁻⁶ M with a detection limit of 1.99 × 10⁻¹³ M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.     

Synthesis,crystal structure,and nuclease activity of planar mono-heterocyclic base copper(II) complexes

A series of mononuclear copper(II) complexes having a 1:1 molar ratio of copper and the planar heterocyclic base like 1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq) and dipyrido[3,2-a:2',3'-c]phenazine (dppz) are prepared from a reaction of copper(II) nitrate.trihydrate and the base (L) in ethanol or aqueous ethanol at different temperatures. The complexes [Cu(dpq)(NO(3))(2)] (2), [Cu(dpq)(NO(3))(H(2)O)(2)](NO(3)) (3), [Cu(dpq)(NO(3))(2)(H(2)O)(2)].2H(2)O (4.2H(2)O) and [Cu(dppz)(NO(3))(2)(H(2)O)].H(2)O (5.H(2)O) have been characterized by X-ray crystallography. The crystal structures show the presence of the heterocyclic base in the basal plane. The coordination geometries of the copper(II) centers are axially elongated square-pyramidal (4+1) in 2, 3 and 5, and octahedral (4+2) in 4. The nitrate anion in the coordination sphere displays unidentate and bidentate chelating bonding modes. The axial ligand is either H(2)O or NO(3) in these structures giving a Cu-L(ax) distance of approximately 2.4 A. The one-electron paramagnetic complexes (mu approximately 1.8 mu(B)) exhibit axial EPR spectra in DMF glass at 77 K giving g(parallel)>g( perpendicular ) with an A(parallel) value of approximately 170G indicating a [d(x)2(-y)2](1) ground state. The complexes are redox active and display a quasireversible cyclic voltammetric response for the Cu(II)/Cu(I) couple near 0.0 V vs. SCE giving an order of the E(1/2) values as 5(dppz)>2-4 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). The complexes bind to calf thymus DNA giving an order 5 (dppz)>2 (dpq)>[Cu(phen)(2)(H(2)O)](2+)>1 (phen). An effect of the extended planar ring in dpq and dppz is observed in the DNA binding. The complexes show nuclease activity with pUC19 supercoiled DNA in DMF/Tris-HCl buffer containing NaCl in presence of mercaptopropanoic acid as a reducing agent. The extent of cleavage follows the order: [Cu(phen)(2)(H(2)O)](ClO(4))(2)>5>2 approximately 3 approximately 4>1. The bis-phen complex is a better cleaver of SC DNA than 1-5 having mono-heterocyclic base. Mechanistic investigations using distamycin reveal minor groove biding for the phen, dpq complexes, and a major groove binding for the dppz complex 5. The cleavage reactions are found to be inhibited in the presence of hydroxyl radical scavenger DMSO and the reactions are proposed to proceed via sugar hydrogen abstraction pathway. The ancillary ligand is found to have less effect in DNA binding but are of importance in DNA cleavage reactions.     

Degradation of Single Stranded Nucleic Acids by the Chemical Nuclease Activity of the Metal Complex [Cu(phen)(nal)]+

The chemical design of metal complexes of the type [Cu(phen)(antib)]⁺ (where antib is a quinolone or a fluoroquinolone) has been carried out in an approach to better understand how the coordination of their components affect the activity of quinolones. The ability of [Cu(phen)(nal)]⁺ to interact with DNA in vivo and its capacity to promote the degradation of plasmid and chromosomal DNA, under reductive conditions has been previously reported. However whether this compound utilizes other intracellular targets to promote bacterial killing was a question that deserved to be answered. In this paper, the studies of the chemical nuclease properties encoded by the metal complex [Cu(phen)(nal)]⁺ were extended by using different types of single chain nucleic acids, i.e, ribosomal and tumor mosaic virus RNAs as well as poly-dA-dT. Our results showed that degradation of the nucleic acids occurred only under reductive conditions. Although MPA and [3-mercaptoethanol were the chemical reducers that best assisted the nuclease reaction, other biological compounds such as citric and succinic acid also were shown to act like reducers in that reaction. All.hough the nuclease activity of [Cu(phen)(nal)]⁺ was comparable to that exhibited by bis copper phenanthroline [Cu(phen)z]²⁺our results showed that none of the individual components of [Cu(phen)(nal)]⁺ was able to promote the degradation of either the RNAs or poly(dA-dT). These results strongly support the hypothesis that the metal complex [Cu(phen)(nal)] uses not only DNA but also RNA as targets to promote bacterial killing.     

Study on the Interaction Between {\mathrm{Cu}}{\left( {{\mathrm{phen}}} \right)}^{{2 + }}_{3} and Bovine Serum Albumin by Spectroscopic Methods

In this work, the interaction between ${\text{Cu}}\left( {{\text{phen}}} \right)_3^{\,\,2 + } In this work, the interaction between Cu(phen)(2+)(3) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy combined with UV-vis absorption and circular dichroism (CD) spectroscopic techniques under physiological conditions. The fluorescence data proved that the fluorescence quenching of BSA by Cu(phen)(2+)(3) was the result of the Cu(phen)(2+)(3) -BSA complex formation. The binding constants (K (a)) between Cu(phen)(2+)(3) and BSA at four different temperatures were calculated according to the modified Stern-Volmer equation. The enthalpy change (DeltaH) and entropy change (DeltaS) were calculated to be 10.74 kJ mol(-1) and 54.35 J mol(-1) K(-1), respectively, which indicated that electrostatic interactions played a major role in the formation of Cu(phen)(2+)(3) -BSA complex. The distance r between the donor (BSA) and acceptor[Cu(phen)(2+)(3)] was obtained to be 3.55 nm based on F?rster's energy transfer theory. The synchronous fluorescence and CD spectroscopy results showed that the polarity of the residues increased and the lost of the alpha-helix content of BSA (from 59.84 to 53.70%). These indicated that the microenvironment and conformation of BSA were changed in the presence of Cu(phen)(2+)(3).     

Mixed-ligand copper(II)-phenanthroline-dipeptide complexes: synthesis, characterization, and DNA-cleavage properties

The mixed-ligand complexes [Cu(II)(HisLeu)(phen)](+) (1) and [Cu(II)(HisSer)(phen)](+) (2; phen=1,10-phenanthroline) were synthesized and characterized. The intercalative interaction of the Cu(II) complexes with calf-thymus DNA (CT-DNA) was probed by UV/VIS and fluorescence titration, as well as by thermal-denaturation experiments, and the intrinsic binding constants (K(b)) for the complexes with 1 and 2 were 4.2x10(3) and 4.9x10(3) M(-1), resp. Both complexes were found to be efficient catalysts for the hydrolytic cleavage of plasmid pUC19 DNA, as tested by gel electrophoresis, converting the DNA from the supercoiled to the nicked-circular form at rate constants of 1.32 and 1.40 h(-1) for 1 and 2, resp.     

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.