Triphenylamine‐Appended Half‐Sandwich Iridium(III) Complexes and Their Biological Applications

Organometallic half‐sandwich Ir^III complexes of the type [(η⁵‐Cp^x)Ir(N^N)Cl]PF₆ (Cp^x: Cp* or its phenyl Cp^xph or biphenyl Cp^xbiph derivatives; N^N: triphenylamine (TPA)‐substituted bipyridyl ligand groups) were synthesized and characterized. The complexes showed excellent bovine serum albumin (BSA) and DNA binding properties and were able to oxidize NADH to NAD⁺ (NAD=nicotinamide adenine dinucleotide) efficiently. The complexes induced apoptosis effectively and led to the emergence of reactive oxygen species (ROS) in cells. All complexes showed potent cytotoxicity with IC₅₀ values ranging from 1.5 to 7.1 μm toward A549 human lung cancer cells after 24 hours of drug exposure, which is up to 14 times more potent than cisplatin under the same conditions.     

Imine‐N‐Heterocyclic Carbenes as Versatile Ligands in Ruthenium(II) p‐Cymene Anticancer Complexes: A Structure–Activity Relationship Study

A family of novel imine‐N‐heterocyclic carbene ruthenium(II) complexes of the general formula [(η⁶‐p‐cymene)Ru(C^N)Cl]PF₆⁻ (where C^N is an imine‐N‐heterocyclic carbene chelating ligand with varying substituents) have been prepared and characterized. In this imine‐N‐heterocyclic carbene chelating ligand framework, there are three potential sites that can be modified, which distinguishes this class of ligand and provides a body of flexibilities and opportunities to tune the cytotoxicity of these ruthenium(II) complexes. The influence of substituent effects of three tunable domains on the anticancer activity and catalytic ability in converting coenzyme NADH to NAD⁺ is investigated. This family of complexes displays an exceedingly distinct anticancer activity against A549 cancer cells, despite their close structural similarity. Complex 9 shows the highest anticancer activity in this series against A549 cancer cells (IC₅₀=14.36 μm ), with an approximately 1.5‐fold better activity than the clinical platinum drug cisplatin (IC₅₀=21.30 μm ) in A549 cancer cells. Mechanistic studies reveal that complex 9 mediates cell death mainly through cell stress, including cell cycle arrest, inducing apoptosis, increasing intracellular reactive oxygen species (ROS) levels, and depolarization of the mitochondrial membrane potential (MMP). Furthermore, lysosomal damage is also detected by confocal microscopy.     

Half‐sandwich Ruthenium (II) complexes with triphenylamine modified dipyridine skeleton and application in biology/luminescence imaging

Four half‐sandwich ruthenium^II (Ru^II) complexes with triphenylamine‐modifed dipyridine frameworks were synthesized and characterized. The cytotoxicity of target complexes toward A549 (lung cancer cells), HeLa (cervical cancer cells) and HepG2 (hepatoma cells) were obtained by the MTT assay, which were superior to cisplatin with the IC₅₀ values changed from 2.4 ± 0.1 μM to 9.2 ± 2.7 μM. Meanwhile, complexes possess the ability of antimetastasis to cancer cells. Ru^II complexes could be transported by serum albumin, catalyze the conversion of NADH (the reduced state of nicotinamide‐adenine dinucleotide) to NAD⁺ and induce the accumulation of reactive oxygen species, which confirmed the antineoplastic mechanism of oxidation. Ru^II complexes could enter A549 cells followed by a non‐energy dependent cellular uptake mechanism, target lysosomes with the Pearson's colocalization coefficient of 0.75, lead to lysosomal damage, disturb the cell cycle (S phase), and eventually induce apoptosis. The results demonstrate that these Ru^II complexes are potential anticancer agents with dual functions, including metastasis inhibition and lysosomal damage.     

Lysosome‐targeted potent half‐sandwich iridium(III) α‐diimine antitumor complexes

Fifteen organometallic Ir(III) half‐sandwich complexes ( 1A – 5C ) having the general formula [(η⁵‐Cp^x)Ir(N^N)Cl]PF₆ (Cp^x = Cp*, tetramethyl(phenyl)cyclopentadienyl (Cp^xph) or tetramethyl(biphenyl)cyclopentadienyl (Cp^xbiph); N^N = diamine) have been synthesized and characterized. The molecular structure of 1A was determined using single‐crystal X‐ray diffraction analysis. The hydrolysis of 1A – 5C was monitored using UV–visible spectra. Complexes 3A – 3C showed catalytic activity for the oxidation of NADH to NAD⁺, where 3C showed the highest turnover number of 29.9 within 450 min. Cytotoxicity examination by MTT assay was carried out against two human cancer cell lines (HeLa and A549) after 24 or 48 h drug treatment. The complexes showed high potency, where the most potent complex ( 3C ; IC₅₀ = 3.4 μM) was six times more active than cisplatin against A549 cells after 24 h drug exposure. Cytotoxic potency towards A549 cells increased with phenyl substitution on Cp ring: Cp^xbiph > Cp^xph > Cp*. In addition, the biological studies showed that 3C caused cell apoptosis and cell cycle arrest at G₁ phase in A549 cancer cells. Moreover, 3C increased the level of reactive oxygen species markedly after 24 h, which may provide an important basis for killing cancer cells. Confocal laser scanning microscopy was used to track 3C in A549 cells. The cellular localization experiment showed that 3C targeted lysosomes and caused lysosomal damage.     

Synthesis,crystal structure,DNA/bovine serum albumin binding and antitumor activity of two transition metal complexes with 4‐acylpyrazolone derivative

Two new complexes, namely [Cu₆L₆] ( 1 ) and [Zn(HL)₂] ( 2 ) (H₂L = N‐(1‐phenyl‐3‐methyl‐4‐propenylidene‐5‐pyrazolone)‐2‐furancarboxylic acid hydrazide), have been synthesized and characterized. Single crystal X‐ray analysis indicates that complex 1 has a hexanuclear structure and complex 2 exhibits a mononuclear structure. The DNA/bovine serum albumin (BSA) binding properties of complexes 1 and 2 were investigated by absorption spectroscopy and fluorescence quenching. Both complexes could effectively intercalate to DNA with calculated quenching constants of 2.6 × 10⁵ and 1.25 × 10⁵ M^?1, respectively. The quenching mechanism of the intrinsic fluorescence of BSA by the complexes was found to be a static one. The cytotoxicities of 1 and 2 were investigated in two human tumor cell lines, human esophageal cancer cells (Eca‐109) and cervical cancer cells (HeLa). Complex 1 exhibits higher antitumor activity than 2 . Furthermore, 1 can inhibit HeLa cells by inducing apoptosis and G0/G1 phase cell cycle arrest. All results demonstrate that 1 and 2 both have DNA/BSA binding capacity and antitumor activity.     

Metal(II) complexes of bioactive aminonaphthoquinone-based ligand: synthesis,characterization and BSA binding,DNA binding/cleavage,and cytotoxicity studies

An aminonaphthoquinone ligand, L, and its metal complexes of general formula [MLCl₂] {M = Co(II), Ni(II), Cu(II) and Zn(II)} have been synthesized and characterized by analytical and spectral techniques. Tetrahedral geometry has been assigned to Ni(II) and Zn(II) complexes and square planar geometry to Co(II) and Cu(II) complexes on the basis of electronic spectral and magnetic susceptibility data. The binding of complexes with bovine serum albumin (BSA) is relatively stronger than that of free ligand and alters the conformation of the protein molecule. Interaction of these complexes with CT-DNA has been investigated using UV-Vis and fluorescence quenching experiments, which show that the complexes bind strongly to DNA through intercalative mode of binding (K_app 10⁵ M^?1). Molecular docking studies reiterate the mode of binding of these compounds with DNA, proposed by spectral studies. The ligand and its complexes cleave plasmid DNA pUC18 to nicked (Form II) and linear (Form III) forms in the presence of H₂O₂ oxidant. The in vitro cytotoxicity screening shows that Cu(II) complex is more potent against MCF-7 cells and Zn(II) complex exhibits marked cytotoxicity against A-549 cells equal to that of cisplatin. Cell imaging studies suggested apoptosis mode of cell death in these two chosen cell lines.     

Recycling of NAD+ using coimmobilized alcohol dehydrogenase andE. coli

The use of immobilized enzymes has opened the possibility of large scale utilization of NAD⁺-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD⁺ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD⁺ from NADH. This NAD⁺ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.     

苯并咪唑衍生的单核钴(Ⅱ)和单核镍(Ⅱ)配合物与DNA和蛋白质的结合反应性及细胞毒活性研究

利用紫外吸收光谱、荧光光谱、圆二色光谱(CD)等各种光谱手段对比地研究了由苯并咪唑衍生的单核钴配合物[Co(EDTB)]2+(1)和单核镍配合物[Ni(EDTB)]2+(2)(这里EDTB为N,N,N′,N′-四(2′-苯并咪唑甲基)-1,2-乙二胺)与小牛胸腺DNA(CTDNA)和牛血清白蛋白(BSA)的相互作用。结果表明,在生理条件下,配合物1和2均能通过插入方式较强的与CT-DNA结合,诱导DNA构象的改变;且配合物1对DNA的结合能力略强于2,其结合常数分别为Kb(1)=3.23×104L·mol-1和Kb(2)=2.40×104L·mol-1。配合物与BSA相互作用的研究表明,1和2均能与BSA发生较强的相互作用,结合常数均处在104~105 L·mol-1;该结合引起了BSA微环境和构象发生变化,且使BSA内源荧光被淬灭,淬灭机理为静态淬灭。利用MTT法研究了配合物1和2对小鼠白血病细胞株P388和人非小细胞肺癌细胞株A-549的体外细胞毒活性,实验结果表明,配合物1和2对P388不敏感,对A-549在高浓度(10-4~10-5 mol·L-1)下表现出与顺铂相当的细胞毒活性。     

稀土-全反式维甲酸-精氨酸三元配合物的合成、表征及抗肿瘤活性研究

合成了4种新型稀土三元固体配合物:REL2L′Cl·nH2O(RE:Nd3+、Eu3+、La3+、Sc3+;L=全反式维甲酸;L′=L-精氨酸阳离子),并对其进行红外光谱、紫外光谱、荧光光谱、元素分析和TG-DTA等测试。利用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)测试方法,初步探讨了配合物对体外培养的人肝癌细胞HepG2、人肺癌细胞A549和人宫颈癌细胞Hela生长的影响。结果表明:4种稀土配合物和稀土硝酸盐、配体全反式维甲酸和L-精氨酸盐酸盐对3种癌细胞株的生长都有一定的抑制作用,但在一定的浓度范围内,三元固体配合物的抑制效果明显优于稀土硝酸盐和2种配体;稀土配合物对3种癌细胞株生长的抑制作用基本上随配合物浓度的增大而增强;在浓度为1 mmol·L-1时,4种配合物对3种肿瘤细胞的抑制率超过60%,ScL2L′Cl配合物表现出最强的作用,抑制率在80%以上。通过荧光,粘度和紫外光谱方法研究了配合物与药物靶分子DNA的相互作用,结果表明配合物与DNA以嵌入的方式相互作用,推测配合物抗肿瘤活性的起效与这种嵌入DNA双螺旋结构的作用方式有关     

Attempting to remove the substrate inhibition ofL-lactate dehydrogenase fromBacillus stearothermophilus by site-directed mutagenesis

L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD⁺ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD⁺-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD⁺. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD⁺-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.     

苯并咪唑衍生的单核钴（Ⅱ）和单核镍（Ⅱ）配合物与DNA和蛋白质的结合反应性及细胞毒活性研究

利用紫外吸收光谱、荧光光谱、圆二色光谱（CD）等各种光谱手段对比地研究了由苯并咪唑衍生的单核钴配合物[Co（EDTB）]²⁺（1）和单核镍配合物[Ni（EDTB）]²⁺（2）（这里EDTB为N,N,N’,N’-四（2-苯并咪唑甲基）-1,2-乙二胺）与小牛胸腺DNA（CT-DNA）和牛血清白蛋白（BSA）的相互作用。结果表明,在生理条件下,配合物1和2均能通过插入方式较强的与CT-DNA结合,诱导DNA构象的改变;且配合物1对DNA的结合能力略强于2,其结合常数分别为K_b（1）=3.23×10⁴L·mol^-1和K_b（2）=2.40×10⁴L·mol^-1。配合物与BSA相互作用的研究表明,1和2均能与BSA发生较强的相互作用,结合常数均处在10⁴~10⁵L·mol^-1;该结合引起了BSA微环境和构象发生变化,且使BSA内源荧光被淬灭,淬灭机理为静态淬灭。利用MTT法研究了配合物1和2对小鼠白血病细胞株P388和人非小细胞肺癌细胞株A-549的体外细胞毒活性,实验结果表明,配合物1和2对P388不敏感,对A-549在高浓度（10^-4~10^-5mol·L^-1）下表现出与顺铂相当的细胞毒活性。     

Fluorescent sensing layer for the determination of<Emphasis Type="SmallCaps"> L</Emphasis>-malic acid in wine

An enzymatic method for determining L-malic acid in wine based on an L-malate sensing layer with nicotinamide adenine dinucleotide (NAD⁺), L-malate dehydrogenase (L-MDH) and diaphorase (DI), immobilized by sol-gel technology, was constructed and evaluated. The sol-gel glass was prepared with tetramethoxysilane (TMOS), water and HCl. L-MDH catalyzes the reaction between L-malate and NAD⁺, producing NADH, whose fluorescence (λ _exc = 340 nm, λ _em = 430 nm) could be directly related to the amount of L-malate. NADH is converted to NAD⁺ by applying hexacyanoferrate(III) as oxidant in the presence of DI. Some parameters affecting sol-gel encapsulation and the pH of the enzymatic reaction were studied. The sensing layer has a dynamic range of 0.1–1.0 g/L of L-malate and a long-term storage stability of 25 days. It exhibits acceptable reproducibility [s _r(%)≈10] and allows six regenerations. The content of L-malic acid was determined for different types of wine, and polyvinylpolypyrrolidone (PVPP) was used as a bleaching agent with red wine. The results obtained for the wine samples using the sensing layer are comparable to those obtained from a reference method based on UV-vis molecular absorption spectrometry, if the matrix effect is corrected for.     

The Potent Oxidant Anticancer Activity of Organoiridium Catalysts

Platinum complexes are the most widely used anticancer drugs; however, new generations of agents are needed. The organoiridium(III) complex [(η⁵‐Cp^xbiph)Ir(phpy)(Cl)] ( 1‐Cl ), which contains π‐bonded biphenyltetramethylcyclopentadienyl (Cp^xbiph) and C^N‐chelated phenylpyridine (phpy) ligands, undergoes rapid hydrolysis of the chlorido ligand. In contrast, the pyridine complex [(η⁵‐Cp^xbiph)Ir(phpy)(py)]⁺ ( 1‐py ) aquates slowly, and is more potent (in nanomolar amounts) than both 1‐Cl and cisplatin towards a wide range of cancer cells. The pyridine ligand protects 1‐py from rapid reaction with intracellular glutathione. The high potency of 1‐py correlates with its ability to increase substantially the level of reactive oxygen species (ROS) in cancer cells. The unprecedented ability of these iridium complexes to generate H₂O₂ by catalytic hydride transfer from the coenzyme NADH to oxygen is demonstrated. Such organoiridium complexes are promising as a new generation of anticancer drugs for effective oxidant therapy.     

3-芳基噻唑烷-4-酮-2-酰胺的合成及其抗肿瘤活性研究

合成了系列新的3-芳基噻唑烷-4-酮-2-酰胺衍生物,并测试了化合物抑制肿瘤细胞增殖活性.部分化合物对A-549和Hela肿瘤细胞有弱的细胞毒性,而对BGC-823没有抑制作用,表现出一定的选择性.其中,化合物7ad对A-549有较强的抑制活性(IC50=21.0μmol·L-1),与阳性对照顺铂的抑制活性(IC50=19.4μmol·L-1)相当.初步的构效关系表明化合物的立体结构可能对其抗肿瘤活性影响较大.     

Improved scope and diastereoselectivity of C–H activation in an expanded supramolecular host

Chiral Ga₄L₆ assembly Ga₄(L^N)₆ encapsulates cationic iridium half-sandwich complexes that activate aldehyde C–H bonds to form chiral, strongly bound piano-stool complexes. Herein, we report the expanded scope of the larger Ga₄(L^P)₆ host in mediating this transformation. The larger assembly significantly improves both the scope and the diastereoselectivity of this organometallic transformation generally, while substrate-specific interactions demonstrate that host size is an important, but not definitive, factor in determining diastereoselectivity.     

Equilibrium (NAD+/NADH) potential on poly(Neutral Red) modified electrode

The electrochemical regeneration of nicotinamide adenine dinucleotide (NAD⁺/NADH) has been one of the central subjects of bioelectrochemistry during past three decades. We report on the unique chemical electrocatalyst for NAD⁺/NADH regeneration based on electropolymerized Neutral Red. Using poly(Neutral Red) modified electrodes, the reversible polarographic waves of nicotinamide adenine dinucleotide reduction–oxidation and the equilibrium (NAD⁺/NADH) potential were observed. This was impossible using all known catalytic and mediator systems. The unique poly(Neutral Red) based electrocatalyst allowed us to determine the standard (NAD⁺/NADH) potential more precisely (E^′≅0.59 V SCE, pH 6.0).     

Cobalt(II) complexes with thiosemicarbazone as potential antitumor agents: synthesis,crystal structures,DNA interactions,and cytotoxicity

Two cobalt(II) complexes [Co(QCT)₂]·Cl·1.5H₂O (1) (QCT = quinoline-2-carboxaldehyde thiosemicarbazone) and [Co(QCMT)(CH₃OH)Cl₂] (2) (QCMT = quinoline-2-carboxaldehyde N⁴-methyl-thiosemicarbazone) have been synthesized and structurally characterized. Complex 1 crystallizes in a triclinic system with space group P–1 and complex 2 crystallizes in a monoclinic system with space group P2(1)/n. In both complexes the cobalt(II) center is six coordinated with distorted octahedral geometry. The interactions of two complexes with CT-DNA were investigated by electronic absorption spectra, circular dichroism (CD) spectra and fluorescence spectra. Results suggest that the complexes bind to DNA via groove binding mode, and complex 2 has stronger binding ability than complex 1. The in vitro cytotoxicity has been tested against the human lung adenocarcinoma cell line A-549, cisplatin-resistant cell line A-549/CDDP, and human breast adenocarcinoma cell line MCF-7. Complex 2 is more cytotoxic than complex 1, and both of them show higher cytotoxicity than the parent ligands alone. Compared with cisplatin, the two cobalt(II) complexes are more active against A-549/CDDP and MCF-7 cell lines at most experimental concentrations. Notably, although complex 2 is found to be less effective than cisplatin against the parent cell line A-549, it is much more effective than cisplatin against the resistant cell A-549/CDDP.     

Electrocatalytic oxidation of the reduced nicotinamide adenine dinucleotide at carbon ionic liquid electrode modified with polythionine/multi-walled carbon nanotubes composite

A carbon ionic liquid electrode (CILE) was modified with a polythionine (PTh)/multi-walled carbon nanotubes (MWCNTs) composite and used for the detection of reduced nicotinamide adenine dinucleotide (NADH). The electrode was prepared by electrochemical polymerization of thionine on the MWCNTs in neutral medium. Cyclic voltammetry indicated that the electrode was capable of mediating the oxidation of NADH at an overpotential as low as 0.03 V. Amperometric experiments showed that a sensitive and stable response towards NADH is obtained within 5 s. The linear range for the determination of NADH is from 0.8 μmol L^?1 to 422 μmol L^?1, with a detection limit of 0.26 μmol L^?1 (S/N = 3). The wide linear range, lower detection limit and faster response towards NADH suggests that the new method potentially is useful for developing NAD⁺-dependent enzyme-based biosensors.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Cooperative Catalysis of an Alcohol Dehydrogenase and Rhodium‐Modified Periodic Mesoporous Organosilica

The combined use of a metal‐complex catalyst and an enzyme is attractive, but typically results in mutual inactivation. A rhodium (Rh) complex immobilized in a bipyridine‐based periodic mesoporous organosilica (BPy‐PMO) shows high catalytic activity during transfer hydrogenation, even in the presence of bovine serum albumin (BSA), while a homogeneous Rh complex exhibits reduced activity due to direct interaction with BSA. The use of a smaller protein or an amino acid revealed a clear size‐sieving effect of the BPy‐PMO that protected the Rh catalyst from direct interactions. A combination of Rh‐immobilized BPy‐PMO and an enzyme (horse liver alcohol dehydrogenase; HLADH) promoted sequential reactions involving the transfer hydrogenation of NAD⁺ to give NADH followed by the asymmetric hydrogenation of 4‐phenyl‐2‐butanone with high enantioselectivity. The use of BPy‐PMO as a support for metal complexes could be applied to other systems consisting of a metal‐complex catalyst and an enzyme.