新型络合物的形成与复合毒性分子机理研究
文献类型：学位论文
| 作者 | 李岩 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 朱本占 |
| 关键词 | 复合毒性 Ternary complex Hydrogen-bonding Crystal structure Supramolecular compounds 三元络合物 氢键复合物 晶体结构 超分子化合物 Combined toxicity |
| 其他题名 | The Formation of Novel Complexes and Molecular Mechanisms of Combined Toxicity |
| 学位专业 | 环境科学 |
| 中文摘要 | 化学品在生活生产中变得越来越重要，它提供了许多的好处，尤其是在提高工业，农业产品产量以及控制疾病方面。然而，这些化学品从生产到处置过程中，同样会对环境或人体造成不良的影响。目前，我们对环境污染物毒性和致癌性的知识主要来源于对某单一化学物质在相对高浓度情况下的研究。多年来，对单独某种物质的研究所获得的作用机理方面的信息，虽然在多数情况下对我们从实验结果推及对人类风险评估方面打下了合理的基础，然而当用于评估复杂环境混合物时却不那么成功。另外，人们在其一生中很少只暴露于单独某一种化学物质，而是同时暴露于多种低浓度的化学物质。从公众健康的角度讲，大家主要担心的是当两种或两种以上化学物质相互作用时，两者能否产生不同寻常的协同毒性，而每一种物质在单独存在时并不产生明显毒性。事实上，最近的研究表明，某些环境化学物质的毒性往往被低估了。本文从两类环境中典型的有机和无机污染物入手，在先前生物毒理学的基础上，系统的研究了它们产生复合毒性的分子机制，为以后研究其它类似的现象提供了理论依据。研究主要分为以下三个部分： 第一部分：多卤酚类化合物-金属-邻菲罗啉三元络合物不同的配位模式与稳定性导致不同的生物效应 当无毒性或亚毒性剂量的五氯酚(Pentachlorophenol, PCP)与铜-邻菲罗啉配合物同时存在的条件下，我们发现其对细菌以及哺乳动物细胞有着显著的协同毒性。这种协同毒性同样存在于其他多氯酚类化合物与金属-邻菲罗啉类配合物的共同暴露体系。从相关化合物的结构特点考虑，我们推测，它们之间发生反应可能生成了亲脂性的三元络合物，从而促进了细胞对铜离子的吸收，产生协同毒性。然而，由于该三元络合物的强亲脂性，导致其在水中的溶解度极差，因此限制了许多经典分析方法如UV-Vis, HPLC, MS等的应用。在本研究中，我们通过IR，固体ESR，X射线晶体衍射等固体分析手段揭示了三元络合物的组成和分子结构，并阐述了PCP与铜-邻菲罗啉配合物产生协同毒性的分子机理。在此基础上，我们还研究了PCP与其他金属离子邻菲罗啉配合物所形成的三元络合物的结构性质，并发现了一类新型的离子对三元络合物。通过比较这两类三元络合物的结构，我们发现生成三元络合物的性质主要取决于金属-邻菲罗啉配合物的稳定性。当配合物本身不稳定时，PCP可以将一分子的邻菲罗啉取代下来，并与铜直接发生配位，生成中性的三元络合物。而当配合物本身很稳定时，PCP则无法取代邻菲罗啉，而是以PCP阴离子的形式存在于金属配合物的外侧，形成离子对形式的三元络合物。由于这两类三元络合物都具有较强的亲脂性，因而皆可透过细胞膜进入到细胞内。进入细胞后，由于PCP作为单齿配体或抗衡阴离子与金属阳离子作用又不是特别强，因此可被细胞内的生物配体取代下来，生成相应的邻菲罗啉-金属-生物配体复合物。如果金属-邻菲罗啉类配合物具有较好的氧化还原活性(如铜-邻菲罗啉)，则可产生大量的活性氧物种，导致较强的协同毒性。反之，则协同毒性较弱, 然而却可表现出其它特殊生物效应，如钌-邻菲罗啉类配合物可与细胞核内的DNA结合产生荧光，从而可被用作活细胞DNA荧光探针。另外，本研究还发现PCP的主要代谢产物之一四氯邻苯二酚(Tetrachlorocatechol, TCC)也可与铜-邻菲罗啉配合物之间生成亲脂性的三元络合物。然而，由于TCC作为双齿配体与铜-邻菲罗啉络合异常紧密，因此其在细胞内不能与生物配体相结合，所以不但未表现出协同毒性，反而有一定的的拮抗效应。本研究通过对三类不同三元络合物的结构和性质的综合分析，发现氯代酚类化合物-金属-邻菲罗啉三元络合物不同的配位模式与稳定性导致其在生物体系中不同的复合毒性或生物效应，为将来研究其它有机-无机化合物之间的相互作用和复合生物效应的分子机制打下了良好的实验和理论基础。 第二部分：一价铜专属络合剂新亚铜灵对多氯邻苯二酚的新型解毒机制：一种具有罕见垂直构象与氧化还原抑制能力的稳定氢键复合物的生成与鉴定 2,9-二甲基-1,10-邻菲罗啉(2,9-Dimethyl-1,10-phenanthroline, 2,9-Me2OP; 也称新亚铜灵(Neocuproine))作为一价铜的专属络合剂，广泛用于研究生物或化学体系中铜所起的作用。先前的研究发现，在与TCC等比例的条件下，2,9-Me2OP可以完全抑制TCC与叠氮化钠(NaN3)反应所产生的协同毒性，而其他金属络合剂则不能。后来证明2,9-Me2OP不是通过络合铜离子而产生抑制作用，而很可能直接和TCC发生反应，进而完全阻止了TCC/NaN3反应的发生。然而，由于2,9-Me2OP与TCC反应产物溶解性很差，一直未能对其进行准确的鉴定。在本研究中，我们运用红外光谱，固体NMR以及X射线晶体衍射等多种固体研究手段，直接对其反应产生的沉淀进行分析，发现2,9-Me2OP与TCC之间生成了一种非同寻常的多重氢键复合物。另外，此复合物由于2,9-Me2OP中甲基的空间位阻效应，还具有一种较为罕见的垂直构象。此构象不仅增强了复合物中的二级静电作用，而且由于空间位阻，构象受到强烈的约束，使得复合物整体处于一个稳定的状态。此稳定复合物的形成，完全阻止了TCC中活泼羟基的去质子化，使得N3-不能继续与之反应，进而阻止了半醌自由基的生成，抑制了协同毒性。然而，其它位置甲基取代的邻菲罗啉虽然也能和TCC之间形成相应的氢键复合物，但由于缺少空间位阻效应，生成的氢键复合物不够稳定，所以只能部分抑制TCC/NaN3反应的协同毒性。通过比较2,9-Me2OP/TCC与[Cu(2,9-Me2OP)2]+的空间结构，我们发现它们的构象惊人的相似，都是由于甲基的空间位阻效应而形成了稳定的构象，进而阻止了中心活泼基团的氧化。因此，2,9-Me2OP不仅可通过配位键络合一价的无机铜离子，还可通过多重氢键“络合”有机的邻苯二酚类化合物，是一种很好的氧化还原抑制剂。除此之外我们还发现，2,9-Me2OP不仅能够和TCC发生反应，而且还能够与其它低卤代邻苯二酚，氯代酚以及天然多酚类化合物反应，生成相对应的稳定的氢键复合物。尤其是通过研究2,9-Me2OP抑制多酚促氧化的反应，我们提出了一种2,9-Me2OP不依赖于铜离子的新型解毒机制，具有潜在的生物学意义。 第三部分：二氯二羟基苯醌/邻菲罗啉氢键复合物的合成与结构研究 前面的研究表明，2,9-Me2OP与TCC之间能够形成具有垂直构象的多重氢键复合物。其中，由于TCC的两个羟基以及2,9-Me2OP中的两个N原子均为邻位，因此其氢键的成键方向是固定的，只能形成1:1的产物。二氯二羟基苯醌(2,5-Dichloro-3,6-dihydroxy-1,4-benzoquinone, DDBQ)是PCP及TCC的一种重要代谢终产物。由于其分子中两个羟基是对位取代的，所以不仅可以和多种含N的有机物反应生成1:1, 1:2的产物，而且还可能通过DDBQ的分子间氢键发生链增长，生成具有特殊构型和功能的超分子有机化合物，因此是一种很好的超分子化合物合成元件。为了扩展之前的2,9-Me2OP/TCC的氢键体系，我们研究了DDBQ与邻菲罗啉类化合物之间的反应，发现它们之间不仅能形成类似的氢键复合物，而且苯基取代的邻菲罗啉与DDBQ之间还能形成两种不同形式的超分子化合物。这些有趣的超分子结构对于研究邻菲罗啉类物质取代基团的作用有着重要的意义。 综上，本研究通过多种分析方法，发现并鉴定了几类新型金属三元络合物和氢键复合物，通过分析研究其确切的分子结构以及相关的化学性质，解释了这些三元络合物及氢键复合物产生复合毒性和其它生物效应的分子机理。本研究为将来研究有机和无机化合物诱导的复合毒性和其它生物效应的分子机理打下了坚实的实验和理论基础。 |
| 英文摘要 | Chemical substances are essential in many economic activities and are a significant part of our daily life. They provide society with a wide range of benefits, and particularly increased agricultural and industrial productivity and improvements in the control of diseases. Nevertheless, chemical compounds have the potential to cause considerable environmental and health problems from production to disposal. Current knowledge on the toxicity of chemicals comes primarily from studies conducted on individual chemicals at relatively high concentrations. However, throughout their lifetimes, people are more commonly exposed to a range of chemicals at low concentrations. From a public health perspective, a major concern is whether toxicity could result from the interaction of two or more chemicals. Based on our previous research, we further investigated the molecular mechanisms for combined toxicity of polyhalogenated phenolic compounds and metal-phenanthroline complexes in this study. It was composed of three major parts: Part 1: Different Coordination Modes and Stability for Polychlorinated Phenolic Compounds and Metal-1,10-phenanthroline Ternary Complexes Result in Different Biological Effects We have shown previously that exposing bacteria and mammalian cell to wood preservatives pentachlorophenol (PCP) and copper-containing compounds together causes synergistic toxicity. The synergism was found to be due to the formation of a lipophilic ternary complex which facilitated copper transport into the cells. However, because of the poor solubility, many typical techniques such as UV-Vis, HPLC and MS could not be applied to identify this ternary complex. In this study, we showed that a neutral PCP2Cu(II)OP complex was formed by complementary application of single crystal X-ray diffraction, IR and solid state ESR. To our surprise, only one molecule of OP was found in the crystal; and more interestingly, not only Cu-O bond, but also Cu-Cl bond, were observed between Cu and one PCP molecule, which may make the ternary complex more stable. Meanwhile, all of the ten Cl atoms together may function as hydrophobic group to further enhance the lipophilicity of the ternary complex, which leads to high synergistic toxicity. We also found that a novel ion-paired ternary complex was formed between PCP and [Fe(OP)3]2+or [Ru(OP)3]2+ complex. Compared with the neutral PCP2Cu(II)OP complex, PCP could not directly coordinate with Fe2+or Ru2+due to the high stability of [Fe(OP)3]2+or [Ru(OP)3]2+complex,just serve ascounter-anions in the complex. Although the form of PCP was different in the two ternary complexes, due to their lipophilicity,both can penetrate through the membrane and get into the cells. Upon entering the cell, the ternary complex may dissociate and form new chelates with cellular ligands, such as DNA and other macromolecules. If the metal-OP complex is redox-active (such as [Cu(OP)2]2+), then reactive oxygen species (ROS)could be generated during the redox cycling of the transition metals, and strong synergistic toxicity could be induced. Otherwise, there will be weak synergistic toxicity, but with other special biogical effects, for example, [Ru(OP)2dppz]2+complex could combine with nucleus DNA to produce fluorescence, which could be used as a DNAfluorescent probe in living cells. In another case, the observed bacterial killing between [Cu(OP)2]2+ and tetrachlorocatechol (TCC, which is one of the major toxic metabolites of pentachlorophenol) was lower than that of either TCC or [Cu(OP)2]2+ alone. We found that a ternary complex TCCCu(II)OP was also formed after theirinteraction. However, compared with PCP, the bidentate nature of TCC could coordinate with Cu2+ tightly, makingthe ternarycomplexextremely stable. We found that TCCCu(II)OP complex was readily partitioned into the 1-octanol phase similar to that of PCP2Cu(II)OP, but the complex was hardly dissolved. These results suggested that although the TCCCu(II)OP complexcould be taken up by the cells, since the complex was so stable that it may not be dissociatedto form new chelates with cellular ligands. Thus, copper was detoxified by the formation of such a strong complex with TCC. In conclusion, the formation of ternary complexes of similar lipophilic character could be ofrelevance as a general mechanism of combined toxicity or other biological effects between the organic and inorganic transition metal complexes. However, different coordination modes and stabilityof theternary complexes mayresult in different biological effects. Part 2: Detoxifying Polyhalogenated Catechols by a Copper Chelating Agent via Forming Stable and Redox-inactive Hydrogen-bonded Complexes with Unusual Perpendicular Structure As we know, 2,9-dimethyl-1,10-phenanthroline (2,9-Me2OP, also called neocuproine) is a well-known copper-specific chelator and has been widely used to study the role of copper in biological systems. However, during our study on the role of transition metal ions in the genotoxicity of the wood preservative pentachlorophenol, we found, unexpectedly, that only 2,9-Me2OP markedly inhibitedthe synergistic toxicity induced by tetrachlorocatehol (TCC) and sodium azide (NaN3). Further studies indicate that the protection by 2,9-Me2OP may not be due to its chelation of copper, but possibly due to the formation of an unknown supramolecular complex with TCC and/or NaN3. However, the exact chemical structure and the composition of such a complex remained unclear because we were unable to detect and identify this complex by the typical techniques applied in the characterization of organic compounds in solutions. In this study, by complementary application of single crystal X-ray diffraction, IR and NMR methods, we found that a multiply hydrogen-bonded 2,9-Me2OP/TCC complex with an unusual perpendicular conformation was formed. The combination of two primary N···H-O H-bonds with two additional attractive secondary H-bonding interactions and two weak C-H···O H-bonds, should provide a high thermodynamic stability to the 2,9-Me2OP/TCC complex. Interestingly, the two methyl groups at the 2, 9 positions in 2,9-Me2OP were found to be critical to stabilize the 2,9-Me2OP/TCC complex via steric hindrance. It allowed the hydroxyl H atom forming H-bond with N atom on the other side and established two secondary attractive H-bonding interactions between the two adjacent primary H-bonds. The steric hindrance also helped to restrict the conformation and make the H-bonds in a stable condition. Therefore, these special features might completely inhibit the deprotonating process of TCC, thus inhibiting further oxidation of TCC (with or without NaN3) to produce toxic semiquinone radical species. In contrast, due to the absence of the two CH3 groups at the 2, 9 positions, although 4,7-Me2OP and 5,6-Me2OP could form similar two primary N···H-O H-bonds with TCC, the secondary attractive interactions in the two 4,7-Me2OP/TCC and 5,6-Me2OP/TCC complexes were markedly reduced because the less perpendicular orientation makes the hydroxyl group far away from the N atom on the other side; and the complex was structurally flexible, which most likely made the complex less stable and more prone to dissociation with competing agents like NaN3 or solvent molecules. Therefore, the synergistic toxicity could only be partially inhibited by these 2,9-Me2OP regioisomers. By comparison between 2,9-Me2OP/TCC and [Cu(2,9-Me2OP)2]+ complexes, we found that two CH3 groups could not only lead 2,9-Me2OP to bind favorably with cuprous ion to form the preferred tetrahedral structure, but also lead 2,9-Me2OP to bind favorably with TCC to form the preferred perpendicular structure. In other words, 2,9-Me2OP could not only chelate the inorganic Cu+ by coordination, but could also “chelate” the organic catecholic compounds via H-bonding. Due to its unique steric hindrance effect of the two CH3 groups at 2,9 positions, both of the two complexes were restricted, and the catechol group and/or Cu+ were also protected against the attack from other competing agents. We found that the formation of the unusual H-bonded 2,9-Me2OP complex is not only limited to TCC, butit is also a general mechanismfor all polyhalogenated catecholic compounds and even some natural polyphenolic compounds. Therefore the formation of H-bonded complexes with these catecholic compounds may not only serve as a general, but previously unrecognized Cu-independent new detoxication mechanism for the widely-used 2,9-Me2OP, but also have interesting biological and environmental implications. Part 3: Preparationand Structural Studies on H-bonded Complexes of 2,5-Dichloro-3,6-dihydroxy-1,4-benzoquinone and Phenanthrolines 2,5-Dichloro-3,6-dihydroxy-1,4-benzoquinone(DDBQ) was the main metabolite, or degradation product of PCP or TCC. Unlike TCC, the two OH groups were para-substituted in DDBQ. Therefore, compared with OPs/TCC, different stoichiometric ratios of OPs/DDBQ complexes could be formed. By thestructural study, we found not only H-bonded complexes between the methyl-substituted OP and DDBQ, but also supramolecular compounds between phenyl-substituted OP and DDBQ. These results suggested DDBQ could be a good chain-building unit in the application of supramolecular chemistry. In conclusion, we identified and characterized several novel ternary metal complexes and H-bonding complexes by complementary application of various analytical methods for solid forms. Through structural analysis of the unique property of each complex, the underlying molecular mechanisms of the combined toxicity and other biological effects between polyhalogenated phenolic compounds and metal-complexes (or ligands) could be reasonably explained. The findings of this study may serve as basis and guidelines for future investigation on molecular mechanisms of the combined toxicity and other biological effects induced by the combination of both organic and inorganic compounds. |
| 公开日期 | 2015-07-07 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/15738]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 李岩. 新型络合物的形成与复合毒性分子机理研究[D]. 北京. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：生态环境研究中心
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