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题名	卤代苯醌与氢过氧化物的反应机制异羟肟酸类药物的保护机理研究
作者	单国强
学位类别	博士
答辩日期	2009
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	烷氧自由基 carbon-centered quinone ketoxy radical(& 二氯苯醌 8226 叔丁基过氧化氢 CBQ=O) 以碳为中心的醌酮自由基 alkoxyl radical 苯甲异羟肟酸 Lossen rearrangement Lossen重排 benzohydox
其他题名	Mechanism of Metal-independent Decompositionof Hydroperoxides by Halogenated Quinones andProtection by hydroxamic Acids against DCBQ/H2O2
中文摘要	1．许多卤代环境污染物如氯代酚类杀虫剂、卤代苯、橙色剂和溴代阻燃剂等，均可在生物体内代谢或化学脱氯生成卤代醌。而卤代醌诱发的毒理机制可以部分解释这些环境污染物的致癌性。关于卤代醌诱发的毒理机制，目前普遍认同的是半醌介导氢醌/醌之间的氧化还原循环产生的活性氧造成生物体氧化损伤。一般认为这一机制中包含了依赖金属离子的Fenton反应。然而，近来的研究发现卤代醌与过氧化氢可以不依赖金属离子产生高破坏力的•OH，卤代醌与有机过氧化氢可以不依赖金属离子产生烷氧自由基，从而呈现潜在的生物毒性。另外，过氧化氢作为绿色安全的环境氧化剂，已经广泛应用于环境污染物如氯代酚类农药的氧化处理。已发现卤代醌是这种氧化处理的中间产物。因此，过氧化氢参与的卤代醌降解机制，可能在这种环境修复或净化系统中具有重要的意义。 近来发现卤代醌与氢过氧化物之间的反应可能是一种新颖的亲核取代及均裂机制：以DCBQ/t-BuOOH为例，首先t-BuOOH亲核攻击DCBQ形成2-氯-5-叔丁过氧基-1,4-苯醌（CBQ-OO-t-Bu）中间体，后者随即均裂分解产生t¬-BuO•和5-氯-对苯醌-2-氧自由基（CBQ-O•）。在过去的研究中t¬-BuO•、•CH3等产物均得到确认，然而，反应产物及中间体CBQ-OO-t-Bu、CBQ-O•等仍没有分离并充分鉴定。因此，本研究继续以DCBQ/t-BuOOH反应体系为研究对象，对中间体和终产物进行分析鉴定，进行机制的深入研究。 研究中首先通过半制备HPLC分离出一种主要产物，采用各种方法鉴定其为2-羟基-3-叔丁氧基-5-氯-1,4-苯醌（CBQ(OH)-O-t-Bu），该物质是先前假设的CBQ-OO-t-Bu中间体的重排异构体。又在使用自旋捕获剂DMPO的实验中发现，DMPO能抑制CBQ(OH)-O-t-Bu的形成，并伴随形成一种新的DMPO加合物（质荷比m/z 268）。在此基础上，进一步采用电子自旋共振（ESR）、核磁共振氢谱（1H-NMR）和高效液相-傅里叶变换离子回旋共振（HPLC/FTICR）等手段研究DMPO/DCBQ/t-BuOOH（或H2O2）体系。研究结果表明，DMPO捕获的并不是CBQ-O•，而是其共振异构体——以碳为中心的醌酮自由基（•CBQ=O）。本研究首次检测并分离出一种新的以碳为中心的•CBQ=O，为先前提出的卤代醌介导的不依赖金属离子的氢过氧化物分解机制提供了直接的实验证据，并丰富和完善了该机制。 本文发现不仅DCBQ，其它α-H卤代醌也都能与氢过氧化物通过不依赖于金属的途径发生反应，产生烷氧基/羟基自由基以及以碳为中心的醌酮自由基。这一发现可能具有重要的生物学意义。因为许多卤代环境污染物等可在生物体内代谢或化学脱氯生成卤取代醌。本研究结果表明，这些卤代醌与氢过氧化物发生反应，不仅可以通过促进烷氧基/羟基自由基的产生发挥其毒性，而且还可以通过形成•CBQ=O而发挥毒性。•CBQ=O可以直接与重要的生物大分子如DNA、蛋白质和脂类发生交联反应。 2．在研究卤代醌与氢过氧化物反应机制的同时，我们又对DCBQ/H2O2反应产生的•OH造成的DNA单链断裂的保护进行了研究。异羟肟酸类药物是强有力的金属离子螯合剂，在生物医学上具有广泛的应用。在本项工作中，我们发现苯甲异羟肟酸（BHA）可以有效地保护DCBQ/H2O2反应产生的•OH造成的DNA单链断裂。这种保护现象既不是•OH清除作用，也不是消除2,5-二氯-1,4-半醌自由基（DCSQ•）作用，而是因为BHA与DCBQ首先发生了亲核取代反应，形成了单或双取代的加合物，从而耗竭了DCBQ，阻断了与H2O2反应产生•OH的途径。两种加合物经分离纯化并得到鉴定。异羟肟酸类药物的保护机理展现了一种新颖的异羟肟酸类药物的作用方式，为异羟肟酸类药物的临床新应用开拓了领域。 3．我们的研究小组曾经发现苯甲异羟肟酸类药物能大大加快四氯苯醌（TCBQ）的水解速度，形成低毒、稳定的二氯二羟基苯醌，而且有意义的是，BHA与TCBQ之间的反应可能是一种新型的氯代醌介导的Lossen重排机制。过去的研究已经分离到支持Lossen重排的产物：O-苯胺基甲酰基苯甲羟肟酸酯、N,N′-二苯基脲、苯胺。但是，在BHA/TCBQ反应体系中，却没有检测到BHA/TCBQ加合物以及Lossen重排的直接证据苯基异氰酸酯。既然本研究2中，在BHA/DCBQ反应体系中已经分离到稳定的BHA/DCBQ加合物，那么有必要以BHA/DCBQ（1:1）加合物为研究对象，进行其分解机制研究，从而清楚阐明这种新型的Lossen重排机制。 本研究利用TLC、HPLC证明BHA/DCBQ（1:1）加合物热解形成N,N′-二苯基脲、苯胺、2-羟基-5-氯对苯醌等，利用GC-MS证明其热解形成苯基异氰酸酯。因此，我们首次发现并报道了一种新型的氯代醌介导的Lossen重排机制，该机制的研究对于丰富经典的Lossen重排反应具有重要的化学意义。 4. 在研究BHA与DCBQ的反应机制基础上，本研究又使用高效液相色谱-电喷雾四级杆飞行时间串联质谱等方法对另一种重要的天然异羟肟酸类金属螯合剂DFO，与DCBQ的反应机制进行了研究。DFO临床多用于急、慢性铁中毒，近来发现它还具有不依赖金属离子的自由基清除作用，可以保护TCHQ导致的纯化DNA单链断裂。研究结果表明DFO与DCBQ的反应与BHA/DCBQ的机理一致，均通过亲核取代反应形成相应的加合物。本研究的结果为全面研究DFO的保护机理提供了实验依据。
英文摘要	1. Many halogenated environmental pollutants such as widely-used biocides chlorinated phenols, the halogenated benzene, the Agent Orange, and the brominated flame-retardants can be metabolized in vivo, or dechlorinated chemically to halogenated quinones. These metabolites could induce toxics, which might partly explain the potential carcinogenicity of these polyhalogenated aromatic pollutants. A generally accepted theory about the toxic mechanism is reactive oxygen species produced by semiquinone-mediated hydroquinones/quinones redox circle, which lead to biological damages. It should be noted that the metal-dependent Fenton reaction exerts crucial effect during the process. However, the past findings show that these halogenated quinones could react with hydroperoxides to form alkoxyl/hydroxyl radicals independent of transition metal ions, thus representing a potential biological toxicity. In addition, hydrogen peroxide as a green and safe environmental oxidant has been widely used in environmental pollutants such as chlorinated phenol pesticides and halogenated quinones are the intermediate during the oxidation process. Therefore, the mechanism of hydrogen peroxide involved in the degradation of halogenated quinones may be important in those environmental treatment system. The recent findings reveal that these halogenated quinones may react with hydroperoxides to product alkoxyl/hydroxyl radicals by a novel nucleophilic substitution and homolytical decomposition mechanism. For example, t-butoxyl radical (t-BuO•) produced by t-BuOOH and halogenated quinones independent of transition metal ions by the nucleophilic attack of t-BuOOH on 2,5-dichloro-1,4-benzoquinone (DCBQ). The attack leads to a chloro-t-butylperoxyl-1,4-benzoquinone (CBQ-OO-t-Bu) intermediate, which can decompose homolytically to produce t-BuO• and 2-chloro-5-hydroxy-1,4-benzoquinone radical (CBQ-O•). However, neither the major reaction products were isolated and fully characterized, nor the proposed quinone phenoxyl radical CBQ-O• and intermediate CBQ-OO-t-Bu were detected. So we continue to investigate the precise molecular mechanism of DCBQ/t¬-BuOOH in this work, In this work, one of the major reaction products of DCBQ/t¬-BuOOH was isolated by semi-preparative HPLC, and identified as 2-hydroxy-3-t-butoxy-5-chloro-1,4-benzoquinone (CBQ(OH)-O-t-Bu), which is the rearranged isomer of the postulated CBQ-OO-t-Bu. The formation of CBQ(OH)-O-t-Bu was found to be inhibited by the spin trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and concurrently, a new DMPO adduct with one-chlorine isotope peak clusters at m/z 268 was observed. Further electron spin resonance (ESR) spin-trapping, 1H-NMR and HPLC/Fourier transform ion cyclotron resonance (FTICR) mass spectrometric studies with O17-labeled and unlabeled hydrogen peroxide strongly suggest that the radical trapped by DMPO is a carbon-centered quinone ketoxy radical, which is the spin isomer of the proposed oxygen-centered quinone enoxy radical. CBQ-O•. This study represents the first detection and identification of an unusual carbon-centered quinone ketoxy radical, which provides direct experimental evidence to further support and expand our previously-proposed mechanism for metal-independent decomposition of hydroperoxides by halogenated quinones. Our observation that not only DCBQ, but also other α-H halogented quinones can react with hydroperoxides to produce carbon-centered quinone ketoxy radicals in addition to alkoxyl/hydroxyl radicals in a metal-independent manner, which may have interesting biological implications. For example, many halogenated environmental pollutants can be metabolized in vivo, or dechlorinated chemically to halogenated quinones. Our data suggest that these halogenated quinones may react with hydroperoxides and exert toxic effects not only through enhanced production of alkoxyl/hydroxyl radicals, but also through the formation of carbon-centered quinone ketoxy radicals which may react directly with critical biological macromolecules such as DNA, protein and lipids. 2. Meanwhile, we also study the protective mechanism of hydroxamic acids such as benzohydoxamic acid (BHA) against DNA single-strand breaks caused by metal-independent •OH production from DCBQ/H2O2. Hydroxamic acids play a variety of important roles in biology and medicine mainly due to their chelating properties with metal ions. In this work, we find that BHA can effectively protect the single-strand breaks caused by metal-independent •OH production from DCBQ/H2O2. We also find that this protection of BHA is neither attributed to •OH scavenging just as DMSO nor to the scavenging of 2,5-dichloro-1,4-semiquinone radical (DCSQ•). A unusual protective mechanism of hydroxamic acids is proposed: a nucleophillic substitution reaction occur between DCBQ and BHA, forming the stable adducts 2-O-benzohydroxamic-5-chloro-1,4-benzoquione and 2,5-di-O-benzohydroxamic-1,4-benzoquione,consuming up DCBQ efficiently, consequently blocked the pathway of •OH production with H2O2. The two adducts was separated and identified by NMR and IR. This new protective mechanism represents new mode of action of hydroxamic acids, which may open up new application of clinical areas of hydroxamic acids. 3. Our research group had recently demonstrated that BHA could dramatically enhance the hydrolysis of TCBQ to form trichlorohydroxyquinone and less toxic and stable chloranilic acid through a mechanism involved a Lossen rearrangement. That is a novel halogenated quinones mediated Lossen rearrangement. Although the products (O-phenethylcarbamyl benzohydromamate, aniline, N,N′-diphenylurea),which are indirect evidences for Lossen rearrangement, were isolated and determinated. However, direct evidences such as BHA/TCBQ adducts and the intermediate phenyl isocyanate were still not detected. In this work, we study the molecular mechanism of halogenated quinones mediated Lossen rearrangement, using the reaction system of BHA/DCBQ. We have demonstrated that BHA/DCBQ can form the stable adducts in part 2. We confirm BHA/DCBQ adduct could decompose thermally to CBQ-OH and phenyl isocyanate by Lossen rearrangement. using HPLC and GC-MS. This report about a novel Lossen rearrangement embodies an important chemical significance for the classic Lossen rearrangement. 4. Based on the work of the mechanism of BHA/DCBQ, we continue to study the reaction of DCBQ with DFO, another important natural hydroxamic acid siderophore DFO, which is used for clinical acute and chronic iron poisoning. it has recently found that DFO do not rely on metal ions to scavenge the free radical TCSQ•. In this work, it is found that DFO could react with DCBQ by the similar mode of BHA/DCBQ, forming the adducts of DFO/DCBQ by the nucleophillic substitution reaction. HPLC/MS data provide an experimental basis for a comprehensive study of the protective mechanism of DFO.
内容类型	学位论文
源URL	[http://ir.rcees.ac.cn/handle/311016/35009]
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	单国强. 卤代苯醌与氢过氧化物的反应机制异羟肟酸类药物的保护机理研究[D]. 北京. 中国科学院研究生院. 2009.

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