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题名: 环状异羟肟酸及衍生物与多卤代醌反应机理研究
作者: 李锋
学位类别: 博士
答辩日期: 2016-11
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: 四氯苯醌,N-羟基吡啶酮,N-羟基吡啶硫酮,N-羟基邻苯二甲酰亚胺, 糖精钠 ; Tetrachloro-1,4-benzoquinone (TCBQ), N-hydroxypyridone (NHP), N-hydroxypyrithione (NHPT), N-hydroxyphthalimide (NHPI), saccharin sodium
其他题名: Molecular Mechanisms of Reactions between Cyclic Hydroxamic Acids and Derivatives and Polyhalogenated Quinones
学位专业: 环境科学
中文摘要:       异羟肟酸类化合物在制药和化工等领域有着广泛的应用,其中最常用的几种是立诺他(抗癌药物)、去铁胺(治疗铁中毒)和N-羟基芳酰亚胺(有机合成催化剂)。N-羟基吡啶硫酮作为一个硫代异羟肟酸,在船舶工业和去屑用品中也有重要应用。四氯苯醌(TCBQ)是持久性有机污染物五氯酚(PCP)的主要代谢产物,可以引起细胞和小鼠DNA 的损伤。在我们前期的工作中,已经对几种链式异羟肟酸针对TCBQ 的解毒机理进行了深入的研究。我们发现,氮原子上取代基团的微小改变可以造成反应机理上很大的不同。在本论文中,我们将聚焦在环状异羟肟酸。我们选取了三种环状异羟肟酸作为模型化合物来测试对TCBQ的解毒能力:N-羟基吡啶酮(NHP)、N-羟基吡啶硫酮(NHPT)和N-羟基芳酰亚胺(NHPI)。有趣而令我们感到意外的是,它们各自有着截然不同的表现和反应机理。

NHP 通过两步自由基重排反应对TCBQ 进行解毒
      NHP 常用作金属络合剂。我们发现,在NHP 存在时,高毒性的TCBQ 很快通过两步水解脱氯而被降解为毒性很低的二羟基产物2,5-二氯-3,6-二羟基-1,4-苯醌(DDBQ)。同时,NHP 被完全转化为产物2-羟基吡啶。有趣的是,在ESR 实验中分别用POBN 和BMPO 作为捕获剂检测到了NHP 的两种自由基中间产物的信号,一种是氮中心自由基,另一种是不同寻常的氧中心自由基。根据以上结果,我们提出NHP 通过亲核取代偶联N-O 键均裂而对TCBQ 产生解毒作用,并且生成了相应的自由基中间体。

NHPT 对TCBQ 的解毒以及意外发现的NHPT/H2O2 反应生成HO•新机制
      NHPT 是硫取代的NHP 类似物,常用作络合剂和抗真菌剂。虽然NHPT 与NHP 有着类似的结构,但是它与TCBQ 反应时却表现出与NHP 截然不同的性质。我们得到了NHPT:TCBQ 为1:1、2:1 和4:1 三种不同比例的加合物,其中4:1 加合物为氢醌形式。令我们感到惊讶的是,即使在很高的浓度条件下,NHPT 也不能完全抑制TCBQ 和H2O2 造成的pBR322 DNA 的损伤。并且随着浓度的增加,NHPT 还可造成更多的DNA 损伤。NHPT 和H2O2 主要反应产物经质谱鉴定为1-氧-吡啶-2-磺酸。据此我们提出了NHPT 和H2O2 生成HO•的可能反应机理:NHPT 首先被H2O2 氧化加两个氧原子生成2-亚磺酸基-吡啶-N-氧化物,随后与H2O2 发生亲核加成反应生成1-氧-吡啶-2-过氧磺酸,继而发生O-O 键的均裂从而生成羟基自由基。

通过一种前所未见的溶剂水介导的两步Lossen 重排反应机理发生两步结构异构以及TCBQ 的解毒
       NHPI 是一种重要的有机催化剂,可以与温和氧化剂作用形成具有催化活性的N-O 自由基进而诱导C-H 键的活化。在本研究中,我们发现NHPI 可以在TCBQ的作用下极为容易地发生异构化生成靛红酸酐,同时TCBQ 经过两步水解脱氯反应降解为低毒性的DDBQ。有趣的是,通过使用氧-18 标记、HPLC-MS 和傅里叶变换高分辨质谱等手段,我们发现溶剂水是靛红酸酐中一个氧原子的来源。根据这些数据,我们提出了NHPI 和TCBQ 的反应机理:通过亲核取代偶联溶剂水辅助的Lossen 重排反应,NHPI 先被转化为异氰酸酯再通过快速分子内加成环化生成同分异构体靛红酸酐。这是首次报道在正常生理条件下NHPI 通过溶剂水促进的Lossen 重排被诱导异构化。

人工甜味剂糖精钠的新作用:通过质子中和作用促进TCBQ/H2O2 的反应
      糖精钠是一种广为人知的人工甜味剂。先前有研究提出糖精钠可以被H2O2氧化为其异羟肟酸形式N-羟基糖精,进而与TCBQ 通过电荷转移形成一种紫红色复合物;以此为基础发展出了一种简便的糖精钠的常规定量分析方法。然而,其确切的分子机理仍不清楚。在本研究中,我们发现在没有糖精钠的条件下TCBQ 仍然可以与H2O2 作用生成紫红色溶液。通过质谱鉴定,这种紫红色物质为TCBQ 的羟基化产物三氯一羟基苯醌(TrCBQ-OH)。其形成速率可被糖精钠显著促进,但是却被其共轭酸糖精抑制。有趣的是,糖精钠的含量在反应过程中并没有改变。进一步的研究表明TCBQ 和H2O2 的反应速率取决于溶液的pH 值,TCBQ 和H2O2 反应生成的酸(HCl 和TrCBQ-OH)降低了溶液的pH 值,但是可在一定程度上被弱碱性的糖精钠中和从而延缓溶液pH 值的下降。糖精钠的两种类似结构的化合物邻苯二甲酰亚胺钾和乙酰磺胺酸钾都表现出了类似的效应。基于以上的发现,我们提出糖精钠通过中和反应生成的酸进而促进TCBQ/H2O2 反应的机理。这是首次报道人工甜味剂糖精钠作为质子中和剂来使用。
      异羟肟酸和卤代醌是化学、环境科学和生物学研究中两类重要的化合物。因此我们的研究结果为进一步研究其他异羟肟酸和醌类化合物提供了广泛的理论基础。
英文摘要:        Hydroxamic acids have broad applications from pharmaceutical to chemical industry, among which suberanilohydroxamic acid (SAHA), deferoxamine and N-hydroxyphthalimide are the most commonly used ones. N-Hydroxypyrithione, a thiohydroxamic acid, also has important applications in shipping industry and antidandruff products. Tetrachloro-1,4-benzoquinone (TCBQ), a major metabolite of the persistent organic pollutant pentachlorophenol (PCP), was found to induce DNA damage in cells and mice. The detoxication mechanisms of TCBQ by several chain hydroxamic acids have been systematically investigated in our previous work, and we found that small changes of the substitution group, especially on the nitrogen atom,lead to dramatic change of the reaction mechanism. In this thesis, we will focus on cyclic hydroxamic acids. Three model cyclic hydroxamic acids, N-hydroxypyridone (NHP), N-hydroxypyrithione (NHPT) and N-hydroxyphthalimide (NHPI), were chosen to test their capability for detoxication of TCBQ. Interestingly and unexpectedly, they showed distinctively different detoxication mechanisms.

Detoxifying TCBQ by NHP via Double Radical Rearrangement
    NHP is well known for its chelating property. Here we found that highly toxic TCBQ was quickly degraded into its much less toxic dihydroxylation product 2,5-dichloro-3,6-dihydroxy-1,4-benzoquinone (DDBQ) through a two-step hydrolytic dechlorination in the presence of NHP. Simultaneously, NHP was found to be completely transformed into its corresponding product 2-hydroxypyridine.Interestingly, two radicals derived from NHP were detected by electron spin resonance (ESR) method, using 4-pyridyl N-oxide-N-tert-butylnitrone (POBN) and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline-N-oxide (BMPO) as spin-trapping agents:One is a nitrogen-centered radical, while another is an unusual oxygen-centered radical. Based on above results, we proposed that nucleophilic attack with a subsequent homolytic cleavage of N-O bond in two consecutive steps was responsible for the hydroxylation/detoxication of TCBQ by NHP, and the formation of the respective radical intermediates.

The Detoxication of TCBQ by NHPT and Unexpected Hydroxyl Radical Generation from NHPT and H2O2
     NHPT, an S-substituted analog of NHP, has been widely used as chelating and antifungal agents. Although NHPT has similar structure to NHP, it shows special characteristics in the reaction with TCBQ: Three adducts were detected and identified with the ratio of NHPT:TCBQ at 1:1, 2:1 and 4:1, in which the 4:1 adduct is a hydroquinone. To our surprise, NHPT cannot completely inhibit the damage of pBR322 DNA caused by TCBQ and H2O2, even at very high concentrations. Moreover, with the concentration increased, NHPT caused more damage to DNA. The major product from the reaction of NHPT and H2O2 was identified to be 1-oxy-pyridine-2-sulfonic acid. Together, we proposed a possible mechanism for hydroxyl radical formation from the reaction between NHPT and H2O2: NHPT was oxidized by H2O2 to form 2-sulfino-pyridine-N-oxide, which could react further with H2O2 to produce its nucleophilic addition product 1-oxy-pyridine-2-sulfonoperoxic acid, which decomposes homolytically to generate hydroxyl radicals.

An Exceptionally Facile Two-Step Structural Isomerization and Detoxication via an Unprecedented Double Water-Assisted Double Lossen Rearrangement
     NHPI, which is best known as an organocatalyst for efficient C-H activation, has been found to be oxidized by quinoid compounds to its corresponding catalytically active nitroxide-radical. Here, we found that NHPI can be isomerized into isatoic anhydride by an unusually facile two-step method using TCBQ, accompanied by a two-step hydrolytic dechlorination of highly toxic TCBQ into the much less toxic DDBQ. Interestingly, through the complementary application of oxygen-18 isotope-labeling, HPLC combined with electrospray ionization quadrupole time-of-flight and high resolution Fourier transform ion cyclotron resonance mass spectrometric studies, we determined that water was the source and origin of oxygen for isatoic anhydride. Based on these data, we proposed that nucleophilic attack with a subsequent water-assisted Lossen rearrangement coupled with rapid intramolecular addition and cyclization in two consecutive steps was responsible for this unusual structural isomerization of NHPI and concurrent hydroxylation/detoxication of TCBQ. This is the first report of an exceptionally facile double-isomerization of NHPI via an
unprecedented water-assisted double-Lossen rearrangement under normal physiological conditions.

A New Mode of Action for the Artificial Sweetener Saccharin Sodium: Acceleration of TCBQ/ H2O2 Reaction via Proton Neutralization
    Saccharin sodium, the well-known artificial non-nutritive sweetener, has been previously suggested to be oxidized into its hydroxamic acid form N-hydroxysaccharin, which may form a purple-red colored charge-transfer complex with TCBQ; and a simple spectroscopic method for its routine analysis was developed based on this finding. However, the exact chemical mechanism underlying such reactions remains unclear. Here we found that the colored species could be produced by the direct interaction between TCBQ and H2O2 in the absence of saccharin sodium, which was unequivocally identified by HPLC/MS as trichlorohydroxy1,4-benzoquinone (TrCBQ-OH), the monohydroxylation product of TCBQ. The rate of its formation was accelerated significantly by saccharin sodium, but was inhibited by its corresponding conjugate acid saccharin. Interestingly and surprisingly, no quantitative change of saccharin sodium was observed during the reaction. Further studies showed that the rate of the reaction between TCBQ and H2O2 is dependent on the pH of the
reaction solution, and TCBQ/H2O2 interaction led to a decline of the pH value of the reaction mixture due to the acids (HCl and TrCBQ-OH) generated during the reaction,which could be partially reversed by the weak base saccharin sodium. Analogous effects were observed with phthalimide potassium and acesulfame potassium, which have similar chemical structures with SacNa. Based on these findings, we proposed that neutralizing the generated acid rather than forming charge transfer complex is responsible for the acceleration of TCBQ hydroxylation by saccharin sodium. This represents the first report of an unexpected new mode of action for the artificial sweetener, which serves as a proton neutralizer.
    Our findings may have broad implications for future research on hydroxamic acids and polyhalogenated quinoid carcinogens, which are two important classes of compounds of major chemical, environmental and biological interest.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/36875
Appears in Collections:环境化学与生态毒理学国家重点实验室_学位论文

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Recommended Citation:
李锋. 环状异羟肟酸及衍生物与多卤代醌反应机理研究[D]. 北京. 中国科学院研究生院. 2016.
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