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题名: 卤代醌/过氧化氢体系产生羟基自由基的荧光监测、反应动力学及分子机理研究
作者: 任福荣
学位类别: 博士
答辩日期: 2015-05
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: 卤代醌 ; 氯代萘醌 ; 荧光法 ; 羟基自由基 ; 动力学,Halogenated Quinones ; Chlorinated 1 ; 4-Naphthoquinone ; Fluorescent method ; Hydroxyl Radical ; Kinetics
其他题名: Hydroxyl Radical Production by Halogenated Quinones and Hydrogen Peroxide: Fluorescent Monitoring, Kinetics and Molecular Mechanism
学位专业: 环境科学
中文摘要:       五氯酚(PCP)是一种重要的杀虫剂,常用作木材保护。PCP经代谢可产生两种主要的醌类化合物,四氯-1,4-氢醌(TCHQ)及四氯-1,4-苯醌 (TCBQ)。先前研究通常使用电子自旋共振(ESR)捕获法来直接检测TCHQ以及 TCBQ与H2O2反应产生的羟基自由基(?OH),但形成的自旋加合物的半衰期一般很短因而不利于进行动力学的研究。另外ESR仪器相对比较昂贵,操作 比较复杂也不利于日常分析检测。因此我们考虑是否可用选择性强、灵敏度高、操作简单及检测仪器相对便宜的荧光法对体系产生的?OH进行动力学监测以研究其 产生的分子机理?本研究使用检测?OH较为经典的香豆素-3-羧酸(3-CCA)作为荧光探针。3-CCA与?OH反应生成的主要产物是具有强荧光特性且 相对稳定的7-羟基-香豆素-3-羧酸(7-OHCCA)。研究分以下三个部分:
    1.采用荧光法监测四氯苯醌/H2O2体系通过两步反应产生羟基自由基的反应动力学
    该研究使用3-CCA作为检测体系产生?OH的荧光探针,成功检测到TCBQ/H2O2体系产生的?OH。并通过荧光动力学研究进一步确认体系产生?OH 是通过两步反应历程,得到的结果与ESR捕获法相同。向TCBQ/H2O2体系加入三种经典羟基清除剂(二甲基亚砜、乙醇及甲酸钠)及两种典型的抗氧化剂 (抗坏血酸及还原型谷胱甘肽)时均能很好地抑制体系产生的?OH。将此方法扩展到其它卤代苯醌(XBQ),同样发现所有XBQ/H2O2体系都能产 生?OH,而 ?OH产生的量与XBQ中卤原子个数和取代位置有关。总的来说,卤原子在苯环上取代的个数越多,产生的?OH越多;无取代的苯醌(BQ)与H2O2不产 生?OH;一氯代苯醌(2-CBQ)及两种二氯代苯醌(2,5-DCBQ、2,6-DCBQ)与H2O2反应产生的?OH量较少,且主要以一级反应动力学 特征为主;2,3-二氯苯醌(2,3-DCBQ)、三氯代苯醌(TrCBQ)及TCBQ与H2O2反应产生的?OH量相对较多,且以二级反应动力学特征为 主。
      2. 四氯氢醌体系并非通过其相应半醌自由基而是其氧化形式产生羟基自由基
      TCHQ是PCP的另一种主要醌类代谢产物,由于其具有强基因毒性而被广泛关注。在之前研究中,使用ESR自旋捕获法成功检测到体系产生的四氯半醌自由基 (TCSQ?)及?OH,但是仍不清楚其准确的分子机理。本研究同样使用3-CCA作为监测体系产生?OH的荧光探针。研究发现单独的TCHQ体系就能产 生?OH,向体系中加入H2O2后,能明显增加体系产生?OH, 而加入Cu2+后,能加速?OH产生。此前研究认为,TCHQ体系产生?OH可能是通过产生的相应的TCSQ?诱导的有机Fenton反应,因此 TCSQ?是TCHQ体系产生?OH所必要的。而我们通过综合利用荧光、紫外动力学及高效液相方法研究发现TCHQ体系产生的?OH并非直接由TCSQ? 产生,而是由TCHQ的氧化产物TCBQ产生,TCBQ才是体系产生?OH的关键,并进一步提出了TCHQ体系产生?OH的新的反应机理是通过亲核取代反 应及均裂分解反应。
      3.氯代萘醌/H2O2体系产生羟基自由基的反应机理
      萘醌类化合物是一类广泛存在于自然界中具有多种生物活性的小分子化合物。氯代萘醌(CNQ)与氯代苯醌(CBQ)具有相似的结构。由先前的研究结果可 知,CBQ在H2O2存在时,可产生?OH。既然这两类物质具有相似的结构,那么CNQ是否也可以产生?OH;如果是,其分子机理如何?我们发现,2-氯 -1,4-萘醌(2-CNQ)在H2O2存在时,3-CCA可以检测到体系产生的?OH,其主要反应产物被鉴定为2-羟基-1,4-萘醌。采用ESR自旋 捕获法发现2-CNQ/H2O2反应体系不仅检测到?OH,同时还观察到以碳为中心的醌酮自由基的生成。在此基础上,提出了CNQ/H2O2体系产 生?OH的新的可能反应机理。
英文摘要:        Pentachlorophenol (PCP) is an important biocide that has been mainly used as a wood preservative. Tetrachloro-1,4-benzoquinone (TCBQ) and tetrachloro-1,4-hydroquinone (TCHQ), the major genotoxic and carcinogenic metabolites of PCP, were found to react with H2O2 to produce hydroxyl radical (?OH) by ESR spin-trapping method. Since the spin-trapping adducts often have short half-time, they are not suitable for long-term detection and monitoring. In addition, the ESR equipment is very expensive and the operation is complex, so it is not suitable for routine analysis. In this work, coumarin-3-carboxylic acid (3-CCA) was used as a fluorescent probe for ?OH, which can produce a stable fluorescent product 7-hydroxy-coumarin-3-carboxylic acid (7-OHCCA). This study was composed of the following three parts:
      1. The Kinetics of the Two-step Hydroxyl Radical Production by TCBQ/H2O2 Monitored by Fluorescent Method
      The metal-independent ?OH production by TCBQ/H2O2 was studied with fluorescent spectrophotometer using 3-CCA as a fluorescent probe. We found that 7-OHCCA, the fluorescent product of 3-CCA and ?OH, can be produced by TCBQ/H2O2. A two-step ?OH production was observed for TCBQ/H2O2 by this fluorescent method, which is similar to other methods such as ESR. ?OH production by TCBQ/H2O2 is markedly inhibited by the three classic ?OH scavenging agents (dimethyl sulfoxide, ethanol and formate) and the two typical antioxidants (ascorbic acid and the reduced form of glutathione). ?OH can also be produced by H2O2 with other halogenated quinones (XBQ). In general, ?OH production by XBQ/H2O2 is determined not only by the number, but also by the position of chlorine substitution. No ?OH production was observed for the non-substituted 1,4-benzoquinone with H2O2. ?OH production with second-order kinetics characteristics was found for 2,3-dichloro-1,4-benzoquinone (2,3-DCBQ), trichloro-1,4-benzoquinone (TrCBQ) and TCBQ, while the others showed first-order kinetics characteristics.
      2. Hydroxyl Radical Production by TCHQ/H2O2 was not Mediated by its Corresponding Semiquinone Radical, but by its Oxidized Quinone
      TCHQ, a major genotoxic and carcinogenic metabolite of PCP, has been implicated in its genotoxicity. In our previous work, both TCSQ? and ?OH, produced by TCHQ, have been successfully detected by ESR spin-trapping method, but the exact molecular mechanism remains not clear. The metal-independent ?OH production by TCHQ was further studied using 3-CCA as a fluorescent probe. We found that 7-OHCCA could be produced by TCHQ, which was greatly enhanced by H2O2, and accelerated by cupric ion. ?OH production by TCHQ/H2O2 was found to be not through the direct interaction of H2O2 with its corresponding semiquinone radical TCSQ?, but rather with its oxidized form TCBQ as demonstrated by the complementary applications of UV-Vis, fluorescence and HPLC methods. These results suggested that ?OH production by TCHQ/H2O2 was not through the so-called “organic Fenton reaction”, but rather through a novel nucleophilic substitution coupled with homolytical decomposition mechanism.
      3. Mechanism of Hydroxyl Radical Production by Chlorinated 1,4-Naphthoquinones (CNQ) and H2O2
      Naphthoquinones are wide-spread quinoid compounds in nature, displaying very significant pharmacological properties. Since ?OH could be produced by chlorinated 1,4-benzoquinone (CBQ) and H2O2, it is interesting to know whether chlorinated -1,4-naphthoquinones (CNQ) can also produce ?OH with H2O2; and if so, what is the underlying mechanism? We found that ?OH could indeed be produced by CNQ such as 2-dichloro-1,4-naphthoquinone (2-CNQ) and 2,3-dichloro-1,4-naphthoquinone (2,3-DCNQ) with H2O2, but not by the non-substituted 1,4-naphthoquinone. The major products produced by 2-CNQ/H2O2 was identified as 2-hydroxy-1,4-naphthoquinone and 2,3-dihydroxy-1,4-naphthoquinone, and a novel carbon-centered quinone ketoxy radical was also observed by the ESR spin-trapping method in 2-CNQ/H2O2 system.Based on these findings, a new molecular mechanism was proposed for OH production by CNQ/H2O2.
内容类型: 学位论文
URI标识: http://ir.rcees.ac.cn/handle/311016/34506
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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任福荣. 卤代醌/过氧化氢体系产生羟基自由基的荧光监测、反应动力学及分子机理研究[D]. 北京. 中国科学院研究生院. 2015.
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