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细胞色素P450酶介导的芳香族化合物代谢机理的研究

发布时间:2018-11-29 13:08
【摘要】:芳香族化合物(Aromatic Compounds)的来源与分布都非常广泛,是环境中典型的有机污染物。该类化合物的分子结构中一般都带有苯环,当其进入生物体后极易引发致畸、致癌、致突变效应,对人类的健康产生巨大的威胁。细胞色素P450酶(Cytochrome P450)作为自然界中的万能氧化酶对进入生物体的外源性化合物尤其是一些间接致癌物的代谢转化(包括代谢解毒和代谢活化)中起着至关重要的作用。这类化合物进入生物体后,经细胞色素P450酶的氧化激活生成极易与体内的DNA、蛋白质等生物大分子上的亲核位点发生共价结合的亲电分子,引起肿瘤,诱发癌症。因此,深入了解这类化合物在P450酶介导下的代谢路径具有极其重要的意义,这有助于诊断环境污染的状况和程度,并为早期环境预警提供理论支撑。但由于常规的实验手段存在诸多限制,例如,P450酶的活性单体CpdⅠ(CompoundⅠ)、Cpd0(Compound 0)等在反应过程中转瞬即逝,极其难以捕捉,给化学反应动力学的研究带来极大的困难。因此我们采用密度泛函理论(Density Functional Theory,DFT)计算的方法来对这些反应进行研究。主要分为以下三部分内容:(1)本位取代反应(ipso-Substitution)是P450酶代谢对位取代苯酚类化合物的一种重要途径,却鲜有用量子化学计算的方法来研究其内在机制的报道。我们以双酚A为模型化合物对其在P450酶的CpdⅠ介导下的本位取代反应路径与氧加成路径进行计算,比较后发现本位取代反应路径为优势反应路径,即:P450酶的CpdⅠ活性中心的氧将酚上的氢提取后反弹到对位或邻位生成相应产物,该路径发生在低自旋二重态下,速率决定步骤为氢原子的提取。同时我们研究了一系列对位不同取代基苯酚发现本位取代反应的过渡态能垒与取代基效应密切相关,随对位取代基特性常数σp的增大而增大。(2)苯并(a)花(Benzo[a]pyrene)作为一种典型的P450酶底物,具有极强的致癌效应。本研究从反应动力学及热力学方面对P450酶的CpdⅠ环氧化苯并(a)芘各活性位点的路径进行比较,结合前人关于离域能的研究进一步解释了该反应中出现的区域选择性现象。并对该反应的主产物之一——9,10-环氧化苯并芘的形成路径进行分析,得出该反应主要发生在低自旋二重态的结论。(3)芳香胺类化合物(Aromatic Amine)可以在P450酶的CpdⅠ作用下代谢活化生成氮正阳离子自由基,与体内的亲核分子发生共价结合诱发癌症。本研究通过DFT方法计算了包括普通芳香胺与氮杂环芳香胺在内的28中芳香胺类化合物氢在P450酶的CpdⅠ介导下的兼并双态下氢提取反应的平均活化能垒AE*,发现该能垒△E**与N-H键的解离能(Bond Dissociation Energy,BDEN-H)之间存在着良好的线性相关。通过BDEN-H与AE*建立的预测模型(简单芳香胺:R2=0.92;氮杂环芳香胺:R2=0.95)具有良好的稳健性和预测能力。这为快速有效预测此类化合物的代谢活性甚至潜在的致癌性风险提供了依据。
[Abstract]:The source and distribution of aromatic compounds are very broad and are typical organic pollutants in the environment. The molecular structure of the compound is generally provided with a benzene ring, and when the benzene ring enters an organism, a teratogenic, carcinogenic and mutagenic effect can be easily induced, and a great threat to the health of the human is generated. Cytochrome P450 (Cytochrome P450), as a universal oxidase in nature, plays a critical role in the metabolic transformation of exogenous compounds, especially some indirect carcinogens, into organisms, including metabolic detoxification and metabolic activation. after the compound enters the organism, the cell pigment P450 enzyme is oxidized and activated to generate the electrophilic molecule which is very easy to be covalently bound to the nucleon site on the biological macromolecule such as the DNA and the protein in the body, and causes the tumor and the cancer to be induced. Therefore, it is of great significance to understand the metabolic pathway of these compounds under the P450 enzyme, which can help to diagnose the condition and extent of environmental pollution and provide theoretical support for early environmental early warning. However, due to the limitations of conventional experimental means, for example, the activity monomer Cpd I (Sound I), Cpd0 (Sound 0), and the like of the P450 enzyme are transient in the course of the reaction, and it is extremely difficult to capture, which brings great difficulty to the study of the chemical reaction kinetics. So we use the density functional theory (DFT) to study these reactions. It is mainly divided into the following three parts: (1) the standard substitution reaction (ipso-subregulation) is an important way for P450 enzyme to metabolize the para-substituted benzene phenolic compound, but few of the methods of quantum chemistry calculation are used to study the report of the mechanism. We use diphenol A as a model compound to calculate the standard substitution reaction path and the oxygen addition path mediated by the Cpd I of the P450 enzyme, and then find that the standard substitution reaction path is the advantage reaction path, namely: The oxygen of the active center of the Cpd I of the P450 enzyme returns to the para-position or the adjacent position to generate a corresponding product after the oxygen on the phenol is extracted, and the path takes place in the low-spin double state, and the rate determination step is the extraction of the hydrogen atom. At the same time, we have studied the transition state energy base of a series of p-substituted-based phenol-based substitution reaction, which is closely related to the substitution-based effect, and increases with the increase of the p-substitution-based property constant (p). (2) Benzo[a] pyrene, as a typical P450 enzyme substrate, has a very strong carcinogenic effect. In this study, from the reaction kinetics and the thermodynamic aspect, the Cpd I-ring of the P450 enzyme was compared with the path of each active site, and the regional selectivity in the reaction was further explained in the light of the previous study on the off-domain energy. The formation path of one of the main products of the reaction is analyzed, and the conclusion that the reaction is mainly in the low-spin double state is obtained. (3) The aromatic amine compound can be metabolically activated under the action of the Cpd I of the P450 enzyme to generate the positive cationic radical of the nitrogen, and is covalently bound to the nucleophile in the body to induce the cancer. In this study, the average activation energy base AE * of the hydrogen extraction reaction of the hydrogen in 28 of the 28 aromatic amine compounds, including the common aromatic amine and the nitrogen heterocyclic aromatic amine, was calculated by the DFT method, and the release energy (Bond Disassociation Energy) of the energy barrier E ** and the N-H bond was found. There is a good linear correlation between BDEN-H. The prediction model established by BDEN-H and AE * (simple aromatic amine: R2 = 0.92; N-heterocyclic aromatic amine: R2 = 0.95) has good robustness and prediction ability. This provides a basis for rapid and effective prediction of the metabolic activity of such compounds and even the potential carcinogenicity risk.
【学位授予单位】:浙江大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:X592

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