P450催化下几种不同类型亚硝胺羟基化代谢机理的理论研究

发布时间：2019-06-09 21:11

【摘要】：N-亚硝胺类化合物(NNCs)是一类重要的环境致癌物，其可以诱发动物包括人类在内的多种肿瘤。N-亚硝胺类化合物结构类型众多，致癌活性具有很大差异，且肿瘤发生部位也具有特异性。N-亚硝胺类化合物结构本身较为稳定，但进入体内经过细胞色素P450的代谢活化后，其代谢产物表现出明显的致癌活性。研究表明，N-亚硝胺的代谢途径主要是在细胞色素P450酶的催化下，首先发生α-羟基化反应后生成α-羟基烷基亚硝胺，接着α-羟基烷基亚硝胺分解后的产物与DNA结合，可以使DNA碱基烷基化，并最终导致肿瘤产生。N-亚硝胺代谢过程中的后半部分已经得到证实，而其羟基化反应过程则可能是其致癌特异性存在的主要原因。因此，本论文通过量子化学的方法对N-亚硝胺的羟基化反应进行了模拟，为N-亚硝胺类化合物致癌性的相关研究提供理论基础。一、在密度泛函理论下，对二甲基亚硝胺(NDMA)在细胞色素P450催化氧化活性中心Cpd I和Cpd0作用下的α-羟基化反应过程代谢机理进行了理论计算，结果表明，Cpd I作为催化活性中心具有优势：NDMA的羟基化反应过程中简并的高自旋态(HS)和低自旋态(LS)均参与反应。反应经过两个特征阶段，首先是NDMA中甲基中的H原子与Cpd I中氧原子通过氢键作用形成高低自旋态初始络合物，氢原子抽提并转移到Cpd I中与Fe相连的氧原子上，形成PorFe-OH与α-烷基自由基中间体络合物。紧接着高自旋态的中间体络合物发生反弹过程，自由基中间体反弹到羟基上，形成羟基化产物；而低自旋中间体不发生反弹过程，直接形成羟基化产物。其中，氢原子的抽提过程为此整个反应过程的控速步骤，反应能垒为17.56/17.46kcal/mol(HS/LS)，反应中没有呈现明显的自旋选择性，与烷烃羟基化反应过程类似，符合双态反应性(TSR)。该结果给出了NDMA在细胞色素P450作用下的羟基化反应过程机理，完善了NDMA的体内代谢过程，建立了亚硝胺类化合物羟基化反应代谢过程的机理模型，为后续相关实验提供了有力的理论支持。二、计算了甲基乙基亚硝胺(MENA)在细胞色素P450的两个催化氧化活性中心Cpd I和Cpd0作用下的羟基化反应过程代谢机理。结果表明，在催化甲基乙基亚硝胺代谢过程中，Cpd I显示出其优势，羟基化反应过程中的氢原子抽提过程为控速步骤。比较了顺反两种异构体两侧α-H的羟基化过程，其中顺式结构下长链端即乙基端C-H键的羟基化反应过程能垒最低，反应最易进行。甲基乙基的羟基化反应过程中没有明显的自旋选择性，，反应符合双态反应性特征。进一步研究了细胞色素P450的两个氧化活性中心Cpd I和Cpd0的催化氧化活性，证明，Cpd I可能是N-亚硝胺在体内代谢活化的催化剂。三、计算了甲基丁基亚硝胺(MBNA)在细胞色素P450的催化氧化活性中心Cpd I作用下的羟基化反应过程代谢机理。结果表明，反应过程具有两个特这阶段，首先是C-H键的活化，氢原子从MBNA中α-甲基上转移到铁卟啉中与Fe原子相连的氧原子上形成ForFe-OH与烷基中间体络合物。接着烷基自由基中间体络合物反弹到在羟基上形成羟基化产物。氢原子抽提过程是反应的限速步骤，反应符合双态反应性。顺式结构下长链端即丁基侧C-H键的羟基化反应过程能垒最低，是优势反应路径。本研究阐明了N-亚硝胺的体内羟基化代谢过程机理，建立了N-亚硝胺化合物的代谢模型，证明Cpd I是N-亚硝胺体内代谢的主要催化剂。同时也得出初步结论，即对于各种不同类型的亚硝胺的体内代谢过程，其长链端的C-H键羟基化反应过程最易进行，为后续相关研究提供了重要的理论基础。
[Abstract]:The N-nitrosamine compound (NNCs) is an important environmental carcinogen that can induce a variety of tumors, including humans. The type of N-nitrosamine compounds has a large structure, and the carcinogenic activity is very different, and the tumor-forming part is also specific. The structure of N-nitrosamine compounds is stable, but after the metabolic activation of cytochrome P450 in the body, its metabolites show significant carcinogenic activity. The research shows that the metabolic pathway of N-nitrosamine is mainly under the catalysis of cytochrome P450 enzyme, firstly, the p-hydroxyalkylnitrosamine is generated after the oxidation-hydroxylation reaction, and the product after the decomposition of the p-hydroxyalkylnitrosamine is combined with the DNA, so that the DNA base can be alkylated, And ultimately results in a tumor production. The latter part of the N-nitrosamine metabolic process has been confirmed, and the hydroxylation reaction process may be the main cause of its carcinogenic specificity. In this paper, the hydroxylation of N-nitrosamine was simulated by quantum chemical method, and the theoretical basis for the study of the carcinogenicity of N-nitrosamine compounds was provided. First, under the theory of density functional, the mechanism of the mechanism of the mechanism of the oxidative-hydroxylation of dimethylnitrosamine (NDMA) under the action of cytochrome P450 in the catalytic oxidation of the active sites of Cpd I and Cpd0 was calculated. The results showed that Cpd I was excellent as the center of catalytic activity. Potential: Both the high spin state (HS) and the low spin state (LS) of the degeneracy in the hydroxylation reaction of NDMA participate in the reaction. the reaction is carried out through two characteristic phases, firstly, the H atom in the methyl in the NDMA and the oxygen atom in the Cpd I form the initial complex of the high-low spin state by the action of hydrogen bonding, the hydrogen atom is extracted and transferred to the oxygen atom which is connected with the Fe in the Cpd I, And the low-spin intermediate does not undergo a rebound process, so that the hydroxylated product is directly formed. And the reaction energy base is 17.56/ 17.46 kcal/ mol (HS/ LS), no obvious spin selectivity is present in the reaction, and the reaction with the hydroxylation of the alkane is similar to that of the alkane hydroxylation reaction (TSR). The mechanism of the hydroxylation reaction of NDMA under the action of cytochrome P450 was given. The mechanism model of the hydroxylation reaction of nitrosamine compounds was established. The hydroxylation reaction process of methyl ethyl nitrosamine (MENA) under the action of Cpd I and Cpd0 in the two catalytic oxidation active sites of cytochrome P450 was calculated. The results show that, in the process of catalyzing the metabolism of methyl ethyl nitrosamine, Cpd I shows its advantages, and the extraction process of hydrogen atom in the process of hydroxylation is the control. the hydroxylation process of the two isomers of cis-H in the cis-structure is compared, wherein the hydroxylation reaction process of the long chain end of the cis structure, namely the ethyl end C-H bond, can be the lowest, and the reaction is the most It is easy to proceed. There is no apparent spin selectivity in the process of hydroxylation of methyl ethyl, and the reaction is in a two-state reaction. The catalytic oxidation activity of Cpd I and Cpd0 in the two oxidative sites of cytochrome P450 was further studied. It was shown that Cpd I might be N-nitrosamine in vivo. The hydroxylation of methylbutyl nitrosamine (MBNA) under the action of Cpd I in the catalytic oxidation of cytochrome P450 was calculated. The results show that the reaction process has two stages, the first is the activation of the C-H bond, the hydrogen atom is transferred from the methyl-methyl group of the MBNA to the oxygen atom which is connected with the Fe atom to form a ForFe-OH and an alkyl group, Interbody complex. The alkyl radical intermediate complex then bounces to form on the hydroxyl group The process of hydrogen atom extraction is the speed-limiting step of the reaction, and the reaction is in line with The reaction of the two-state reaction is the lowest in the process of the hydroxylation of the C-H bond at the long chain end of the cis structure, that is, the butyl-side C-H bond, which is excellent. The mechanism of the hydroxylation of N-nitrosamines in the body of N-nitrosamine was established, and the metabolic model of N-nitrosamine compounds was established. At the same time, a preliminary conclusion is also drawn that the C-H bond of the long chain end is most important for the in vivo metabolic process of various types of nitrosamines, which is provided for the follow-up studies.
【学位授予单位】：北京工业大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：O623.737;R114

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