邻酰氨基苯甲酰肼（胺）类化合物的合成及其同大分子蛋白的相互亲和作用研究

发布时间：2018-06-07 06:23

本文选题：邻酰氨基苯甲酰肼类化合物 + 合成　；参考：《中国农业大学》2016年博士论文

【摘要】：作用于昆虫鱼尼丁受体的邻甲酰氨基苯甲酰胺类化合物氯虫苯甲酰胺、溴氰虫酰胺是具有作用机理独特、低毒高效、与环境友好的新型高效杀虫剂。随着近年的大面积使用,田间害虫对氯虫苯甲酰胺、溴氰虫酰胺的抗性逐渐显现,因此发现作用于鱼尼丁受体的新化合物是解决害虫对该两个化合物抗性的方法之一。昆虫鱼尼丁受体是由4个分子量为560 Kd亚基构成的大分子跨膜蛋白,难以获得其三维空间结构,限制了作用于鱼尼丁受体同活性化合物之间的相互作用研究。因此,建立合适的方法和技术研究活性化合物同昆虫鱼尼丁受体的相互亲和作用,也是目前杀虫剂活性小分子发现研究的热点之一1、本论文通过活性亚结构拼接的方法将酰肼结构引入邻甲酰氨基苯甲酰胺结构中,同时在母体苯环上引入碘原子和氰基,设计合成了系列邻酰氨基苯甲酰肼类化合物,所有化合物的结构通过了核磁共振氢谱的确认。初步的生物活性研究,发现部分化合物对粘虫具有一定的致死活性,其中化合物cau-Ikc1、cau-lkc2和cau-lkc5(如图1)在100 mg门L的浓度下对粘虫的致死率达到100%。2、通过匀浆、离心等方法提取了美洲大蠊腿部含鱼尼丁受体的膜蛋白,并通过Bradford法对膜蛋白提取物中的鱼尼丁受体蛋白进行了确认和含量的测定,结果显示美洲大蠊提取物中鱼尼丁受含量为69 μg/mL。3、利用已有的荧光配体,基于荧光偏振技术建立邻甲酰氨基苯甲酰胺化合物同昆虫肌肉中鱼尼丁受体膜蛋白的亲和作用研究。结果显示(如图2)氯虫苯甲酰胺和溴氰虫酰胺均可竞争性抑制氯虫苯甲酰胺荧光配体和昆虫鱼尼丁受体的结合,氯虫苯甲酰胺和溴氰虫酰胺抑制荧光配体和昆虫鱼尼丁受体结合的IC50分别为135.65 nM和146.36 nM；而结构骨架不同的氟苯虫酰胺则不能抑制氯虫苯甲酰胺荧光配体和昆虫鱼尼丁受体的结合。该结果说明氯虫苯甲酰胺和溴氰虫酰胺作用于昆虫鱼尼丁受体的同一位点,而氟苯虫酰胺作用于昆虫鱼尼丁受体的其他位点。同时,建立的邻甲酰氨基苯甲酰胺化合物同昆虫肌肉鱼尼丁受体相互亲和作用方法的Z’因子达到0.76,说明该方法适用于高通量筛选作用于昆虫鱼尼丁受体邻甲酰氨基苯甲酰胺(氯虫苯甲酰胺和溴氰虫酰胺)结合位点的活性化合物,对作用于鱼尼丁受体高效新化合物的创制具有重要意义。4、根据半抗原的特性要求,利用氨基丁酸取代氯虫苯甲酰胺和溴氰虫酰胺母体结构中的甲基氨基,设计并合成了含氯虫苯甲酰胺结构和溴氰虫酰胺结构的半抗原(如图3)。然后经过免疫抗原的制备、小鼠免疫、抗体评价等成功制备了氯虫苯甲酰胺抗单克隆抗体1D2和溴氰虫酰胺单克隆抗体3B2.5、利用ELISA技术建立了研究双酰胺类化合物同氯虫苯甲酰胺抗体和溴氰虫酰胺抗体亲和作用的方法,进一步研究抗体模拟鱼尼丁受体的结合特性。测试结果显示,氯虫苯甲酰胺间接竞争ELISA的IC50为1.60 ng/mL溴氰虫酰胺间接竞争ELISA的1C50为1.57mg/mL活性化合物同抗体的亲和作用结果表明,抗体和受体蛋白一样能够识别性结合半抗原中的母体结构,但是具有很强的特异性,对于其它类型的化合物也有一定的识别。该研究对促进研究昆虫鱼尼丁受体和活性化合物的亲和作用研究具有一定意义。6、基于溴氰虫酰胺单克隆抗体的特异性,利用ELISA技术建立了溴氰虫酰胺icELISA检测方法。该方法的IC50为1.57 ng/mL,检测范围为0.43-6.15 ng/mL(如图4),检测溴氰虫酰胺在小白菜和自来水中的添加回收率分别为93.7-101.0%和94.4%-101.6%。建立的间接竟孝ELISA方法可以对小白菜样品和水样中的溴氰虫酰胺残留进行快速准确测定。
[Abstract]:It is a new type of high efficient insecticide, which has a unique mechanism, low toxicity, high efficiency and environmentally friendly insecticides. With the large area use in recent years, the resistance of field pests to chloroacetamide and bromo cyanamide gradually appears. The new compound which is currently acting on the receptor is one of the methods to solve the resistance of the insect to the two compounds. The insect fish neidine receptor is a large molecular transmembrane protein composed of 4 molecular weights of 560 Kd subunits. It is difficult to obtain its three-dimensional spatial structure and restricts the interaction between the active compounds and the active compounds of the fish. Therefore, the establishment of appropriate methods and techniques to study the interaction of active compounds with the insect fish neidin receptor is also one of the hot spots in the discovery of insecticide active small molecules. In this paper, the structure of hydrazine was introduced into the structure of anoylbenzamidoamide by the method of substructure splicing by the active substructure, and at the same time in the mother benzene ring. A series of ortho amyl benzoyl hydrazine compounds were designed and synthesized by the introduction of iodine atoms and cyanogen groups. The structure of all the compounds was confirmed by the nuclear magnetic resonance spectrum. Preliminary biological activity studies found that some compounds have certain lethal activity to the armyworm, of which compounds cau-Ikc1, cau-lkc2 and cau-lkc5 (such as figure 1) are in the 100 mg gate L The membrane protein of the fish netin receptor in the leg of Periplaneta americana was extracted by homogenate and centrifugation by homogenate and centrifugation. The protein of the receptor protein in the membrane protein extract was confirmed and determined by Bradford method. The results showed that the content of the fish was 69 u g/mL.3 in the extract of Periplaneta americana. The affinity of phthalamidbenzamide compounds with the membrane proteins in the insect muscles was established on the basis of fluorescence polarization, and the results showed that (as shown in Figure 2) chloroacetamide and brominamide can inhibit the binding of chloranaclamide fluorescence ligand and insect nidin receptor. The combination of benzamide and cyanamide of chloroacetamide and brominamide inhibited the binding of IC50 to 135.65 nM and 146.36 nM, respectively, while the different structural skeletal fluoroacetamide could not inhibit the binding of the benzamide fluorescent ligand and the insect neidin receptor. It acts on the same site of the insect fish nidin receptor, while fluoranamide acts on other loci of the insect fish nidin receptor. At the same time, the Z 'factor of the mutual affinity between the phthalamidamide compound and the insect muscle fish nidin receptor is 0.76, indicating that this method is suitable for high throughput screening for insect fish. The active compounds of the binding site of the nidin receptor, phthalamidamidamide (benzamidamide and brominacamide), have important significance for the creation of a new highly efficient compound of the fish nidin receptor,.4, which is based on the characteristics of the hapten, and uses aminobutyric acid to replace the methyl chloroacetamide and the methyl bromide matrix in the structure of the methyl group. Amino, design and synthesis of the benzamide structure of chloroinsects and the hapten of the structure of bromocyanamide (Figure 3). After the preparation of immune antigen, immunization, and antibody evaluation, the monoclonal antibody 1D2 of chloroacetanilide and the monoclonal anti body 3B2.5 of bromocyanamide were prepared, and the study of diamides was established by ELISA Technology. The affinity of the compound with benzamide and brominamide antibodies was further studied. The results showed that the IC50 of the indirect competitive ELISA of chloroacetamide was 1.60 ng/mL, the 1C50 of the indirect competitive ELISA of the cyanamide amide was the affinity of the 1.57mg/mL active compound with the antibody. The results show that the antibody and the receptor protein can identify the parent structure of the hapten, but it has a strong specificity and has a certain recognition for other types of compounds. The study has a certain significance in promoting the study of the affinity of the research insect fish neidine receptor and active compounds, based on.6. The specificity of amido monoclonal antibody was established by ELISA technology. The IC50 of the method was 1.57 ng/mL and the detection range was 0.43-6.15 ng/mL (Figure 4). The recovery rate of bromo cyanamide in pakchoi and tap water was detected by 93.7-101.0% and 94.4%-101.6%., respectively. It can be used for rapid and accurate determination of residues of bromincyanamide in Chinese cabbage samples and water samples.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ453

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