【作者】 解先业；

【导师】 姜林；

【作者基本信息】 山东农业大学 ， 应用化学， 2008， 硕士

【摘要】 酚氧化酶(phenoloxidase, PO)(EC 1.14.18.1)又称酪氨酸酶,是结构复杂的多亚基的含铜氧化还原酶。它广泛地存在于动物、植物、微生物、昆虫及人体内。酚氧化酶在昆虫的变态发育和免疫系统中起着重要作用,在昆虫变态发育过程中,它先将L-酪氨酸羟基化,产生邻二羟基苯丙氨酸(L-多巴),然后再将L-多巴氧化成多巴醌,醌与蛋白质结合导致硬化。N-乙酰多巴胺在酚氧化酶作用下形成醌的衍生物,该衍生物再脱乙酰或羧基后,非酶促缩合成为一个吲哚结构,该吲哚结构又以多种方式聚合形成黑色素。酚氧化酶是昆虫体内的一种重要酶类,在昆虫的正常发育过程中具有重要的生理功能:(1)参与表皮的硬化和黑化;(2)对卵壳的鞣化作用;(3)参与伤害防御;(4)加速伤口的愈合。以酚氧化酶为作用靶标,寻找超高效、低毒、杀虫谱广的目标化合物,已经成为化学和农药界研究的热点领域之一。研究发现苯甲醛类、苯甲酸类、槲皮素、芦丁、曲酸、黄酮类等天然源化合物中大部分能与酚氧化酶活性中心的铜离子螯合使该酶活性降低。鉴于铜离子是酚氧化酶生理功能的重要因子,向该酶中加入与铜离子具有强螯合能力的配体,使之与铜离子螯合来降低酚氧化酶的活性。本文合成了多个系列的酚氧化酶抑制剂并测试了其活性,主要研究内容和结果如下。1.合成了21种取代苯甲醛缩氨基硫脲、21种取代水杨醛席夫碱、15种α-巯基-β-芳基丙烯酸等3个系列的57种目标化合物,其中新化合物37种。它们的结构经红外光谱、核磁共振氢谱及元素分析等进行了确证。并对目标化合物的理化性质及波谱性质进行了系统的分析和讨论。2.对菜青虫5龄幼虫体内的酚氧化酶进行了提取及初纯化,研究了目标化合物对该酶的抑制活性。初步的生物活性测定结果表明:这3个系列目标化合物都表现出良好的抑制活性。其中取代苯甲醛缩氨基硫脲类化合物的抑制活性最好,IC50在0.26 ~ 100μmol·L-1范围内;α-巯基-β-芳基丙烯酸类化合物的抑制活性IC50在0.37 ~ 6.30 mmol·L-1范围内;取代水杨醛席夫碱部分化合物抑制活性相对较好,在0.10 mmol·L-1和0.20 mmol·L-1下,化合物IIn与IIo可使该酶的活性降低55% ~ 70%以上。本文还对3个系列目标化合物的结构与活性的关系进行了初步的分析和讨论,不仅为进一步设计新的高生物活性、低毒、副作用小的酚氧化酶抑制剂提供了一定的参考依据,并且部分目标化合物可望在医疗和农用杀虫剂领域具有应用价值。更多还原

【Abstract】 Phenoloxidase(PO, EC.1.14.18.1), also known as tyrosinase, is a copper-containing enzyme that widely distributed in plants, microorganisms animals and human. The enzyme possesses an important function in meta- morphism developing and immunity system. This multifunctional enzyme catalyzes two distinct reactions, the hydroxylation of monophenol to o-diphenol(monophenolase activity) and the conversion of o-diphenol to the corresponding o-quinone(diphenolase activity). Further, it is involved in formation of pigments such as melanin. In insects, phenoloxidase is also a widely distributed enzyme playing important roles in normal developmental processes, such as cuticular tanning, scleration, wound healing, production of opsonins, encapsulation and nodule formation for defense against foreign pathogens. This is a useful basis for designing effective, selective, and environment-friendly PO inhibitors.In previous studies, we investigated the inhibition of compounds such as substituted benzaldehydes, substituted benzoic acids, kojic acid, apigenin, quercetin, rutin and flavonoids against PO activity. Most of them inhibit the PO by chelating with Cu(II) ions. Two Cu(II) ions are defined in the active site of the PO. The contents and results were summarized as follows:1. Three series of 57 compouds containing 21 substituted benzaldehyde thiosemicarbazones, 21 substituted salicyl schiff bases and 15α-mercapto-β-aryl acrylic acids were synthesized, including 37 new compounds, which were characterized by IR, 1HNMR and elemental analysis. Synthesis condition, physicochemical properties, and spectrum properties of the compounds were reported.2. PO was extracted and primary purified from the 5th instar Pieris rapae(L.), then, target compounds against the phenoloxidase were tested. Primary results showed that the compounds displayed good inhibitory activity. The inhibitory effects on the PO activity by substituted benzaldehyde thiosemicarbazones andα-mercapto-β-aryl acrylic acids were determined, the IC50 were 0.26 ~ 100μmol·L-1 and 0.37 ~ 6.30 mmol·L-1, respectively. At the concentration of 0.10 mmol·L-1 and 0.20 mmol·L-1 of substituted salicyl schiff bases, inhibitory activity of the IIn and IIo were 55% ~ 70%.At the same time, the relationships of the activity and structure of three series target compounds were analyzed and discussed. These not only supported some reffences to more design on new high biological activity and low toxicity inhibitors of the PO, but also some of the compounds possibly have the practical application in the fields of medical treatments and agriculturel insecticides.更多还原