【作者】 刘友涛；

【导师】 王高学；

【作者基本信息】 西北农林科技大学 ， 化学生物学， 2011， 硕士

【摘要】 随着我国水产养殖业的不断的扩大和快速的发展,由细菌、病毒、寄生虫引起的疾病已经成为制约我国水产养殖业健康发展的瓶颈之一。目前,鱼类病害防治仍然以抗生素,化学治疗剂等化学药物为主,但是由于化学药物的诸多负面影响,例如药物在组织内的残留,病原体耐药性的提高以及环境污染等一系列问题,许多传统药物已经被禁止在水产养殖中使用。免疫增强剂可以提高动物机体的免疫功能,提高动物的防病抗病能力,其来源包括了动物、植物、微生物。近年来,随着微生物代谢产物中具有免疫增强活性的物质的发现,使得以微生物源的活性物质为先导,开发新型免疫增强剂成为了渔药创制的有效途径。本研究以此为出发点,参照具有免疫增强活性的天然活性物质环(L-脯氨酸-甘氨酸),以L-脯氨酸,甘氨酸,L-苯丙氨酸等氨基酸为原料,人工合成了一些列2,5-二酮哌嗪类化合物,通过对草鱼肾脏细胞CIK的免疫活性测定,分析其潜在的受体及信号通路,为开发微生物源的免疫增强剂提供基础资料。1.以L-脯氨酸,甘氨酸,L-缬氨酸,L-苯丙氨酸,L-丙氨酸为原料,采用固相合成的方法通过对氨基的保护(Fmoc)、与Wang树脂的缩合、脱氨基保护基、氨基酸的缩合、树脂的洗脱及环化等步骤合成了环(L-脯氨酸-甘氨酸),环(甘氨酸-甘氨酸),环(L-缬氨酸-甘氨酸),环(L-丙氨酸-甘氨酸),环(L-苯丙氨酸-甘氨酸)及环(L-脯氨酸-L-脯氨酸)等六种2,5-二酮哌嗪类化合物。所有产物均经波谱学鉴定。2.以L-苯丙氨酸,L-亮氨酸,L-丙氨酸,L-脯氨酸,L-缬氨酸为原料,采用液相合成的方法通过对氨基的保护(Fmoc)、羧基的保护(甲酯化)、缩合、脱氨基保护基、在甲醇中回流成环等步骤合成了环(L-苯丙氨酸-L-缬氨酸),环(L-缬氨酸-L-亮氨酸),环(L-脯氨酸-L-脯氨酸),环(L-苯丙氨酸-L-丙氨酸),环(L-苯丙氨酸-L-苯丙氨酸)和环(L-苯丙氨酸-L-亮氨酸)等六种2,5-二酮哌嗪类化合物。所有产物均经波谱学鉴定。3.实时荧光定量PCR表明以环(L-脯氨酸-甘氨酸)为代表的2,5-二酮哌嗪类化合物在细胞内的潜在受体为TLR2,并确定了一条信号通路,通过此通路,机体可在下游表达出抗炎症细胞因子,能起到提高机体的非特异性免疫能力的作用,也从侧面反应出了2,5-二酮哌嗪类化合物具有免疫增强活性。更多还原

【Abstract】 As aquaculture activities of our country intensify and expand, the disease problems caused by viruses, bacteria, parasites and other undiagnosed and emerging pathogens has become a primary constraint to the culture of many aquatic species. Traditionally, antibiotics have been generally adopted to treat diseases of aquatic animals. But these are no longer recommended practices due to the accumulation of drugs in tissues, development of drug resistance and enviromental pollution. Immunostimulants can improve the immune system of animals body and also enhance the ability to prevent diseases. The resorce of the immunostimulants includs aimal, plant and microorganisms. At present as the discovery of bioactive compounds from secondary metabolites of microorganisms, it is feasible to develop new immunostimulants aquaculture which leaded by the active compound from microorganisms. In the previous study, cyclo-(L-Pro-Gly) which come from the secondary metabolites of Anoxybacillus kamchatkensis XA-1 was determined to have the immunostimulatory activities to carp. So, in the present study, we synthesis the several diketopiperazines with the raw material of L-Pro, Gly, L-Phe et al. and evaluate their immunostimulatory activities on CIK cells to develop potential medicines for the control of fish disease.In this work, six diketopiperazines (cyclo-(L-Pro-Gly), cyclo-(Gly-Gly), cyclo-(L-Val-Gly), cyclo-(L-Ala-Gly), cyclo-(L-Phe-Gly) and cyclo-(L-Pro-L-Pro)) were synthesis through the solid-phase synthesis by the protection of amino group (Fmoc), the protection of carboxy (methyl esterification), condensation with Wang resin, deprotection of of amino group, condensation with amino acid, elution off the resin and cyclization with the raw material of L-Pro, Gly, L-Val, L-Ala and L-Phe. All the compounds were identified by 1H NMR and 13C NMR.At tha same time, six diketopiperazines (cyclo-(L-Phe-L-Val), cyclo-(L-Val-L-Leu), cyclo-(L-Pro-L-Pro), cyclo-(L-Phe-L-Ala), cyclo-(L-Phe-L-Phe), cyclo-(L-Phe-L-Leu) through the liquid-phase synthesis by the protection of amino group (Fmoc), the protection of carboxy (methyl esterification), condensation with amino acid, deprotection of of amino group and cyclization in methanol with the raw material of L-Pro, L-Val, L-Ala, L-Leu and L-Phe. All the compounds were identified by 1H NMR and 13C NMR.Real-time qPCR indicated that the potential receptor inside the cell of diketopiperazines represented by cyclo-(L-Pro-Gly) is Toll-like receptor 4 and identify a signaling pathway by which the body can express the cytokines in the downstream to improve the non-specific immune system and it also reflected that diketopiperazines can produce the immunostimulatory activities.更多还原