RGD 三肽的高效合成及其缀合物的制备

保密级别：内部 学位级别：理学 论文提交日期：五月三十日 论文答辩日期：六月九日　论文题目：RGD三肽的高效合成及其缀合物的制备　Title: An Efficient Synthesis Method of RGDTripeptide and Studies on its Conjugation作者： 张严冬 所在单位：分子酶学工程实验室　指导教师：张学忠 所在单位：分子酶学工程实验室　分类标识：Q55 关键词：RGD，整合素，抗癌细胞转移　Keywords: RGD, integrin , anti-metastasis总页数：六十二页 开本：十六开 有图表 摘　要　 RGD三肽（精氨酰甘氨酰天冬氨酸）序列是细胞外基质及血液中的许多粘附蛋白共有的细胞识别位点。

早在1984年，pierschbacher　和Ruoslahti首次证明了它的细胞粘附作用。

由RGD介导的粘附蛋白与受体的相互作用构成了一个广泛多功能的识别体系作为细胞附着、转移及分化生长的信号。

这些粘附蛋白包括纤粘连蛋白、玻连蛋白、骨桥蛋白、胶原蛋白、血小板反应蛋白、血纤蛋白原及V on Willebrand　因子等。

它们参与了人体许多重要的病理生理过程如血栓生成、肿瘤转移、炎症发生、急性肾衰及骨质疏松等。

近些年来，对RGD及其衍生物的研究成为一个热点，就是因为发现含RGD的肽及蛋白质在许多病理学过程中具有医疗价值。

它们可以竞争结合于靶部位，阻断由粘附蛋白介导的一些病理过程的发生，缓解一些疾病的出现。

比如，抗癌细胞转移、抗血栓、治疗骨质疏松、抗急性肾衰及在烧伤病人的术后痊愈等中都能发挥重要的作用，具有极大的医药开发潜力。

　正是基于RGD肽类的这种有着极大开发价值的考虑，我们设计了一种高效率、低成本的肽合成方法便于它的应用。

目前对于多肽合成的方法，主要有化学合成、酶促合成及重组DNA技术。

总体来说，都是需要大量投入的，成本也很高。

考虑到RGD肽这种特殊的氨基酸种类和次序。

我们试图找到一种最适合于生产的低成本合成路线。

现在所设计的这种方法就是针对RGD肽这种特殊的序列而设计的一种特殊方法。

　首先用氯乙酰氯同天冬氨酸在弱碱下反应生成氯乙酰化的天冬氨酸，再将这一产物用浓氨水进行氨代，即可生成甘氨酸－天冬氨酸二肽。

三肽的合成采用N－羧基内酸酐法。

首先将CBZ－精氨酸用催化剂使其生成Ｎ－羧基内酸酐－精氨酸。

这种NCA-精氨酸可以在碱性条件下与游离GD二肽发生缩合，再将其酸化即可生成三肽。

　 这种合成方法不需要进行氨基酸的保护，可直接合成出RGD游离三肽。

相对于其他方法而言，具有简便、快速、便宜的特点，而且产率也较高，是　24.1%。

很适合于工业的开发，并可作为专利开发的项目。

　 Sigma产品目录中10毫克RGD的售价为51.50$。

而采用我们的合成路线，采用一些廉价的原料，辅以适当的分离手段，就可制备大量的RGD游离三肽。

所以这一肽的合成路线很适合于工业开发，并可作为专利开发的项目。

同时，这一路线也可作为进一步合成含RGD的四肽及五肽的基础工作。

这是本论文的第一部分工作。

除此之外，我们还对二肽及三肽的分离纯化进行了探讨，建立了用Sephadex G-10、Amberlite　IR120　阳离子交换树脂相结合的一套分离路线，并对各种分离条件进行了摸索。

用离子交换树脂进行阶段洗脱确定了二肽及三肽的阶段洗脱的pH值，二肽是pH 3-4之间，三肽是pH7-8之间。

进行阶段洗脱的二肽及三肽可达HPLC分析单峰的纯度，而后再用Sephadex　G-10除盐。

我的论文后期工作是对RGD脂质体载药进行了初步的探讨。

因为单独的RGD　很快被从体内清除，而若将其连于载体则可延长其体内的存留时间。

以从卵黄中提取粗卵磷脂为原料，加入适量胆固醇以提高脂质体的稳定性，以及维生素E作为抗氧化剂。

采用了三种方法制备脂质体，即薄膜法、反相蒸发法及摇床法。

对这三种　脂质体的显微结构，包封率及稳定性进行了探讨。

发现反相蒸发法具有包封率高，稳定等特点。

用电子显微镜观察其显微结构，其直径为100nm、350nm、750nm不等，但多为几百纳米，属于较大的脂质体。

一般还应将其通过微孔滤膜使其均一化。

反相蒸发法的包封率为 41.2%左右　。

　 总之，我的实验建立了一套RGD三肽的低成本合成路线，及其相应的检测方法，并对其脂质体载药作了初步尝试，便于以后将其应用于生理实验，并进行药物的开发。

ABSTRACTThe tripeptide Arginyl-Glycyl-Aspartic acid (RGD) is a common cell recognition site of many adhesive proteins contained in extracellular matrices and in the blood . As early as 1904, Pierschbacher and Ruoslahti firstly proved its cell adhesive function. RGD-containing adhesive proteins include fibronectin, vitronectin, osteopontin, collagens, thrombospondin, fibrigogen and von Willebrand factor. These proteins have important function in pathology and physiology, such as thrombosis, metastasis, inflammation, acute renal failure a nd osteoporosis. The adhesion proteins and their receptors constitute a versatile recognition system providing cells with anchorage, traction for migration and signals for polarity, position, differentiation and growth. In recent years, researchon RGD and its derivatives has become a hot spot, mainly because RGD-containing peptides and proteins have therapeutic value in pathology, they can bind competitively to target sites, block the adhesive proteins-induced generation of some diseases, and can alleviate some diseases as well. For instance, they have been found to be useful in anti-metastasis, anti-thrombosis, and treating osteoporosis, they also have therapeutic value in acute renal failure and in the recovery from burn after operation. Taking consideration in all of the above medical values, we need to design an efficient and low-cost peptide synthesis route for its production and application. As we know, there are a few methods to synthesize peptides, including chemical synthesis , enzyme-catalyzed synthesis and recombinant DNA technology. Although there is some difference between them, the cost are all not low, they all need large amount of research or money. We now try another way, according to the specialty of its amino acid composition and its sequence, we succeed in developing a low-cost route for batch production. It is a special one for the special tripeptide.At first, we make aspartic acid react with cholracetyl chloride in weakly basic aqueous solution to get the intermediate chloracetyl aspartic acid. Then ammoniate it with dense ammonium solution to obtain the dipeptide glycine-aspartic acid(GD). To synthesize the whole tripeptide, we choose N-carboxyanhydrides(NCA) method. In this reaction, we first use catalyst to change CBZ-arginine to NCA-arginine. Secondly, condense it with free GD dipeptide in basic aqueous solution, after acidification, we can obtain the final product, free RGDtripeptide.The exceptional point in this route is that we don’t need any protected amino acids, and we get directly free RGD tripeptide as well. Compared to other methods, this route is much simpler, quicker and cheaper, besides, the final yield is fairly well. Concluding from above steps, we find that the overall yield reach 24.1%.Searching the product index of Sigma, we find that the price for 10 mg RGD is 51.50 dollars, If one use our route, he only need some cheap materials such as aspartic acid, chloracetyl cholride, and a comparably more expensive material, CBZ-Arg, and some other commonly –used reagent, plus a proper separation and purification method. They can get fairly amount of RGD tripeptide. Therefore, it is very proper for industry application and to be developed as a patent. At the same time, on the basis of our work, we can further synthesize four-amino acid- peptide, and even five –amino acid-peptide.This is one of the main parts of my work, in addition, I also study the separation and purification method of the dipeptide and tripeptide and set up a systematic separation method by combination of the column SephadexG-10 and cation exchange resin. Through step-elution, I determine the pH scope for GD and RGD separately, with GD pH 3 to 4, with RGD pH 7to 8. In HPLC analysis they are both unique leak except the salt leaks. After this step, I further purify them with SephadexG-10 to get rid of salt. Although there is some absorption when using G-10, this method can be seen as a preliminary work.The other part of my work is initially research on how toencapsulate RGD into liposome. Because RGD itself can be clear off very quickly in vivo. Only if they combine with other materials, can they stay longer. To make the liposome more stable. I add proper amount of cholesterol as additive to proveits stability and vitamin E as antioxidant. I try three methods, including thin film method, reverse-phase evaporation(REV)and the swing bed method. We study the product from its microscopic structure、stability and yield. As a result, we findthat REV method is the best. Because it is more stable and hasa higher yield. We watch its microscopic structure with electron microscopic to determine its diameter. They are mainly a few hundred nanometers.On the whole, I set up a low-cost synthetic route for RGD tripeptide and the corresponding separation and assay method.At last, I study initially about its liposome to make basis for its later application in physiological experiment and to be further developed as medicine.提　要　　在细胞外基质及血液中的许多粘附蛋白都含有RGD（精氨酰甘氨酰天冬氨酸）三肽序列作为它们的细胞识别位点。