23F血清型肺炎链球菌和艰难梭菌相关多糖抗原的寡糖类似物合成研究

发布时间：2018-01-19 00:28

本文关键词： 23F血清型肺炎链球菌艰难梭菌荚膜多糖脂磷壁酸化学合成　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：糖类化合物是地球上结构最为丰富的一类有机物质,广泛地分布于自然界各种动植物以及微生物中,与核酸、蛋白质一起并称为生命体三大基本物质。糖通常与蛋白质或脂类物质缀合,以糖蛋白或糖脂等糖缀合物的形式存在。糖类化合物作为生命体所必需的物质,在生物合成、细胞识别、细胞黏附等生命活动中发挥着重要的作用,与疾病的发生、发展关系密切。目前,临床上治疗细菌感染的方式主要是使用抗菌药物,特别是抗生素。但随着抗生素及抗菌药物的长期使用和不合理应用,细菌耐药性问题已变得日益突出。为寻找和发展新的治疗和预防方法,疫苗作为一种预防细菌感染的重要手段以及控制耐药菌扩散的重要措施之一,开始备受人们的重视。长期的研究发现,细菌细胞表面被一层糖质所覆盖,被称作细胞糖萼。糖类分子作为细菌表面结构的重要组成物质,在细菌的生存、生长、感染、致病及宿主免疫反应过程中都发挥着重要作用。同时,细菌细胞表面的糖类分子暴露在细胞表面,并具有高度的保守性和一定的免疫原性,因此成为了疫苗研制的理想靶标抗原。然而,糖类物质的免疫原性通常较弱,且不能刺激T细胞发生免疫应答,只能与B细胞作用诱导出较弱的免疫应答反应,且不具有免疫记忆效应。这些问题严重阻碍了以细菌表面多糖为靶标抗原的糖疫苗的研发。随着科学技术的发展和研究的深入,上述一些问题得到了有效的解决。例如,研究发现将多糖分子与具有较强免疫原性的蛋白进行共价链接所得到的糖缀合物,能刺激机体产生T细胞依赖的免疫反应,从而发挥良好的免疫效果。目前临床上使用的糖类疫苗多为多糖蛋白缀合物疫苗。目前用于制备糖缀合物疫苗的糖分子大多是从细菌的细胞提取获得的。其结构的微观不均一性和杂质的不确定性为糖类缀合物疫苗的构效关系研究、质量控制和安全性研究都带来了困难。此外,天然多糖往往需要通过化学降解方法活化之后,才能与载体分子结合,这进一步提高了用于制备糖缀合疫苗的糖分子的结构不均一性。为克服上述问题,化学合成法已成为一种获得糖抗原的有效途径。本论文对23F血清型肺炎链球菌荚膜多糖和艰难梭菌细胞表面磷酸酯多糖LTA重复单元及其衍生物进行了设计与合成,以便进一步研究这些多糖的结构-免疫学活性之间的关系,以利于寡糖缀合疫苗的研发。论文主要包括以下三部分内容:一、首先简要介绍了肺炎链球菌和艰难梭菌的流行病学研究、诊断、治疗和疫苗研发的现状,并对它们细胞表面多糖重复单元的具体化学结构以及其重复单元的衍生物的合成与性能研究工作进行了详细的综述。二、23F血清型肺炎链球菌荚膜多糖生物学重复单元的3-氨基丙基寡糖苷的化学合成。目标化合物SP-2由一个分支四糖和一个磷酸甘油侧链组成。首先,我们应用连续糖苷化法策略构建得到了重要四糖中间体SP-5,即:以SP-6作为受体,依次与单糖供体SP-7、SP-8和SP-9进行糖基化反应,从而构建得到四糖骨架。在四糖SP-5的合成过程中,分子内的β-鼠李糖苷键的构建是通过2-甲基萘基(NAP)参与的分子内糖苷配基转移反应来实现的;同时利用苯甲酰基在糖基化过程中的邻基参与作用来实现1,2-trans糖昔键的高效构键。接着,我们又应用亚磷酸三酯法将磷酸甘油侧链SP-4链接到四糖骨架SP-5上,得到全保护四糖SP-3。最后,脱除SP-3分子内的苄基和苄叉基,并将叠氮基还原为氨基,得到目标产物SP-2。目标化合物SP-2的还原端基上含有一个游离氨基,为后续基于SP-2糖缀合物制备及生物学研究提供了便利。三、艰难梭菌细胞表面磷酸酯多糖脂磷壁酸(LTA)衍生物的化学合成。基于艰难梭菌细胞表面磷酸酯多糖LTA的具体结构,我们设计了[2+3]和[2+2+3]的合成策略来高效地构建两个LTA目标衍生物,即LTA五糖CD-1和七糖CD-2。其中,重复单元二糖CD-5和CD-6中α-氨基葡萄糖苷键都是通过使用2-叠氮基取代的葡萄糖胺三氯乙酰亚胺酯作为供体与相应的受体反应得到;而在还原端三糖CD-7的合成中,分别尝试了连续糖苷化法和预活化一锅法来合成CD-7,其分子内的β-糖苷键是通过苯甲酰基的邻基参与作用实现的。目标分子中不同寡糖模块间的磷酸二酯键的构建是经由H-膦酸酯法实现的。同样,目标化合物CD-1和CD-2的还原端三糖末端含有一个游离氨基,为后续制备基于CD-1和CD-2的寡糖缀合物奠定了基础。
[Abstract]:Carbohydrate is the earth structure one of the most abundant organic matter, are widely distributed in nature all kinds of animals and plants and microorganisms, and nucleic acid, protein and called the three basic material life. Usually proteins or lipids and sugar conjugated to glycoproteins or glycolipids such glycoconjugates in the form of sugar. Compound as life necessary for the material, identification of cells in biosynthesis, cell adhesion plays an important role in life activities, and the incidence of the disease, closely related to the development. At present, the clinical treatment of bacterial infection is the main way of using antibacterial drugs, especially antibiotics. But with the long-term use of antibiotics and antibacterial drugs and the unreasonable application, the problem of bacterial resistance has become increasingly prominent. In order to find and develop new methods of prevention and treatment, as a vaccine to prevent bacterial infection is important One of the important measures and means of control of drug resistant bacteria spread, began to attract much attention. The study found that the bacterial cell surface was covered with a layer of sugar, called the cell glycocalyx. Carbohydrate molecules as an important component of material structure in the surface of bacteria, bacterial survival, growth, infection, pathogenic and host immune responses in the play an important role. At the same time, sugar molecules in bacterial cell surface exposed on the cell surface, and is highly conserved and immunogenicity, thus become the ideal target antigen for vaccine development. However, the immunogenicity of carbohydrates are often weak, and can not stimulate T cell immune response, only the role of B cells and induce the immune response is weak, and does not have immune memory effect. These problems seriously hindered by the bacterial surface polysaccharides as research target antigen of sugar vaccine. With the development of science and technology in depth, some of these problems have been solved. For example, the study found that the polysaccharide molecules and the covalent link with protein strong immunogenicity of glycoconjugates, can stimulate the immune T cell dependent response, which play a good immune effect. Carbohydrate vaccines used in clinical for polysaccharide protein conjugate vaccine. Currently used for sugar molecule preparation of glycoconjugate vaccines are mostly from the bacterial cells obtained by extraction. The structure of the microheterogeneity and impurities of uncertainty for the QSAR study of carbohydrate conjugates vaccine research, control and safety the quality is difficult. In addition, natural polysaccharides are often required by chemical degradation method after activation, and to carrier molecules, which further improves the preparation of glycoconjugate vaccine for sugar The structure of heterogeneity. To overcome the above problems, chemical synthesis has become an effective way to obtain the carbohydrate antigen. This thesis repeat unit of 23F serotypes of Streptococcus pneumoniae capsular polysaccharide and Clostridium difficile cell surface polysaccharides LTA phosphate and its derivatives were designed and synthesized, in order to further study the relationship between the the structure of the sugar - immunological activity, in order to facilitate the development of oligosaccharide conjugate vaccines. The paper includes the following three parts: first, briefly introduces the epidemiology of Streptococcus pneumoniae and Clostridium difficile, diagnosis, treatment and vaccine development status, and the specific chemical structure on their cell surface polysaccharides and its repeating unit the repeat unit of derivatives: synthesis and properties research were reviewed in detail. Two, 3- amino 23F serotypes of Streptococcus pneumoniae capsular polysaccharide biological repeating unit The chemical synthesis of propyl oligoglycosides. Target compounds SP-2 by a branch of the four sugar and a glycerol phosphate side chain. Firstly, we use continuous glycosidation strategy constructed four important sugar intermediates SP-5, namely: to SP-6 as a receptor, in turn with the monosaccharide donor SP-7, SP-8 and SP-9 glycosylation thus, to construct the four sugar skeleton. In the synthesis of four sugar SP-5, construct the intramolecular beta key rhamnoside is through 2- (NAP) methyl naphthyl participate in intramolecular aglycon transfer reaction to realize; at the same time using benzoyl in glycosylation in the process of neighboring group participation the key to achieve efficient 1,2-trans glycoside bond. Then, we also used three phosphite ester method, glycerol phosphate side chain link to SP-4 to four SP-5 sugar backbone, get full protection four sugar SP-3. finally, the removal of SP-3 in molecule and benzyl benzylidene, and azide Reduced to amino terminal reduction to obtain the target product SP-2. target compound SP-2 containing a free amino group, for the subsequent preparation and biological research of SP-2 system based on glycoconjugates provides convenience. Three, Clostridium difficile cell surface polysaccharide lipid phosphate teichoic acid (LTA) synthesis of derivatives. The specific structure of Clostridium difficile cell surface phosphate polysaccharide based on LTA, we designed [2+3] and [2+2+3] synthesis strategy to efficiently construct two LTA derivatives, namely LTA five CD-1 and seven CD-2. sugar sugar, sugar and two CD-5 repeat units of CD-6 alpha amino glucoside bond is through the use of 2- stack nitrogen substituted three glucosamine chloride ethylidenimide ester as the donor and the corresponding reaction; and in the synthesis of reduced sugar CD-7 in the end of three, we tried to synthesize CD-7 by one pot method and pre activation continuous glycosylation, the intramolecular beta glycosidic bond The neighboring group participation to achieve the role of benzoyl. Target molecules construct different oligosaccharides between modules two phosphate ester bond is achieved via H- phosphonate method. Similarly, reducing end target compounds CD-1 and CD-2 three at the end of a sugar containing free amino groups, for the subsequent preparation of oligosaccharide conjugates and CD-1 based on the CD-2 Foundation.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R392;O629.1

【参考文献】