本文關(guān)鍵詞： 23F血清型肺炎鏈球菌 艱難梭菌 莢膜多糖 脂磷壁酸 化學(xué)合成　出處：《山東大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：糖類化合物是地球上結(jié)構(gòu)最為豐富的一類有機(jī)物質(zhì),廣泛地分布于自然界各種動(dòng)植物以及微生物中,與核酸、蛋白質(zhì)一起并稱為生命體三大基本物質(zhì)。糖通常與蛋白質(zhì)或脂類物質(zhì)綴合,以糖蛋白或糖脂等糖綴合物的形式存在。糖類化合物作為生命體所必需的物質(zhì),在生物合成、細(xì)胞識(shí)別、細(xì)胞黏附等生命活動(dòng)中發(fā)揮著重要的作用,與疾病的發(fā)生、發(fā)展關(guān)系密切。目前,臨床上治療細(xì)菌感染的方式主要是使用抗菌藥物,特別是抗生素。但隨著抗生素及抗菌藥物的長(zhǎng)期使用和不合理應(yīng)用,細(xì)菌耐藥性問(wèn)題已變得日益突出。為尋找和發(fā)展新的治療和預(yù)防方法,疫苗作為一種預(yù)防細(xì)菌感染的重要手段以及控制耐藥菌擴(kuò)散的重要措施之一,開始備受人們的重視。長(zhǎng)期的研究發(fā)現(xiàn),細(xì)菌細(xì)胞表面被一層糖質(zhì)所覆蓋,被稱作細(xì)胞糖萼。糖類分子作為細(xì)菌表面結(jié)構(gòu)的重要組成物質(zhì),在細(xì)菌的生存、生長(zhǎng)、感染、致病及宿主免疫反應(yīng)過(guò)程中都發(fā)揮著重要作用。同時(shí),細(xì)菌細(xì)胞表面的糖類分子暴露在細(xì)胞表面,并具有高度的保守性和一定的免疫原性,因此成為了疫苗研制的理想靶標(biāo)抗原。然而,糖類物質(zhì)的免疫原性通常較弱,且不能刺激T細(xì)胞發(fā)生免疫應(yīng)答,只能與B細(xì)胞作用誘導(dǎo)出較弱的免疫應(yīng)答反應(yīng),且不具有免疫記憶效應(yīng)。這些問(wèn)題嚴(yán)重阻礙了以細(xì)菌表面多糖為靶標(biāo)抗原的糖疫苗的研發(fā)。隨著科學(xué)技術(shù)的發(fā)展和研究的深入,上述一些問(wèn)題得到了有效的解決。例如,研究發(fā)現(xiàn)將多糖分子與具有較強(qiáng)免疫原性的蛋白進(jìn)行共價(jià)鏈接所得到的糖綴合物,能刺激機(jī)體產(chǎn)生T細(xì)胞依賴的免疫反應(yīng),從而發(fā)揮良好的免疫效果。目前臨床上使用的糖類疫苗多為多糖蛋白綴合物疫苗。目前用于制備糖綴合物疫苗的糖分子大多是從細(xì)菌的細(xì)胞提取獲得的。其結(jié)構(gòu)的微觀不均一性和雜質(zhì)的不確定性為糖類綴合物疫苗的構(gòu)效關(guān)系研究、質(zhì)量控制和安全性研究都帶來(lái)了困難。此外,天然多糖往往需要通過(guò)化學(xué)降解方法活化之后,才能與載體分子結(jié)合,這進(jìn)一步提高了用于制備糖綴合疫苗的糖分子的結(jié)構(gòu)不均一性。為克服上述問(wèn)題,化學(xué)合成法已成為一種獲得糖抗原的有效途徑。本論文對(duì)23F血清型肺炎鏈球菌莢膜多糖和艱難梭菌細(xì)胞表面磷酸酯多糖LTA重復(fù)單元及其衍生物進(jìn)行了設(shè)計(jì)與合成,以便進(jìn)一步研究這些多糖的結(jié)構(gòu)-免疫學(xué)活性之間的關(guān)系,以利于寡糖綴合疫苗的研發(fā)。論文主要包括以下三部分內(nèi)容:一、首先簡(jiǎn)要介紹了肺炎鏈球菌和艱難梭菌的流行病學(xué)研究、診斷、治療和疫苗研發(fā)的現(xiàn)狀,并對(duì)它們細(xì)胞表面多糖重復(fù)單元的具體化學(xué)結(jié)構(gòu)以及其重復(fù)單元的衍生物的合成與性能研究工作進(jìn)行了詳細(xì)的綜述。二、23F血清型肺炎鏈球菌莢膜多糖生物學(xué)重復(fù)單元的3-氨基丙基寡糖苷的化學(xué)合成。目標(biāo)化合物SP-2由一個(gè)分支四糖和一個(gè)磷酸甘油側(cè)鏈組成。首先,我們應(yīng)用連續(xù)糖苷化法策略構(gòu)建得到了重要四糖中間體SP-5,即:以SP-6作為受體,依次與單糖供體SP-7、SP-8和SP-9進(jìn)行糖基化反應(yīng),從而構(gòu)建得到四糖骨架。在四糖SP-5的合成過(guò)程中,分子內(nèi)的β-鼠李糖苷鍵的構(gòu)建是通過(guò)2-甲基萘基(NAP)參與的分子內(nèi)糖苷配基轉(zhuǎn)移反應(yīng)來(lái)實(shí)現(xiàn)的;同時(shí)利用苯甲酰基在糖基化過(guò)程中的鄰基參與作用來(lái)實(shí)現(xiàn)1,2-trans糖昔鍵的高效構(gòu)鍵。接著,我們又應(yīng)用亞磷酸三酯法將磷酸甘油側(cè)鏈SP-4鏈接到四糖骨架SP-5上,得到全保護(hù)四糖SP-3。最后,脫除SP-3分子內(nèi)的芐基和芐叉基,并將疊氮基還原為氨基,得到目標(biāo)產(chǎn)物SP-2。目標(biāo)化合物SP-2的還原端基上含有一個(gè)游離氨基,為后續(xù)基于SP-2糖綴合物制備及生物學(xué)研究提供了便利。三、艱難梭菌細(xì)胞表面磷酸酯多糖脂磷壁酸(LTA)衍生物的化學(xué)合成�；谄D難梭菌細(xì)胞表面磷酸酯多糖LTA的具體結(jié)構(gòu),我們?cè)O(shè)計(jì)了[2+3]和[2+2+3]的合成策略來(lái)高效地構(gòu)建兩個(gè)LTA目標(biāo)衍生物,即LTA五糖CD-1和七糖CD-2。其中,重復(fù)單元二糖CD-5和CD-6中α-氨基葡萄糖苷鍵都是通過(guò)使用2-疊氮基取代的葡萄糖胺三氯乙酰亞胺酯作為供體與相應(yīng)的受體反應(yīng)得到;而在還原端三糖CD-7的合成中,分別嘗試了連續(xù)糖苷化法和預(yù)活化一鍋法來(lái)合成CD-7,其分子內(nèi)的β-糖苷鍵是通過(guò)苯甲�；泥徎鶇⑴c作用實(shí)現(xiàn)的。目標(biāo)分子中不同寡糖模塊間的磷酸二酯鍵的構(gòu)建是經(jīng)由H-膦酸酯法實(shí)現(xiàn)的。同樣,目標(biāo)化合物CD-1和CD-2的還原端三糖末端含有一個(gè)游離氨基,為后續(xù)制備基于CD-1和CD-2的寡糖綴合物奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R392;O629.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉楊;艱難梭菌相關(guān)性腹瀉的治療和預(yù)防[J];中國(guó)抗感染化療雜志;2001年02期

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本文編號(hào)：1441863