本文關(guān)鍵詞： 髓磷脂相關(guān)糖蛋白(MAG) 神經(jīng)節(jié)苷脂 Thomsen-Friedenreich抗原(TF或T抗原) 唾液酸糖苷化 化學(xué)酶法合成 內(nèi)酯　出處：《山東大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：雙唾液酸化神經(jīng)節(jié)苷酯四糖抗原表位存在于多種細胞表面,并在眾多生理和病理過程中發(fā)揮著至關(guān)重要的作用。它是人紅細胞表面高度糖基化的血型糖蛋白(glycophorin)上最主要的糖鏈,這一糖鏈除了可以避免紅細胞的聚集外,還參與了紅細胞所介導(dǎo)的眾多生理過程。這一四糖結(jié)構(gòu)也是腫瘤細胞過度表達的黏蛋白MUCⅡ上的特異性腫瘤相關(guān)糖抗原。此外,這一四糖也是神經(jīng)節(jié)苷脂GDla、GTlaα和GQ1bα非還原末端特征結(jié)構(gòu),是髓磷脂相關(guān)糖蛋白(myelin-associated glycoprotein, MAG)與神經(jīng)節(jié)苷脂結(jié)合時所能識別的最小結(jié)構(gòu)單元,且這一四糖特征結(jié)構(gòu)為其結(jié)合活性最高的天然受體。這一特征四糖結(jié)構(gòu)還是促紅細胞生成素(EPO)上的唯一O-聚糖鏈。 大量合成這種富含唾液酸的寡糖用于在分子水平上研究其功能是非常有價值的,而且發(fā)展一種高效、便捷的合成方法將極大地促進以其為先導(dǎo)化合物的藥物發(fā)現(xiàn)進程。然而,由于這類天然唾液酸糖苷結(jié)構(gòu)的復(fù)雜性及其不穩(wěn)定性,給分離純化帶來了極大困難。為了在分子水平上研究和評價其生物學(xué)意義,尋找一種快速、高效合成雙唾液酸化四糖及其拮抗劑的合成方法是目前亟待解決的問題。 雖然有文獻報道用化學(xué)法或化學(xué)酶法對其及相關(guān)拮抗劑的合成進行研究,但是化學(xué)法合成需要進行反復(fù)的保護與脫保護操作,并且收率較低、立體選擇性不高；而且,九碳糖唾液酸由于其自身獨特結(jié)構(gòu),使得唾液酸糖苷鍵的生成成為糖合成領(lǐng)域的經(jīng)典挑戰(zhàn)。酶法合成中使用的唾液酸糖基轉(zhuǎn)移酶具有高效性、立體選擇性和區(qū)域選擇性,避免了化學(xué)法中反復(fù)的保護與脫保護操作,簡化了合成步驟,提高了效率。酶法合成這類雙唾液酸化四糖需要使用α2-3唾液酸轉(zhuǎn)移酶和α2-6唾液酸轉(zhuǎn)移酶,其作用是分別將唾液酸引入到二糖Galβ1-3GalNAc的C3’和C6位。目前,僅有一例酶法合成的報道,采用的是雞來源的重組a2-6唾液酸轉(zhuǎn)移酶I(chST6GalNAc I)和豬來源的重組a2-3唾液酸轉(zhuǎn)移酶I(pST3Gal I)成功合成了雙唾液酸化的Thomsen-Friedenreich抗原(Galβ1-3GalNAcaSer/Thr)。但是,利用哺乳動物來源的唾液酸轉(zhuǎn)移酶面臨以下兩方面的困難： (1)哺乳動物來源的唾液酸轉(zhuǎn)移酶均為II型跨膜蛋白,現(xiàn)有的技術(shù)手段很難實現(xiàn)其可溶性蛋白的大量表達； (2)該系列酶往往具有嚴格的底物專一性,底物適用性窄,例如報道中的唾液酸轉(zhuǎn)移酶只對糖肽有較高的反應(yīng)活性。 近年來發(fā)現(xiàn)的細菌來源的唾液酸轉(zhuǎn)移酶能在重組大腸桿菌中大量表達,而且容易純化,具有表達量高、底物適應(yīng)性寬等優(yōu)點。因此,我們考慮充分結(jié)合化學(xué)合成和酶法合成的各自優(yōu)點,運用化學(xué)酶法合成策略來合成雙唾液酸化四糖抗原表位。針對上述這些問題,本論文的研究工作主要包括以下幾個方面： (1)“一釜多酶”合成體系的構(gòu)建 利用合作者發(fā)展的“一釜多酶”體系,使用相應(yīng)的幾種糖基轉(zhuǎn)移酶,我們高效地完成了p1-3半乳糖苷鍵、a2-3唾液酸糖苷鍵、a2-6唾液酸糖苷鍵的酶法合成。 (2)隨機糖苷化法合成雙唾液酸化四糖抗原表位 分別在二糖Galβ1-3GalNAc和三糖Neu5Acα2-3Galβ-3GalNAc水平上,進行隨機唾液酸化的酶法合成考察。結(jié)果顯示,P. damsela a2-6唾液酸轉(zhuǎn)移酶,不能區(qū)分二糖結(jié)構(gòu)Galβ1-3GalNAc中的半乳糖和N-乙酰氨基半乳糖,既能將唾液酸加到N-乙酰氨基半乳糖的C6位羥基,也能將其加到半乳糖的C6'位羥基。 (3)化學(xué)操縱下的區(qū)域選擇性酶法唾液酸化 由于隨機糖苷化的方法不能得到天然四糖抗原表位,我們嘗試使用化學(xué)手段,首先在三糖Neu5Aca2-3Galβ1-3GalNAc結(jié)構(gòu)中引入內(nèi)酯結(jié)構(gòu),使原本能自由旋轉(zhuǎn)的Neu5Acα2-3Gal二糖結(jié)構(gòu)單元構(gòu)型鎖定,然后使用a2-6唾液酸轉(zhuǎn)移酶在內(nèi)酯三糖的N-乙酰半乳糖C6位選擇性地引入唾液酸,實現(xiàn)了天然四糖抗原表位的高效合成。 (4)雙唾液酸化四糖抗原表位衍生物的合成 此前的構(gòu)效關(guān)系表明,在雙唾液酸化神經(jīng)節(jié)苷脂四糖抗原表位的a2-3連接的唾液酸C9位引入疏水性基團,可極大地提高該類化合物與MAG受體的結(jié)合。因此,以9N3Neu5Ac為底物,應(yīng)用我們所發(fā)展的新方法,成功高效地合成了含有非天然的唾液酸單元9N3Neu5Ac的雙唾液酸四糖。這一新合成策略具有較為廣泛的底物適應(yīng)性。運用此策略,我們也成功的合成了包括含有Neu5Gc的雙唾液酸四糖的系列衍生物。 綜上,我們利用細菌來源的兩種唾液酸轉(zhuǎn)移酶Pasteurella multocida a2-3唾液酸轉(zhuǎn)移酶(PmSTl)和Photobacterium damselae a2-6唾液酸轉(zhuǎn)移酶(Pd2-6ST),運用含有內(nèi)酯結(jié)構(gòu)的三糖進行“一釜多酶”體系的化學(xué)酶法的合成,通過化學(xué)手段干預(yù)Pd2-6ST的底物選擇性,實現(xiàn)了雙唾液酸化神經(jīng)節(jié)苷脂四糖抗原表位的高效合成。本文所發(fā)展的方法解決了目前化學(xué)或酶法合成該類糖鏈結(jié)構(gòu)中所存在不足,而且相似的策略和思路同樣可以用來嘗試其他的底物和底物適應(yīng)性廣泛的酶類。該論文的研究成果具有原創(chuàng)性和重要的科學(xué)意義,同時也具有廣闊的應(yīng)用前景。 本研究取得的成果和結(jié)論： (1)本文全面考察了唾液酸a2-6唾液酸轉(zhuǎn)移酶Pd2-6ST對Galβ1-3GalNAcβProN3、Galβ1-3GalNAcaProN3,、Neu5Acα2-3Galβ1-3GalNAcβProN3、 Neu5Aca2-3Galβ1-3GalNAcaProN3、9N3Neu5Aca2-3Galβ1-3GalNAcβProN3、Neu5Gcα2-3Ga1β1-3GalNAcβProN3和Neu5Aca2-3Galβ1-3GalSEt及含有唾液酸內(nèi)酯的系列寡糖的底物選擇性,發(fā)現(xiàn)了Pd2-6ST對上述底物的結(jié)合特點。 (2)針對細菌來源的a2-6唾液酸轉(zhuǎn)移酶Pd2-6ST的底物選擇性差,不能大量、高效合成雙唾液酸化四糖結(jié)構(gòu)的不足,創(chuàng)新性地發(fā)展了一個化學(xué)操縱的策略,改變了該酶的底物選擇性。首次應(yīng)用細菌來源的a2-6唾液酸轉(zhuǎn)移酶實現(xiàn)了這類四糖抗原表位的高效合成。這一策略將化學(xué)法的靈活性和酶法的高效性有機結(jié)合起來,而且本實驗使用的酶類均能在重組大腸桿菌中大量、可溶性地表達,易于純化,為合成這類雙唾液酸化復(fù)雜寡糖提供了新的解決途徑。 (3)首次利用所發(fā)展的化學(xué)操縱策略高效合成了包括含有疊氮基團的MAG天然受體拮抗劑的系列衍生物。疊氮基團的引入,方便后續(xù)運用“點擊化學(xué)”的方法,迅速擴增化合物庫,以期發(fā)現(xiàn)新的具有更好活性的先導(dǎo)化合物。 (4)首次利用所發(fā)展的化學(xué)操縱策略實現(xiàn)了含有Neu5Gc的雙唾液酸四糖的合成。這一化學(xué)操縱策略為其它酶的底物適應(yīng)性的改造和應(yīng)用提供了一個新的思路。
[Abstract]:The tetraose structure is the most important sugar chain of glycophorin on the surface of human red blood cells . In order to study and evaluate its biological significance at molecular level , it is urgent to study and evaluate its biological significance in order to study and evaluate its biological significance at molecular level . The synthesis of sialyltransferase I ( chST6GalNI ) and porcine derived recombinant a2 - 3 sialyltransferase I ( pST3Gal I ) have been successfully synthesized . ( 1 ) the sialyltransferase of mammalian origin is type II transmembrane protein , and the prior art is difficult to realize the large expression of soluble protein ; ( 2 ) The enzyme often has strict substrate specificity and narrow substrate applicability , for example , the sialyltransferase in the report has high reactivity to glycopeptides . The sialyltransferase , which has been discovered in recent years , can be expressed in recombinant E . coli , and is easy to purify . It has the advantages of high expression quantity and wide substrate adaptability . Therefore , we consider the advantages of chemical synthesis and enzymatic synthesis , and then synthesize double sialylated tetraose antigen epitope by chemical enzymatic synthesis strategy . In view of these problems , the research work of this paper mainly includes the following aspects : ( 1 ) Construction of " one - pot multi - enzyme " synthesis system Using the " one - pot multi - enzyme " system developed by the cooperators , the corresponding glycosyltransferase was used , and we efficiently completed the enzymatic synthesis of the p1 - 3 galactosidase , the a2 - 3 sialosidase bond , and the a2 - 6 sialosidase . ( 2 ) Synthesis of Double Sialic Acid Tetraose Antigen Epitopes by Random Glycosides Method The results showed that P . damsela 2 - 6 sialyltransferase could not distinguish galactose and N - acetylgalactosamine from the disaccharide structure Gal - 3GalN and add sialic acid to the C6 - hydroxyl group of N - acetylgalactosamine , and it can also be added to the C6 ' hydroxyl group of galactose . ( 3 ) Selective enzymatic sialylation under chemical manipulation Due to the method that the natural tetraose antigen epitope cannot be obtained by the method of randomization , the lactone structure is firstly introduced into the structure of the trisaccharide Neu5Aca2 - 3Gal - 3Galnac , the structure of the Neu5Ac . alpha . 2 - 3Gal disaccharide structural unit which can be freely rotated is firstly locked , and then the sialic acid is selectively introduced into the N - acetylgalactoC6 position of the lactone triose by using the a2 - 6 sialyltransferase , and the high - efficiency synthesis of the epitope of the natural tetraose antigen is realized . ( 4 ) Synthesis of Bisialylated Tetraose Antigen Epitope Derivatives In this paper , we have successfully synthesized the tetraose containing non - natural sialic acid unit 9N3Neu5Ac with 9N3Neu5Ac as a substrate . Therefore , we have successfully synthesized a series of derivatives including tetraose of disialic acid containing Neu5Gc . In this paper , we use two sialyltransferase ( Pd2 - 6ST ) of the bacterial origin to synthesize the sialyltransferase ( Pd2 - 6ST ) and Photobacterium damselae a2 - 6 sialyltransferase ( Pd2 - 6ST ) . By the chemical means , the synthesis of the enzyme method of the " one - pot multi - enzyme " system is carried out . The results and conclusions of this study are as follows : ( 1 ) Sialic acid 2 - 6 sialyltransferase Pd2 - 6ST was investigated in this paper . The substrate selectivity of Pd2 - 6ST to the substrate was investigated . The substrate selectivity of Pd2 - 6ST to the substrate was investigated . ( 2 ) The substrate selectivity of the 2 - 6 sialyltransferase Pd2 - 6ST for bacterial origin is poor , and a chemical manipulation strategy can not be produced in a large amount and efficiently . A chemical manipulation strategy is developed , which changes the substrate selectivity of the enzyme . This strategy combines the flexibility of the chemical method and the high efficiency of the enzyme method . The enzyme used in the experiment can be expressed in a large amount and soluble in the recombinant Escherichia coli , and is easy to purify , and provides a new solution for the synthesis of such disialic acid complex oligosaccharides . ( 3 ) For the first time , a series of derivatives of MAG natural receptor antagonists containing azide groups are efficiently synthesized using the developed chemical manipulation strategies . The introduction of azide groups facilitates the subsequent use of the " click chemistry " method to rapidly amplify the compound library with a view to finding new precursor compounds with better activity . ( 4 ) The synthesis of tetraose containing Neu5Gc is achieved for the first time with the developed chemical manipulation strategy . This chemical manipulation strategy provides a new idea for the adaptation and application of substrate adaptability of other enzymes .

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R914

【參考文獻】

相關(guān)期刊論文 前1條

1 ;Chemoenzymatic synthesis of α2 3-sialylated carbohydrate epitopes[J];Science China(Chemistry);2011年01期

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本文編號：1458673