双唾液酸化四糖抗原表位的化学酶法合成研究

发布时间：2018-01-24 00:37

本文关键词： 髓磷脂相关糖蛋白(MAG) 神经节苷脂 Thomsen-Friedenreich抗原(TF或T抗原) 唾液酸糖苷化化学酶法合成内酯　出处：《山东大学》2014年博士论文　论文类型：学位论文

【摘要】：双唾液酸化神经节苷酯四糖抗原表位存在于多种细胞表面,并在众多生理和病理过程中发挥着至关重要的作用。它是人红细胞表面高度糖基化的血型糖蛋白(glycophorin)上最主要的糖链,这一糖链除了可以避免红细胞的聚集外,还参与了红细胞所介导的众多生理过程。这一四糖结构也是肿瘤细胞过度表达的黏蛋白MUCⅡ上的特异性肿瘤相关糖抗原。此外,这一四糖也是神经节苷脂GDla、GTlaα和GQ1bα非还原末端特征结构,是髓磷脂相关糖蛋白(myelin-associated glycoprotein, MAG)与神经节苷脂结合时所能识别的最小结构单元,且这一四糖特征结构为其结合活性最高的天然受体。这一特征四糖结构还是促红细胞生成素(EPO)上的唯一O-聚糖链。大量合成这种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价值的,而且发展一种高效、便捷的合成方法将极大地促进以其为先导化合物的药物发现进程。然而,由于这类天然唾液酸糖苷结构的复杂性及其不稳定性,给分离纯化带来了极大困难。为了在分子水平上研究和评价其生物学意义,寻找一种快速、高效合成双唾液酸化四糖及其拮抗剂的合成方法是目前亟待解决的问题。虽然有文献报道用化学法或化学酶法对其及相关拮抗剂的合成进行研究,但是化学法合成需要进行反复的保护与脱保护操作,并且收率较低、立体选择性不高；而且,九碳糖唾液酸由于其自身独特结构,使得唾液酸糖苷键的生成成为糖合成领域的经典挑战。酶法合成中使用的唾液酸糖基转移酶具有高效性、立体选择性和区域选择性,避免了化学法中反复的保护与脱保护操作,简化了合成步骤,提高了效率。酶法合成这类双唾液酸化四糖需要使用α2-3唾液酸转移酶和α2-6唾液酸转移酶,其作用是分别将唾液酸引入到二糖Galβ1-3GalNAc的C3’和C6位。目前,仅有一例酶法合成的报道,采用的是鸡来源的重组a2-6唾液酸转移酶I(chST6GalNAc I)和猪来源的重组a2-3唾液酸转移酶I(pST3Gal I)成功合成了双唾液酸化的Thomsen-Friedenreich抗原(Galβ1-3GalNAcaSer/Thr)。但是,利用哺乳动物来源的唾液酸转移酶面临以下两方面的困难： (1)哺乳动物来源的唾液酸转移酶均为II型跨膜蛋白,现有的技术手段很难实现其可溶性蛋白的大量表达； (2)该系列酶往往具有严格的底物专一性,底物适用性窄,例如报道中的唾液酸转移酶只对糖肽有较高的反应活性。近年来发现的细菌来源的唾液酸转移酶能在重组大肠杆菌中大量表达,而且容易纯化,具有表达量高、底物适应性宽等优点。因此,我们考虑充分结合化学合成和酶法合成的各自优点,运用化学酶法合成策略来合成双唾液酸化四糖抗原表位。针对上述这些问题,本论文的研究工作主要包括以下几个方面： (1)“一釜多酶”合成体系的构建利用合作者发展的“一釜多酶”体系,使用相应的几种糖基转移酶,我们高效地完成了p1-3半乳糖苷键、a2-3唾液酸糖苷键、a2-6唾液酸糖苷键的酶法合成。 (2)随机糖苷化法合成双唾液酸化四糖抗原表位分别在二糖Galβ1-3GalNAc和三糖Neu5Acα2-3Galβ-3GalNAc水平上,进行随机唾液酸化的酶法合成考察。结果显示,P. damsela a2-6唾液酸转移酶,不能区分二糖结构Galβ1-3GalNAc中的半乳糖和N-乙酰氨基半乳糖,既能将唾液酸加到N-乙酰氨基半乳糖的C6位羟基,也能将其加到半乳糖的C6'位羟基。 (3)化学操纵下的区域选择性酶法唾液酸化由于随机糖苷化的方法不能得到天然四糖抗原表位,我们尝试使用化学手段,首先在三糖Neu5Aca2-3Galβ1-3GalNAc结构中引入内酯结构,使原本能自由旋转的Neu5Acα2-3Gal二糖结构单元构型锁定,然后使用a2-6唾液酸转移酶在内酯三糖的N-乙酰半乳糖C6位选择性地引入唾液酸,实现了天然四糖抗原表位的高效合成。 (4)双唾液酸化四糖抗原表位衍生物的合成此前的构效关系表明,在双唾液酸化神经节苷脂四糖抗原表位的a2-3连接的唾液酸C9位引入疏水性基团,可极大地提高该类化合物与MAG受体的结合。因此,以9N3Neu5Ac为底物,应用我们所发展的新方法,成功高效地合成了含有非天然的唾液酸单元9N3Neu5Ac的双唾液酸四糖。这一新合成策略具有较为广泛的底物适应性。运用此策略,我们也成功的合成了包括含有Neu5Gc的双唾液酸四糖的系列衍生物。综上,我们利用细菌来源的两种唾液酸转移酶Pasteurella multocida a2-3唾液酸转移酶(PmSTl)和Photobacterium damselae a2-6唾液酸转移酶(Pd2-6ST),运用含有内酯结构的三糖进行“一釜多酶”体系的化学酶法的合成,通过化学手段干预Pd2-6ST的底物选择性,实现了双唾液酸化神经节苷脂四糖抗原表位的高效合成。本文所发展的方法解决了目前化学或酶法合成该类糖链结构中所存在不足,而且相似的策略和思路同样可以用来尝试其他的底物和底物适应性广泛的酶类。该论文的研究成果具有原创性和重要的科学意义,同时也具有广阔的应用前景。本研究取得的成果和结论： (1)本文全面考察了唾液酸a2-6唾液酸转移酶Pd2-6ST对Galβ1-3GalNAcβProN3、Galβ1-3GalNAcaProN3,、Neu5Acα2-3Galβ1-3GalNAcβProN3、 Neu5Aca2-3Galβ1-3GalNAcaProN3、9N3Neu5Aca2-3Galβ1-3GalNAcβProN3、Neu5Gcα2-3Ga1β1-3GalNAcβProN3和Neu5Aca2-3Galβ1-3GalSEt及含有唾液酸内酯的系列寡糖的底物选择性,发现了Pd2-6ST对上述底物的结合特点。 (2)针对细菌来源的a2-6唾液酸转移酶Pd2-6ST的底物选择性差,不能大量、高效合成双唾液酸化四糖结构的不足,创新性地发展了一个化学操纵的策略,改变了该酶的底物选择性。首次应用细菌来源的a2-6唾液酸转移酶实现了这类四糖抗原表位的高效合成。这一策略将化学法的灵活性和酶法的高效性有机结合起来,而且本实验使用的酶类均能在重组大肠杆菌中大量、可溶性地表达,易于纯化,为合成这类双唾液酸化复杂寡糖提供了新的解决途径。 (3)首次利用所发展的化学操纵策略高效合成了包括含有叠氮基团的MAG天然受体拮抗剂的系列衍生物。叠氮基团的引入,方便后续运用“点击化学”的方法,迅速扩增化合物库,以期发现新的具有更好活性的先导化合物。 (4)首次利用所发展的化学操纵策略实现了含有Neu5Gc的双唾液酸四糖的合成。这一化学操纵策略为其它酶的底物适应性的改造和应用提供了一个新的思路。
[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 .

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R914

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