双唾液酸化四糖抗原表位的化学酶法合成研究
|
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。
Di-sialylated ganglioside tetrasaccharide epitopes Neu5Aca2-3Galβ1-3(Neu5Aca2-6)GalNAc widely distribute on the outermost position of cell-surface and play important roles in many physiological and pathological processes. The disialyl tetrasaccharide is found to be the major O-glycan of glycophorin (heavily glycosylated erythrocyte membrane glycoprotein), which prevents the red cells from aggregation and mediates many physiological processes. It is also the special O-glycan of mucin MUC Ⅱ which overexpressed in tumor cells. The disialyl tetrasaccharide is a special component at the non-reducing end of gangliosides GD1α, GT1aa, and GQ1bα and the minimal binding epitope for high-affinity myelin-associated glycoprotein (MAG) ligands. It is also the only O-glycan of erythropoietin (EPO).
It is of great value to synthesize the di-sialyl tetrasaccharide in large quantities in order to study its functions at the molecular levels. Developing an efficient and practical synthesis approach will promote the process of drug discovery of lead compounds. However, these sialosides related to its complexity and instability make it difficult to separatie and purify. In order to evaluate its biological functions and significance at the molecular levels, developing an simple and efficient synthetic approach for synthesis of disialyl tetrasaccharides and their derivatives is imperative.
Several elegant chemical or chemoenzymatic synthetic methods for these disialyl tetrasaccharides and related antagonists have been reported. However, strategies for pure chemical synthesis must address a host of issues, including tedious blocking and deprotection manipulations, low yield and low stereoselectivities. Furthermore, for the unique structure of nine-carbon sugar N-acetylneuraminic acid, the formation of the glycosidic bond is still considered one of the most difficult problems in synthesizing carbohydrates.
Two distinct sialyltransferases (a2-3-sialyltransferase, PmST1and a2-6-sialyltransferase, Pd2-6ST) are required to introduce two N-acetylneuraminic acid (Neu5Ac) to the C3'and C6positions of Galβ1-3GalNAc disaccharide core. Thus far, only a few recombinant N-acetylgalactosamine a2-6sialyltransferases (ST6GalNAc) from mammalian sources have been employed to the synthesis of Neu5Aca2-6GalNAc sequence. A recombinant α2-6-sialyltransferase from chicken (chST6GalNAc I) and a recombinant a2-3-sialyltransferase from porcine (pST3Gal I) have been successfully utilized for enzymatic production of di-sialyl TF-antigen.
However, two major problems encountered in these approaches:
(1) Mammalian sialyltransferases are type II transmembrane protein and it is diffcult to realize high yield expression using existing technologies.
(2) These mammalian sialyltransferases have strict acceptor substrates specificity. For example, the two mammalian sialyltransferases in related report only have activity for glycopeptide substrates.
In contrast to mammalian sialyltransferases, several bacterial sialyltransferases that have been cloned and expressed in E. coli can be produced in sufficient amounts and conveniently purificated, owning remarkable expression amount and promiscuous substrate specificities. So, considering the full advantages of chemical synthesis and enzymatic synthesis, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives. In order to solve these problems, we carried out the research from the following aspects:
(1) One-pot multienzyme (OPME) system
We have efficiently synthesized β1-3linked galactosides,α2-3sialosides and a2-6sialosides using OPME system developed by our collaborators.
(2) Synthesis of disialyl tetrasaccharide epitopes in glycorandomization
Random sialylation of disaccharide Galβ1-3GalNAc and trisaccharide Neu5Aca2-3Galβ1-3GalNAc were investigated. Our previous finding showed that both terminal Gal and GalNAc can be recognized by Pd2-6ST to form Neu5Aca2-6Gal and Neu5Aca2-6GalNAc, respectively. Pd2-6ST was able to add Neu5Ac to both C6-OH of the internal GalNAc and C6'-OH of the terminal Gal.
(3) Regioselective sialylation of conformationally constrained trisaccharide acceptors
To circumvent the issue of substrate promiscuity of Photobacterium damselae (Pd2-6ST), we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing a bacteria a2-6-sialyltransferase from Photobacterium damselae (Pd2-6ST).
(4) Chemoenzymatic synthesis of disialylated tetrasaccharide derivatives
Previous structure-activity relationship (SAR) studies of MAG and disialyl tetrasaccharide epitopes have demonstrated that modification of Neu5Ac by introducing hydrophobic substituents at the C9position in the Neu5Aca2-3GalNAc sequence can significantly increase the binding affinity of the glycan and MAG. Encouraged by these results, chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing a non-natural sialic acid9N3Neu5Aca2-3-linked to the Gal was carried out using the efficient lactone method described above. Meanwhile, the method has the general applicability. A similar high efficiency was achieved for the chemoenzymatic synthesis of disialyl tetrasaccharide containing the sialic acid N-glycolylneuraminic acid (Neu5Gc) and related analogues.
In summary, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing bacteria Pasteurella multocida a2-3-sialyltransferase (PmST1) and Photobacterium damselae α2-6-sialyltransferase (Pd2-6ST). This strategy provides a new route for easy access of disialyl tetrasaccharide epitopes and their derivatives, solving the problems exsiting in chemical and chemoenzymatic methods. The application of similar stratigies can be explored for other substrates and for other acceptor substrate promiscuous enzymes. The research results are provided with originity and significance, revealing promising application.
The main conclusions in this paper were as follows:
(1) This manuscript investigated the substrate selectivities of a2-6-sialyltransferase on sialylation of Galpl-3GalNAcβProN3,Galβ1-3GalNAcaProN3,Neu5Aca2-3Galβ1-3GalNAcβProN3xNeu5Aca2-3Galβ1-3GalNAcaProN3,9N3Neu5Aca2-3Galβ1-3GalNAcβProN3,Neu5Gca2-3Galβ1-3GalNAcβProN3,Neu5Aca2-3Galβ1-3GalSEt and trisaccharide lactone and found the characteristics of substrate specificities of Pd2-6ST.
(2) To circumvent the issues with the promiscuous substrate specificities of bacterial sialyltransferases Pd2-6ST and low yield in synthesis of natural disialyl tetrasaccharide, we herein report a chemical controlled strategy for the synthesis of disialyl tetrasaccharide through changing the promiscuous substrate specificities of Pd2-6ST. This strategy binds flexibilities of chemical synthesis and efficiency of enzymatic synthesis. In addition, bacterial sialyltransferases can be produced in sufficient amounts in convenient bacterial expression systems and easy purification. Therefore, this strategy provides a new route for easy access to disialyl complex oligosaccharide.
(3) The chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing the non-naturalsialic acid9N3Neu5Aca2-3-linked to the Gal using the efficient lactone method was firstly described. The9N3-group can be used as a chemical handle for easy derivatization to expand compound libraries and also lead to the development of lead compounds.
(4) The α2-3-linked trisaccharide containing N-glycolylneuraminic acid (Neu5Gc), a nonhuman sialic acid form with an additional hydroxyl group at C5-NHAc, was also compatible with the regioselective sialylation approach. The general applicability of the method can be further explored for other substrates and for other acceptor substrate promiscuous enzymes.
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。
Di-sialylated ganglioside tetrasaccharide epitopes Neu5Aca2-3Galβ1-3(Neu5Aca2-6)GalNAc widely distribute on the outermost position of cell-surface and play important roles in many physiological and pathological processes. The disialyl tetrasaccharide is found to be the major O-glycan of glycophorin (heavily glycosylated erythrocyte membrane glycoprotein), which prevents the red cells from aggregation and mediates many physiological processes. It is also the special O-glycan of mucin MUC Ⅱ which overexpressed in tumor cells. The disialyl tetrasaccharide is a special component at the non-reducing end of gangliosides GD1α, GT1aa, and GQ1bα and the minimal binding epitope for high-affinity myelin-associated glycoprotein (MAG) ligands. It is also the only O-glycan of erythropoietin (EPO).
It is of great value to synthesize the di-sialyl tetrasaccharide in large quantities in order to study its functions at the molecular levels. Developing an efficient and practical synthesis approach will promote the process of drug discovery of lead compounds. However, these sialosides related to its complexity and instability make it difficult to separatie and purify. In order to evaluate its biological functions and significance at the molecular levels, developing an simple and efficient synthetic approach for synthesis of disialyl tetrasaccharides and their derivatives is imperative.
Several elegant chemical or chemoenzymatic synthetic methods for these disialyl tetrasaccharides and related antagonists have been reported. However, strategies for pure chemical synthesis must address a host of issues, including tedious blocking and deprotection manipulations, low yield and low stereoselectivities. Furthermore, for the unique structure of nine-carbon sugar N-acetylneuraminic acid, the formation of the glycosidic bond is still considered one of the most difficult problems in synthesizing carbohydrates.
Two distinct sialyltransferases (a2-3-sialyltransferase, PmST1and a2-6-sialyltransferase, Pd2-6ST) are required to introduce two N-acetylneuraminic acid (Neu5Ac) to the C3'and C6positions of Galβ1-3GalNAc disaccharide core. Thus far, only a few recombinant N-acetylgalactosamine a2-6sialyltransferases (ST6GalNAc) from mammalian sources have been employed to the synthesis of Neu5Aca2-6GalNAc sequence. A recombinant α2-6-sialyltransferase from chicken (chST6GalNAc I) and a recombinant a2-3-sialyltransferase from porcine (pST3Gal I) have been successfully utilized for enzymatic production of di-sialyl TF-antigen.
However, two major problems encountered in these approaches:
(1) Mammalian sialyltransferases are type II transmembrane protein and it is diffcult to realize high yield expression using existing technologies.
(2) These mammalian sialyltransferases have strict acceptor substrates specificity. For example, the two mammalian sialyltransferases in related report only have activity for glycopeptide substrates.
In contrast to mammalian sialyltransferases, several bacterial sialyltransferases that have been cloned and expressed in E. coli can be produced in sufficient amounts and conveniently purificated, owning remarkable expression amount and promiscuous substrate specificities. So, considering the full advantages of chemical synthesis and enzymatic synthesis, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives. In order to solve these problems, we carried out the research from the following aspects:
(1) One-pot multienzyme (OPME) system
We have efficiently synthesized β1-3linked galactosides,α2-3sialosides and a2-6sialosides using OPME system developed by our collaborators.
(2) Synthesis of disialyl tetrasaccharide epitopes in glycorandomization
Random sialylation of disaccharide Galβ1-3GalNAc and trisaccharide Neu5Aca2-3Galβ1-3GalNAc were investigated. Our previous finding showed that both terminal Gal and GalNAc can be recognized by Pd2-6ST to form Neu5Aca2-6Gal and Neu5Aca2-6GalNAc, respectively. Pd2-6ST was able to add Neu5Ac to both C6-OH of the internal GalNAc and C6'-OH of the terminal Gal.
(3) Regioselective sialylation of conformationally constrained trisaccharide acceptors
To circumvent the issue of substrate promiscuity of Photobacterium damselae (Pd2-6ST), we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing a bacteria a2-6-sialyltransferase from Photobacterium damselae (Pd2-6ST).
(4) Chemoenzymatic synthesis of disialylated tetrasaccharide derivatives
Previous structure-activity relationship (SAR) studies of MAG and disialyl tetrasaccharide epitopes have demonstrated that modification of Neu5Ac by introducing hydrophobic substituents at the C9position in the Neu5Aca2-3GalNAc sequence can significantly increase the binding affinity of the glycan and MAG. Encouraged by these results, chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing a non-natural sialic acid9N3Neu5Aca2-3-linked to the Gal was carried out using the efficient lactone method described above. Meanwhile, the method has the general applicability. A similar high efficiency was achieved for the chemoenzymatic synthesis of disialyl tetrasaccharide containing the sialic acid N-glycolylneuraminic acid (Neu5Gc) and related analogues.
In summary, we herein report a chemoenzymatic approach for the synthesis of disialyl tetrasaccharide epitopes and their derivatives through regioselective sialylation of conformationally constrained trisaccharide acceptors by utilizing bacteria Pasteurella multocida a2-3-sialyltransferase (PmST1) and Photobacterium damselae α2-6-sialyltransferase (Pd2-6ST). This strategy provides a new route for easy access of disialyl tetrasaccharide epitopes and their derivatives, solving the problems exsiting in chemical and chemoenzymatic methods. The application of similar stratigies can be explored for other substrates and for other acceptor substrate promiscuous enzymes. The research results are provided with originity and significance, revealing promising application.
The main conclusions in this paper were as follows:
(1) This manuscript investigated the substrate selectivities of a2-6-sialyltransferase on sialylation of Galpl-3GalNAcβProN3,Galβ1-3GalNAcaProN3,Neu5Aca2-3Galβ1-3GalNAcβProN3xNeu5Aca2-3Galβ1-3GalNAcaProN3,9N3Neu5Aca2-3Galβ1-3GalNAcβProN3,Neu5Gca2-3Galβ1-3GalNAcβProN3,Neu5Aca2-3Galβ1-3GalSEt and trisaccharide lactone and found the characteristics of substrate specificities of Pd2-6ST.
(2) To circumvent the issues with the promiscuous substrate specificities of bacterial sialyltransferases Pd2-6ST and low yield in synthesis of natural disialyl tetrasaccharide, we herein report a chemical controlled strategy for the synthesis of disialyl tetrasaccharide through changing the promiscuous substrate specificities of Pd2-6ST. This strategy binds flexibilities of chemical synthesis and efficiency of enzymatic synthesis. In addition, bacterial sialyltransferases can be produced in sufficient amounts in convenient bacterial expression systems and easy purification. Therefore, this strategy provides a new route for easy access to disialyl complex oligosaccharide.
(3) The chemoenzymatic synthesis of disialyl tetrasaccharide epitope containing the non-naturalsialic acid9N3Neu5Aca2-3-linked to the Gal using the efficient lactone method was firstly described. The9N3-group can be used as a chemical handle for easy derivatization to expand compound libraries and also lead to the development of lead compounds.
(4) The α2-3-linked trisaccharide containing N-glycolylneuraminic acid (Neu5Gc), a nonhuman sialic acid form with an additional hydroxyl group at C5-NHAc, was also compatible with the regioselective sialylation approach. The general applicability of the method can be further explored for other substrates and for other acceptor substrate promiscuous enzymes.
引文
[1]A. Varki, Glycobiology,1993,3,2,97-130.
[2]蔡孟深,李中军,糖化学.化学工业出版社：2006.
[3]Z. Zhang, I. R.Ollmann, X. S.Ye, R.Wischnat, T. Baasov, C, H.Wong, J. Am. Chem. Soc.1999, 121,4,734-753.
[4]O. J. Plante, E. R. Palmacci, P. H. Seeberger, Science 2009,1523-1527.
[5]P. H. Seeberger, Chem. Soc. Rev.2008,37,1,19-28.
[6]H. A. Orgueira, A. Bartolozzi, P. Schell, R. E. Litjens, E. R. Palmacci, P. H. Seeberger, Chem. Eur.J.2002,1,140-169.
[7]T. K. K Mong, H. K. Lee, S. G. Duron, C. H.Wong, PNAS 2003,100,3,797-802.
[8]F. Burkhart, Z. Zhang, S. Wacowich-Sgarbi, C. H. Wong, Angew. Chem. Int. Ed.2001,40,7, 1274-1276.
[9]X. S. Ye, C. H. Wong. J. Org Chem.2000,65,2410-2431.
[10]X. Huang, L. Huang, H.Wang, X. S. Ye, Angew. Chem. Int. Ed.2004,43,5221-5224.
[11]C. H. Wong, X. S. Ye, Z. Zhang, J. Am. Chem. Soc.1998,120,7137-7138.
[12]Z. Wang, Y. M. Xu, B.Yang, G. Tiruchinapally, B. Sun, R. P. Liu, S. Dulaney, J. A. Liu, X. F. Huang, Chem. Eur. J.2010,16,8365.
[13]S.J. Danishefsky, K. F. McClure, J. T. Randolph, R.B. Ruggeri, Science 1993,260,1307.
[14]S. P. Douglas, D. M. Wh itfield, J. J. KrePinsky, J. Am. Chem. Soc.1991,113,5095.
[15]G H.Veeneman, H. F. Brugghe, H. van den Elst, J. H. van Boom, Carbohydr. Res.1990,195,2 C1-4.
[16]L. L. Lairson, B. Henrissat, G. J. Davies, Annu. Rev. Biochem.2008,77,521-555.
[17]P. M. Coutinho, E. Deleury, G J. Davies, B. Henrissat and S.G.Withers, J. Mol. Biol.2003,328, 2,307-317.
[18]B. R. Griffith, J. M. Langenhan, J. S. Thorson, Curr. Opin. Biotechnol.2005,16,6,622-630.
[19]W. A. Barton, J. Lesniak, J. B. Biggins, P. D. Jeffrey, J. Jiang, K.R. Rajashankar, J. S. Thorson and D. B. Nikolov, Nat. Struct. Biol.2001,8,6,545-551.
[20]J. M. Langenhan, B. R. Griffith, and J. S. Thorson, J. Nat. Prod,2005,68,1696-1711.
[21]K. Emami, T. Nagy, C. M. G A. Fontes, L. M. A. Ferreira and H. J. Gilbert, J. Bacteriol.2002, 184,15,4124.
[22]K. A. Stubbs, M. Balcewich, B. L. Mark, and D. J. Vocadlo, J. Biol. Chem.2007,282,29, 21382-21391.
[23]D. Hogg, G. Pell, P. Dupree, F. Goubet, S. M Martin-Orue, S. Armand, and Harry J Gilbert, Biochem. J.2003,371,1027-1043.
[24]R. V. Stick and K. A. Stubbs, Tetrahedron:Asymmetry 2005,16,321-335.
[25]S. J. Williams, S. G Withers, Carbohydr. Res.2000,327,2,27-46.
[26]S. G. Withers, Carb. Polymers.2001,44,4,325-337.
[27]卢丽丽,肖敏,赵晗,王鹏,钱新民.生物化学与生物物理进展,2006,33,4,310-320.
[28]Raymond A Dwek, Chem. Rev.1996,96,683-720.
[29]P. M. Rudd, T. Elliott, P. Cresswell, I.A. Wilson, R. A. Dwek, Science 2001,291,2370-2375.
[30]S. J. Hwang, C. M. Ballantyne, A. R. Sharrett, L. C. Smith, Circulation 1997,96,4219-4225.
[31]A. E. Koch, M. M. Halloran, C. J. Haskell, M. R. Shah, P. J. Polverini, Nature 1995,376,6540, 517-519.
[32]F. Austrup, D. Vestweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, R. Hallmann, A.r Scheffold, A. Radbruch, A. Hamann, Nature 1997,385,81-83.
[33]M. Steegmaler, A. Levinovitz, S. Isenmann, E. Borges, M. Lenter, H. P. Kocher, B. Kleuser, D. Vestweber, Nature 1995,373,615-620.
[34]H. Park, K. Y. Hwang, K. H. Oh et al, Bioorganic Medicihal Chemistry 2008,16,1,284.
[35]Y. Wu, J.H. Yang, G. F. Dai, et al, Bioorganic Medicinal Chemistry 2009,17,4,1464.
[36]K. Matsumoto, K. Takeraata, Analytical Science 2002,18,12,1315.
[37]R. Pili, J. Chang, R.A. Partis, et al, Cancer Res 1995,55,13,2920.
[38]T. M. Block, X. Lu, A. S. Mehta, B. S. Blumberg, B. Tennant, M. Ebling, B. Korba, D. M. Lansky, G. S. Jacob, R. A. Dwek, Nat. Med.1998,4,610-614.
[39]L. F. Steel, D. Shumpert, M. Trotter, S. H. Seeholzer, A. A. Evans, W. T. London, R. Dwek and T. M. Block, Protromics 2003,3,5,601-609.
[40]N. Zitzmann, A. S. Mehta, S. Carrouee, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, T. M. Block 1999,96,21,11878-11882.
[41]E. B. Ave, S. Carrouee, B.T. Butters, R. A. Dwek and T. M. Block, Hepatology 2001,33,6, 1488-1495.
[42]R. B. Parekh, R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D. Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, F. Takeuchi, Y. Nagano, T. Miyamoto & A. Kobata, Nature 1985,316,452-457.
[43]T. Taniguchi, T. Mizuochi, M. Beale, R. A. Dwek, T.W. Rademacher, A. Kobata, Biochemistry 1985,24,20,5551-5557.
[44]F. Kiyoshi, K. Akira, Mol Immunol 1991,28,12,1333-1340.
[45]Y. J. Chen, D. R. Wing, G R. Guile, R. A. Dwek, D. J. Harvey, S. Zamze, Eur. J. Biochem.1998, 251,3,691-703.
[46]S.W. Homans, R.A. Dwek, D.L. Fernandes, T.W. Rademacher, FEBS Lett 1982,150,2, 503-506.
[47]K. Furukawa, K. Matsuta, F. Takeuchi, E. Kosuge, T. Miyamoto and A. Kobata, Int. Immunol 1990,2,1,105-112.
[48]A. Youings, S. C. Chang, R. A. DweK and I. G Scragg, Biochem. J.1996,314,621-630.
[49]Y. Abe, E. Takashita, K. Sugawara, et al, International Congress Series 2004,1263,2,214-217.
[50]T. Lin, G Wang, A. Li, et al, Virology 2009,392,1,73-81.
[51]J. Stevens, O. Blixt, T. M. Tumpey, Science 2006,312,5772,404.
[52]D. Xu, E. I. Newhouse, R. E. Amaro, J Mol Biol 2009,387,2,465-491.
[53]L. S. C Kreisman and B. A. Cobb, Glycobiology 2012,22 (8),1019-1030.
[54]J. Stevens, O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, I.A. Wilson, Science 2006,312,404.
[55]R. R. Schumann, S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison, P. S. Tobias, R. J. Ulevitch, Science,1990,249,4975,1429-1431.
[56]J. C. Chow, D. W. Young, D. T. Golenbock, W. J. Christ, F. Gusovsky, J. Biol. Chem.1999,274, 10689-10692.
[57]M. J. Kiefel, M. von Itzstein, Prog. Med. Chem.1999,36,1-28.
[58]A. Dondoni, A. Marra, P. Merino, J. Am. Chem. Soc.1994,116,3324-3336.
[59]S. J. Danishefsky, M. P. DeNinno, S. H. Chen, J. Am. Chem. Soc.1988,110,3929-3940.
[60]L. S. Li, Y. L. Wu, Y. Wu, Org. Lett 2000,2,891-894.
[61]J. M. Haberman, D. Y. Gin, Org. Lett.2001,3,1665.
[62]G J. Boons, A. V. Demchenko, Chem. Rev.2000,100,4539-4566.
[63]A. V. Demchenko, G. J. Boons, Tetrahedron Lett.1998,39,3065-3068.
[64]A. V. Demchenko, G. J. Boons, Chem.OEur. J.1999,5,1278-1283.
[65]K. Matsuokaa, T. Onagaa, T. Moria, J. I. Sakamotoa, T. Koyamaa, N. Sakairic, K. Hatanoa, D.Terunumaa, Tetrahedron Letters 2004,45,51,9383-9386.
[66]K. Matsuokaa, C. Takitaa, T. Koyamaa, D. Miyamotob, S. Yingsakmongkond, K. I. P. J. Hidarib, W. Jampangerne, T. Suzukib, Y. Suzukic, K. Hatanoa, D. Terunumaa, Bioorg. Med. Chem. Lett.2007,17,3826-3830.
[67]T. T. Martin, R. R. Schmidt, Tetrahedron Lett 1992,33,6123.
[68]H. Kondo, Y. Ichikawa, C. H. Wong, J. Am. Chem. Soc,1992,114,8748.
[69]梁芬芬,陈力,邢国文.有机化学,2009,29,9,1317-1324.
[70]T. K. K. Mong, H. K. Lee, S. G. Duron, C. H.Wong, Proc. Natl. Acad. Sci.2003,100,797.
[71]S. Cai, and B. Yu,, Org. Lett.2003,5,3827-3830.
[72]Y. Ito, T. Ogawa, Tetrahedron 1990,46,89.
[73]D. Ye, W. Liu, D. Zhang, E. Feng, H. Jiang, and H. Liu,J. Org. Chem.2009,74,1733-1735.
[74]H. Tanaka, Y. Nishiura, and T. Takahashi, J. Am. Chem. Soc.2008,130,17244-17245.
[75]D. Crich, and W. Li, J. Org. Chem.2007,72,2387-2391.
[76]D. Crich, and B. Wu, Org. Lett.2008,10,4033-4035.
[77]H. Guo, L. Li, and P. G. Wang, Biochemistry 2006,45,13760-13768.
[78]G L. Huang, D.W. Zhang, H. J. Zhao, H. C. Zhang and P. G. Wang, Bioorg. Med. Chem.2006, 14,2446-2449.
[79]V. Pozsgay, J. R. Brisson, H. J. Jennings, J. Org. Chem.,1991,56,3377-3385.
[80]H. A. Chokhawala, H. Cao, Hai Yu, X. Chen,J. Am. Chem. Soc.2007,129,10630-10631.
[81]Z. Liu, J. Zhang, X. Chen, P. G. Wang, Chem. Bio. Chem.2002,3,348-355.
[82]H. Yu, X. Chen, Org. Lett.2006,8,11,393-2396.
[83]T. J. Morley, S. G. Withers, J. Am. Chem. Soc.2010,132,9430-9437.
[84]S. Muthana, H.Yu, H. Cao, J. Cheng, X. Chen, J. Org. Chem.2009,74,2928-2936.
[85]H. Yu, J. Cheng, L. Ding, Z. Khedri, Y. Chen, S. Chin, K. Lau, V. K. Tiwari, X. Chen, J. Am. Chem. Soc.2009,131,18467-18477.
[86]J. T. Seto, R. Rott, Virology,1966,30,4,731-737.
[87]R. H. Quarles, J. Neurochem.2007,100,1431-1448.
[88]N. R. Mehta, T. Nguyen, J. W. Bullen, Jr., J. W. Griffin, R. L. Schnaar, ACS Chem. Neurosci. 2010,1,215-222.
[89]J. Chen, G. Chen, B. Wu, Q. Wan, Z. Tan, Z. Huaa, S. J. Danishefsky, Tetrahedron Lett 2006, 47,8013-8016.
[90]P. A. Orlandi, F. W. Klotz, J. D. Haynes, J. Cell Biol.1992,116,4,901-909.
[91]T. Nguyen, N. R. Mehta, K. Conant, K. J. Kim, M. Jones, P. A. Calabresi, G. Melli, A. Hoke, R. L. Schnaar, G L. Ming, H. Song, S. C. Keswani, J. W. Griffin, J. Neurosci.2009,29,3: 630-637.
[92]R. L. Schnaar, FEBS Letters 2010,584,1741-1747.
[93]P. H.H. Lopez, R. L Schnaar, Curr Opin Struct Biol 2009,19,549-557.
[94]J. B. Schwarz, S. D. Kuduk, X. T.Chen, D. Sames, P. W. Glunz, S. J. Danishefsky, J. Am. Chem. Soc.1999,121,2662-2673.
[95]O. Blixt, K. Allin, L. Pereira, A. Datta, J. C. Paulson, J. Am. Chem. Soc.2002,124,5739-5746.
[96]S. Huang, H. Yu, X. Chen, Sci China Chem.2011,54,1,117-128.
[97]K. Lau, H. Yu, V. Thon, Z. Khedri, M. E. Leon, B. K. Tran, X. Chen, Org. Biomol. Chem.2011, 9,2784-2789.
[98]H. A. Chokhawala, H. Cao, H. Yu, X. Chen, J.'Am. Chem. Soc.2007,129,10630-10631.
[99]H. Yu, S. Huang, H. Chokhawala, M. Sun, H. Zheng, X. Chen, Angew. Chem. Int. Ed. Engl. 2006,45,24,3938-3944.
[100]L. Ding, H. Yu, K. Lau, Y. Li, S. Muthana, J. Wanga, X. Chen, Chem. Commun.2011,47, 8691-8693.
[101]H. Yu, V. Thon, K.Lau, L. Cai, Y. Chen, S. Mu, Y. Li, P. G. Wang, X. Chen, Chem Commun, 2010,46,40,7507-7509.
[102]J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev.2007,36,1249-1262.
[103]H. C. Kolb, K. B. Sharpless, Drug Discov Today 2003,8,24,1128-1137.
[104]H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed,2001,40,2004-2021.
[105]S. J. Danishefsky, K. Koseki, D. A. Griffith, J. Gervay, J. M. Peterson, F. E. McDonald, T. Oriyama, J. Am. Chem. Soc.1992,114,8331-8333.
[106]Y. P. Yu, M. C. Cheng, H. R. Lin, C. H. Lin, S. H. Wu, J. Org. Chem.2001,66,5248-5251.
[107]Y. Zhang, Y. C. Lee, JBiol Chem 1999,274,10,6183-6189.
[108]N. M. Xavier, A. P. Rauter, and Y. Queneau, Top. Cur.r Chem.2010,295,19-62.
[109]P. Allevi, P. Rota, R. Scaringi, R. Colombo, M. Anastasia, J. Org. Chem.2010,75, 5542-5548.
[110]T. Eckert, C. P. Lu, C. S. Chen, S. H. Wu, J. Gervay-Hague, Tetrahedron Lett 2011,52, 2250-2253.
[111]P. Allevi, M. Anastasia, M. L. Costa, P. Rota, Tetrahedron:Asymmetry 2011,22,338-344.
[112]M. E. DeBellard, S. Tang, G. Mukhopadhyay, Mol Cell Neurosci.1996,7,2,89-101.
[113]B. E. Collins, Lynda J. S. Yang, G. Mukhopadhyayi, M. T. Filbini, M. Kiso, A. Hasegawa, R. L. Schnaar, J. Biol. Chem.1997,272,1248-1255.
[114]Hubli Prabhanjan, Kumiko Aoyama, Makoto Kiso, Akira Hasegawa, Carbohydr. Res.1992, 233,87-99.
[115]B. Ernst, J. L. Magnani, Nat. Rev. Drug Discov.2009,8,661-677.
[116]R. L. Schnaar, P. H.H. Lopez, J. Neurosci. Res.2009,87:3267-3276.
[117]A. Varki, Nature 2007,446,1023-1029.
[118]O. Schwardt, H. Gathje, A.Vedani, S. Mesch, G P Gao, M. Spreafico, J. von Orelli, S. Kelm, B. Ernst, J. Med. Chem.2009,52,989-1004.
[119]D. Schwizer, H. Gathje, S. Kelm, M. Porro, O. Schwardta, B. Ernst, Bioorg. Med. Chem.2006, 14,4944.4957.
[120]A. Bhunia, O. Schwardt, H. Gathje, G P. Gao, S. Kelm, A. J. Benie, M. Hricovini, T. Peters, Beat Ernst, Chem. Bio. Chem.2008,9,2941-2945.
[121]P. K. Vered, H. Yu, H. Cao, H. Chokhawala, F. Karp, N. Varki, X. Chen, A. Varki, Glycobiology 2008,18,10,818-830.
[122]I. O. Nasonkin, V. E. Koliatsos, Exp. Neurol.2006,201,525-529.
[2]蔡孟深,李中军,糖化学.化学工业出版社：2006.
[3]Z. Zhang, I. R.Ollmann, X. S.Ye, R.Wischnat, T. Baasov, C, H.Wong, J. Am. Chem. Soc.1999, 121,4,734-753.
[4]O. J. Plante, E. R. Palmacci, P. H. Seeberger, Science 2009,1523-1527.
[5]P. H. Seeberger, Chem. Soc. Rev.2008,37,1,19-28.
[6]H. A. Orgueira, A. Bartolozzi, P. Schell, R. E. Litjens, E. R. Palmacci, P. H. Seeberger, Chem. Eur.J.2002,1,140-169.
[7]T. K. K Mong, H. K. Lee, S. G. Duron, C. H.Wong, PNAS 2003,100,3,797-802.
[8]F. Burkhart, Z. Zhang, S. Wacowich-Sgarbi, C. H. Wong, Angew. Chem. Int. Ed.2001,40,7, 1274-1276.
[9]X. S. Ye, C. H. Wong. J. Org Chem.2000,65,2410-2431.
[10]X. Huang, L. Huang, H.Wang, X. S. Ye, Angew. Chem. Int. Ed.2004,43,5221-5224.
[11]C. H. Wong, X. S. Ye, Z. Zhang, J. Am. Chem. Soc.1998,120,7137-7138.
[12]Z. Wang, Y. M. Xu, B.Yang, G. Tiruchinapally, B. Sun, R. P. Liu, S. Dulaney, J. A. Liu, X. F. Huang, Chem. Eur. J.2010,16,8365.
[13]S.J. Danishefsky, K. F. McClure, J. T. Randolph, R.B. Ruggeri, Science 1993,260,1307.
[14]S. P. Douglas, D. M. Wh itfield, J. J. KrePinsky, J. Am. Chem. Soc.1991,113,5095.
[15]G H.Veeneman, H. F. Brugghe, H. van den Elst, J. H. van Boom, Carbohydr. Res.1990,195,2 C1-4.
[16]L. L. Lairson, B. Henrissat, G. J. Davies, Annu. Rev. Biochem.2008,77,521-555.
[17]P. M. Coutinho, E. Deleury, G J. Davies, B. Henrissat and S.G.Withers, J. Mol. Biol.2003,328, 2,307-317.
[18]B. R. Griffith, J. M. Langenhan, J. S. Thorson, Curr. Opin. Biotechnol.2005,16,6,622-630.
[19]W. A. Barton, J. Lesniak, J. B. Biggins, P. D. Jeffrey, J. Jiang, K.R. Rajashankar, J. S. Thorson and D. B. Nikolov, Nat. Struct. Biol.2001,8,6,545-551.
[20]J. M. Langenhan, B. R. Griffith, and J. S. Thorson, J. Nat. Prod,2005,68,1696-1711.
[21]K. Emami, T. Nagy, C. M. G A. Fontes, L. M. A. Ferreira and H. J. Gilbert, J. Bacteriol.2002, 184,15,4124.
[22]K. A. Stubbs, M. Balcewich, B. L. Mark, and D. J. Vocadlo, J. Biol. Chem.2007,282,29, 21382-21391.
[23]D. Hogg, G. Pell, P. Dupree, F. Goubet, S. M Martin-Orue, S. Armand, and Harry J Gilbert, Biochem. J.2003,371,1027-1043.
[24]R. V. Stick and K. A. Stubbs, Tetrahedron:Asymmetry 2005,16,321-335.
[25]S. J. Williams, S. G Withers, Carbohydr. Res.2000,327,2,27-46.
[26]S. G. Withers, Carb. Polymers.2001,44,4,325-337.
[27]卢丽丽,肖敏,赵晗,王鹏,钱新民.生物化学与生物物理进展,2006,33,4,310-320.
[28]Raymond A Dwek, Chem. Rev.1996,96,683-720.
[29]P. M. Rudd, T. Elliott, P. Cresswell, I.A. Wilson, R. A. Dwek, Science 2001,291,2370-2375.
[30]S. J. Hwang, C. M. Ballantyne, A. R. Sharrett, L. C. Smith, Circulation 1997,96,4219-4225.
[31]A. E. Koch, M. M. Halloran, C. J. Haskell, M. R. Shah, P. J. Polverini, Nature 1995,376,6540, 517-519.
[32]F. Austrup, D. Vestweber, E. Borges, M. Lohning, R. Brauer, U. Herz, H. Renz, R. Hallmann, A.r Scheffold, A. Radbruch, A. Hamann, Nature 1997,385,81-83.
[33]M. Steegmaler, A. Levinovitz, S. Isenmann, E. Borges, M. Lenter, H. P. Kocher, B. Kleuser, D. Vestweber, Nature 1995,373,615-620.
[34]H. Park, K. Y. Hwang, K. H. Oh et al, Bioorganic Medicihal Chemistry 2008,16,1,284.
[35]Y. Wu, J.H. Yang, G. F. Dai, et al, Bioorganic Medicinal Chemistry 2009,17,4,1464.
[36]K. Matsumoto, K. Takeraata, Analytical Science 2002,18,12,1315.
[37]R. Pili, J. Chang, R.A. Partis, et al, Cancer Res 1995,55,13,2920.
[38]T. M. Block, X. Lu, A. S. Mehta, B. S. Blumberg, B. Tennant, M. Ebling, B. Korba, D. M. Lansky, G. S. Jacob, R. A. Dwek, Nat. Med.1998,4,610-614.
[39]L. F. Steel, D. Shumpert, M. Trotter, S. H. Seeholzer, A. A. Evans, W. T. London, R. Dwek and T. M. Block, Protromics 2003,3,5,601-609.
[40]N. Zitzmann, A. S. Mehta, S. Carrouee, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, T. M. Block 1999,96,21,11878-11882.
[41]E. B. Ave, S. Carrouee, B.T. Butters, R. A. Dwek and T. M. Block, Hepatology 2001,33,6, 1488-1495.
[42]R. B. Parekh, R. A. Dwek, B. J. Sutton, D. L. Fernandes, A. Leung, D. Stanworth, T. W. Rademacher, T. Mizuochi, T. Taniguchi, K. Matsuta, F. Takeuchi, Y. Nagano, T. Miyamoto & A. Kobata, Nature 1985,316,452-457.
[43]T. Taniguchi, T. Mizuochi, M. Beale, R. A. Dwek, T.W. Rademacher, A. Kobata, Biochemistry 1985,24,20,5551-5557.
[44]F. Kiyoshi, K. Akira, Mol Immunol 1991,28,12,1333-1340.
[45]Y. J. Chen, D. R. Wing, G R. Guile, R. A. Dwek, D. J. Harvey, S. Zamze, Eur. J. Biochem.1998, 251,3,691-703.
[46]S.W. Homans, R.A. Dwek, D.L. Fernandes, T.W. Rademacher, FEBS Lett 1982,150,2, 503-506.
[47]K. Furukawa, K. Matsuta, F. Takeuchi, E. Kosuge, T. Miyamoto and A. Kobata, Int. Immunol 1990,2,1,105-112.
[48]A. Youings, S. C. Chang, R. A. DweK and I. G Scragg, Biochem. J.1996,314,621-630.
[49]Y. Abe, E. Takashita, K. Sugawara, et al, International Congress Series 2004,1263,2,214-217.
[50]T. Lin, G Wang, A. Li, et al, Virology 2009,392,1,73-81.
[51]J. Stevens, O. Blixt, T. M. Tumpey, Science 2006,312,5772,404.
[52]D. Xu, E. I. Newhouse, R. E. Amaro, J Mol Biol 2009,387,2,465-491.
[53]L. S. C Kreisman and B. A. Cobb, Glycobiology 2012,22 (8),1019-1030.
[54]J. Stevens, O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C. Paulson, I.A. Wilson, Science 2006,312,404.
[55]R. R. Schumann, S. R. Leong, G. W. Flaggs, P. W. Gray, S. D. Wright, J. C. Mathison, P. S. Tobias, R. J. Ulevitch, Science,1990,249,4975,1429-1431.
[56]J. C. Chow, D. W. Young, D. T. Golenbock, W. J. Christ, F. Gusovsky, J. Biol. Chem.1999,274, 10689-10692.
[57]M. J. Kiefel, M. von Itzstein, Prog. Med. Chem.1999,36,1-28.
[58]A. Dondoni, A. Marra, P. Merino, J. Am. Chem. Soc.1994,116,3324-3336.
[59]S. J. Danishefsky, M. P. DeNinno, S. H. Chen, J. Am. Chem. Soc.1988,110,3929-3940.
[60]L. S. Li, Y. L. Wu, Y. Wu, Org. Lett 2000,2,891-894.
[61]J. M. Haberman, D. Y. Gin, Org. Lett.2001,3,1665.
[62]G J. Boons, A. V. Demchenko, Chem. Rev.2000,100,4539-4566.
[63]A. V. Demchenko, G. J. Boons, Tetrahedron Lett.1998,39,3065-3068.
[64]A. V. Demchenko, G. J. Boons, Chem.OEur. J.1999,5,1278-1283.
[65]K. Matsuokaa, T. Onagaa, T. Moria, J. I. Sakamotoa, T. Koyamaa, N. Sakairic, K. Hatanoa, D.Terunumaa, Tetrahedron Letters 2004,45,51,9383-9386.
[66]K. Matsuokaa, C. Takitaa, T. Koyamaa, D. Miyamotob, S. Yingsakmongkond, K. I. P. J. Hidarib, W. Jampangerne, T. Suzukib, Y. Suzukic, K. Hatanoa, D. Terunumaa, Bioorg. Med. Chem. Lett.2007,17,3826-3830.
[67]T. T. Martin, R. R. Schmidt, Tetrahedron Lett 1992,33,6123.
[68]H. Kondo, Y. Ichikawa, C. H. Wong, J. Am. Chem. Soc,1992,114,8748.
[69]梁芬芬,陈力,邢国文.有机化学,2009,29,9,1317-1324.
[70]T. K. K. Mong, H. K. Lee, S. G. Duron, C. H.Wong, Proc. Natl. Acad. Sci.2003,100,797.
[71]S. Cai, and B. Yu,, Org. Lett.2003,5,3827-3830.
[72]Y. Ito, T. Ogawa, Tetrahedron 1990,46,89.
[73]D. Ye, W. Liu, D. Zhang, E. Feng, H. Jiang, and H. Liu,J. Org. Chem.2009,74,1733-1735.
[74]H. Tanaka, Y. Nishiura, and T. Takahashi, J. Am. Chem. Soc.2008,130,17244-17245.
[75]D. Crich, and W. Li, J. Org. Chem.2007,72,2387-2391.
[76]D. Crich, and B. Wu, Org. Lett.2008,10,4033-4035.
[77]H. Guo, L. Li, and P. G. Wang, Biochemistry 2006,45,13760-13768.
[78]G L. Huang, D.W. Zhang, H. J. Zhao, H. C. Zhang and P. G. Wang, Bioorg. Med. Chem.2006, 14,2446-2449.
[79]V. Pozsgay, J. R. Brisson, H. J. Jennings, J. Org. Chem.,1991,56,3377-3385.
[80]H. A. Chokhawala, H. Cao, Hai Yu, X. Chen,J. Am. Chem. Soc.2007,129,10630-10631.
[81]Z. Liu, J. Zhang, X. Chen, P. G. Wang, Chem. Bio. Chem.2002,3,348-355.
[82]H. Yu, X. Chen, Org. Lett.2006,8,11,393-2396.
[83]T. J. Morley, S. G. Withers, J. Am. Chem. Soc.2010,132,9430-9437.
[84]S. Muthana, H.Yu, H. Cao, J. Cheng, X. Chen, J. Org. Chem.2009,74,2928-2936.
[85]H. Yu, J. Cheng, L. Ding, Z. Khedri, Y. Chen, S. Chin, K. Lau, V. K. Tiwari, X. Chen, J. Am. Chem. Soc.2009,131,18467-18477.
[86]J. T. Seto, R. Rott, Virology,1966,30,4,731-737.
[87]R. H. Quarles, J. Neurochem.2007,100,1431-1448.
[88]N. R. Mehta, T. Nguyen, J. W. Bullen, Jr., J. W. Griffin, R. L. Schnaar, ACS Chem. Neurosci. 2010,1,215-222.
[89]J. Chen, G. Chen, B. Wu, Q. Wan, Z. Tan, Z. Huaa, S. J. Danishefsky, Tetrahedron Lett 2006, 47,8013-8016.
[90]P. A. Orlandi, F. W. Klotz, J. D. Haynes, J. Cell Biol.1992,116,4,901-909.
[91]T. Nguyen, N. R. Mehta, K. Conant, K. J. Kim, M. Jones, P. A. Calabresi, G. Melli, A. Hoke, R. L. Schnaar, G L. Ming, H. Song, S. C. Keswani, J. W. Griffin, J. Neurosci.2009,29,3: 630-637.
[92]R. L. Schnaar, FEBS Letters 2010,584,1741-1747.
[93]P. H.H. Lopez, R. L Schnaar, Curr Opin Struct Biol 2009,19,549-557.
[94]J. B. Schwarz, S. D. Kuduk, X. T.Chen, D. Sames, P. W. Glunz, S. J. Danishefsky, J. Am. Chem. Soc.1999,121,2662-2673.
[95]O. Blixt, K. Allin, L. Pereira, A. Datta, J. C. Paulson, J. Am. Chem. Soc.2002,124,5739-5746.
[96]S. Huang, H. Yu, X. Chen, Sci China Chem.2011,54,1,117-128.
[97]K. Lau, H. Yu, V. Thon, Z. Khedri, M. E. Leon, B. K. Tran, X. Chen, Org. Biomol. Chem.2011, 9,2784-2789.
[98]H. A. Chokhawala, H. Cao, H. Yu, X. Chen, J.'Am. Chem. Soc.2007,129,10630-10631.
[99]H. Yu, S. Huang, H. Chokhawala, M. Sun, H. Zheng, X. Chen, Angew. Chem. Int. Ed. Engl. 2006,45,24,3938-3944.
[100]L. Ding, H. Yu, K. Lau, Y. Li, S. Muthana, J. Wanga, X. Chen, Chem. Commun.2011,47, 8691-8693.
[101]H. Yu, V. Thon, K.Lau, L. Cai, Y. Chen, S. Mu, Y. Li, P. G. Wang, X. Chen, Chem Commun, 2010,46,40,7507-7509.
[102]J. E. Moses and A. D. Moorhouse, Chem. Soc. Rev.2007,36,1249-1262.
[103]H. C. Kolb, K. B. Sharpless, Drug Discov Today 2003,8,24,1128-1137.
[104]H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed,2001,40,2004-2021.
[105]S. J. Danishefsky, K. Koseki, D. A. Griffith, J. Gervay, J. M. Peterson, F. E. McDonald, T. Oriyama, J. Am. Chem. Soc.1992,114,8331-8333.
[106]Y. P. Yu, M. C. Cheng, H. R. Lin, C. H. Lin, S. H. Wu, J. Org. Chem.2001,66,5248-5251.
[107]Y. Zhang, Y. C. Lee, JBiol Chem 1999,274,10,6183-6189.
[108]N. M. Xavier, A. P. Rauter, and Y. Queneau, Top. Cur.r Chem.2010,295,19-62.
[109]P. Allevi, P. Rota, R. Scaringi, R. Colombo, M. Anastasia, J. Org. Chem.2010,75, 5542-5548.
[110]T. Eckert, C. P. Lu, C. S. Chen, S. H. Wu, J. Gervay-Hague, Tetrahedron Lett 2011,52, 2250-2253.
[111]P. Allevi, M. Anastasia, M. L. Costa, P. Rota, Tetrahedron:Asymmetry 2011,22,338-344.
[112]M. E. DeBellard, S. Tang, G. Mukhopadhyay, Mol Cell Neurosci.1996,7,2,89-101.
[113]B. E. Collins, Lynda J. S. Yang, G. Mukhopadhyayi, M. T. Filbini, M. Kiso, A. Hasegawa, R. L. Schnaar, J. Biol. Chem.1997,272,1248-1255.
[114]Hubli Prabhanjan, Kumiko Aoyama, Makoto Kiso, Akira Hasegawa, Carbohydr. Res.1992, 233,87-99.
[115]B. Ernst, J. L. Magnani, Nat. Rev. Drug Discov.2009,8,661-677.
[116]R. L. Schnaar, P. H.H. Lopez, J. Neurosci. Res.2009,87:3267-3276.
[117]A. Varki, Nature 2007,446,1023-1029.
[118]O. Schwardt, H. Gathje, A.Vedani, S. Mesch, G P Gao, M. Spreafico, J. von Orelli, S. Kelm, B. Ernst, J. Med. Chem.2009,52,989-1004.
[119]D. Schwizer, H. Gathje, S. Kelm, M. Porro, O. Schwardta, B. Ernst, Bioorg. Med. Chem.2006, 14,4944.4957.
[120]A. Bhunia, O. Schwardt, H. Gathje, G P. Gao, S. Kelm, A. J. Benie, M. Hricovini, T. Peters, Beat Ernst, Chem. Bio. Chem.2008,9,2941-2945.
[121]P. K. Vered, H. Yu, H. Cao, H. Chokhawala, F. Karp, N. Varki, X. Chen, A. Varki, Glycobiology 2008,18,10,818-830.
[122]I. O. Nasonkin, V. E. Koliatsos, Exp. Neurol.2006,201,525-529.