二糖侧链FG的确切结构特征及其对FXase抑制活性的影响研究

Fucosylated glycosaminoglycan (FG) is the only glycosaminoglycan derivative containing glycosyl branches found to date. It possesses unique structure and potent anticoagulant activity, especially intrinsic FXase inhibition activity. It is agreed that FG has a chondroitin sulfate-like backbone and the side chains composed of fucose, while its precise structure is still under controversy and remains to be clarified. Previously, our researches have elucidated the precise structures of the native FGs from various sea cucumbers, by glycosidic bond-selective depolymerization, purified oligosaccharides preparation and structural analysis technology. Particularly, an octasaccharide was discovered to be the minimal structure fragment with potent anti-FXase activity. In the recent study of the FGs from Thelenota ananas and Ludwigothurea grisea (TaFG and LgFG), a disaccharide branch and a tetrasaccharide which contains such disaccharide were obtained from their depolymerized products. This work may firstly demonstrate the presence of a glycosaminoglycan (GAG) substituted with unique disaccharide branches in nature. This project intends to apply the glycosidic bond-selective method of β-eliminative depolymerization, combined with gel permeation chromatography (GPC) and ion exchange chromatography (IEC), to obtain purified oligosaccharides with various sequences from TaFG and LgFG, and further analyze the precise structural characteristics of the novel FG. Moreover, the anti-FXase activity and structure-activity relationship studies may lead to the discovery of FXase inhibitors with more simple structure.

岩藻糖化糖胺聚糖(FG)是目前仅见的含支链糖基的糖胺聚糖衍生物，具有特殊化学结构和强效抗凝活性，尤其是内源性因子X酶(FXase)抑制活性。目前，学术界一致认为FG具有类似硫酸软骨素的主链，岩藻糖(Fuc)以侧链形式连接于主链，但其确切结构尚存疑问和争议。本课题前期通过糖苷键选择性解聚和纯化寡糖制备与结构解析技术阐明了多种海参来源的天然FG的确切结构，发现了强效抑制FXase的最小结构片段—八糖。新近发现Thelenota ananas和Ludwigothurea grisea来源FG(TaFG和LgFG)的解聚产物中存在侧链二糖和含侧链二糖的四糖，可能首次证明自然界存在特殊的二糖侧链取代的糖胺聚糖(GAG)。本项目拟采用糖苷键选择性的β-消除解聚技术，结合凝胶渗透色谱(GPC)和离子交换色谱(IEC)法从TaFG和LgFG中获得不同序列的纯化寡糖，解析新颖FG的确切结构特征；同时通过构效关系研究，尝试发现更简结构的FXase抑制剂。

岩藻糖化糖胺聚糖(FG)是目前仅见的含支链糖基的糖胺聚糖衍生物，具有特殊化学结构和强效抗凝活性，尤其是内源性FXase抑制活性。前期研究发现，Thelenota ananas和Ludwigothurea grisea来源FG(TaFG和LgFG)可能存在二糖侧链。本项目采用糖苷键选择性的β-消除解聚技术，结合GPC和IEC法从低聚TaFG和低聚LgFG中获得不同序列的纯化寡糖，首次证明自然界存在二糖侧链取代的FG，且其二糖侧链结构与文献推测Fuc二糖不同。TaFG所含二糖侧链为Gal-α1,2-Fuc，而LgFG中二糖侧链为GalNAc-α1,2-Fuc，二糖侧链均为杂多糖，且还原端糖基存在多样性（Gal/GalNAc）；此外，天然FG二糖侧链或单糖侧链上不同硫酸酯基取代类型进一步增加了天然FG结构多样性特点。同时，不同海参来源FG主链存在保守结构特征，均类似于硫酸软骨素E。基于纯化寡糖的构效关系分析显示，在FG来源的Fuc单糖侧链的寡糖中，八糖为具有anti-FXase活性的最小结构片段，且Fuc2S4S侧链的八糖活性最强；在FG来源的含二糖侧链的寡糖中，寡糖结构达到三个重复结构单元时，二糖侧链(Gal/GalNAc-α1,2-Fuc)对寡糖的anti-FXase活性无显著影响，但二糖侧链端基糖(Gal/ GalNAc)的硫酸酯基取代类型可明显影响其anti-FXase活性强度；二糖侧链可显著增强含两个重复结构单元的寡糖活性，含二糖侧链的七糖具有强效anti-FXase活性及强效抗血栓活性，同时显示出低出血倾向优势特征，将为新型FXase抑制剂的设计及研发提供借鉴。