Chemoenzymatic Synthesis of Heparin and Heparan Sulfate Oligosaccharides

描述（由申请人提供）：肝素（HP）和硫酸肝素（HS）参与多种生理和病理事件，包括病毒感染、血液凝固、细胞分化和癌症转移。其多方面的生物学活性表明，HP/HS作为新型治疗药物具有巨大的潜力。然而，获得结构明确的HP/HS低聚糖非常困难，这严重阻碍了详细结构活性关系的建立。在此应用中，提出了一种基于化学酶方法的合成策略，以获得结构多样，精确设计的HP/HS低聚糖面板。目的1：研究基于预活化的一锅糖基化法化学合成HP/HS低聚糖。目标结构将系统地变化，包括葡萄糖醛酸和伊杜醛酸在主链中，多样化的o -磺化模式和差异化的氮修饰。大小接近多糖的寡糖也将被组装。基于预活化的一锅糖基化法具有快速合成HP/HS低聚糖的优势。在目标2中，化学合成将与酶修饰相结合。化学制备的HP/HS低聚糖将被硫转移酶以不同的方式修饰，从而进一步增加其序列多样性。此外，糖基转移酶将被用于延长功能化的HP/HS低聚糖，从而提供获得由不同结构域组成的低聚糖的途径。在目标3中，将检测精确设计的HP/HS低聚糖的肝素酶抑制活性以及生长因子和血小板因子4的结合。将评估主链序列、氮取代和o -硫酸化的影响，以开发具有低不良生物相互作用的高特异性肝素酶抑制剂。这些研究结果将为HP/HS的结构-功能关系研究奠定基础和工具，为发现基于HP/HS的新型治疗剂提供令人兴奋的机会。

DESCRIPTION (provided by applicant): Heparin (HP) and heparan sulfate (HS) participate in a wide variety of physiological and pathological events, including viral infection, blood coagulation, cell differentiation and cancer metastasis. Their multi-faceted biological activities suggest that there are tremendous potential in using HP/HS as novel therapeutics. However, access to structurally well defined HP/HS oligosaccharides has been very difficult, which severely hinders the establishment of detailed structure activity relationships. In this application, a new synthetic strategy based on chemoenzymatic methods is proposed to acquire a panel of structurally diverse, precisely designed HP/HS oligosaccharides. In aim 1, chemical synthesis of HP/HS oligosaccharides using the pre-activation based one pot glycosylation method will be studied. The target structures will be systematically varied to include both glucuronic acid and iduronic acid in the backbone, diversified O-sulfation patterns, and differentiated nitrogen modifications. Oligosaccharides with sizes approaching those of polysaccharides will also be assembled. The pre-activation based one pot glycosylation method is highly advantageous as it allows rapid synthesis of HP/HS oligosaccharides with great sequence diversity. In aim 2, chemical synthesis will be integrated with enzymatic modification. The chemically prepared HP/HS oligosaccharides will be modified by sulfo transferases in a divergent manner, thus further increasing their sequence diversity. Moreover, glycosyl transferases will be used to elongate the functionalized HP/HS oligosaccharides, providing access to oligosaccharides composed of distinct domains. In aim 3, the precisely designed HP/HS oligosaccharides will be assayed for their heparanase inhibitory activities as well as growth factor and platelet factor 4 binding. The effects of backbone sequence, nitrogen substitution and O-sulfation will be evaluated to develop a highly specific heparanase inhibitor with low undesired biological interactions. The results of the proposed studies will establish the basis and tools for the structure-function relationship studies of HP/HS, leading to exciting opportunities for discovery of HP/HS based novel therapeutic agents. PUBLIC HEALTH RELEVANCE: Heparin (HP) and heparan sulfate (HS) interact with many proteins involved in important biological processes such as cell differentiation, pathogen infection, cancer metastasis, and blood coagulation. The long term goal of this project is to prepare structurally well defined HP/HS oligosaccharides, and use these precisely designed compounds to establish the structure and activity relationship of HP/HS. This can potentially lead to the development of novel therapeutic agents against blood coagulation, cancer and viral infection.