Chemoenzymatic Synthesis of Heparin and Heparan Sulfate Oligosaccharides

说明(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作为新的治疗药物具有巨大的潜力。然而，获得结构明确的HP/HS寡糖一直是非常困难的，这严重阻碍了建立详细的结构活性关系。在这一应用中，提出了一种基于化学酶方法的新的合成策略，以获得一组结构多样、设计精确的HP/HS寡糖。在目标1中，我们将研究基于预活化的一锅法化学合成HP/HS低聚糖。目标结构将被系统地改变，以包括主干上的葡萄糖醛酸和艾杜糖酸，多样化的O-硫化模式，以及不同的氮修饰。大小接近多糖的低聚糖也将被组装。基于预活化的一锅法糖基化方法具有很大的优势，因为它可以快速合成具有很大序列多样性的HP/HS寡糖。在目标2中，将把化学合成和酶修饰结合起来。化学合成的HP/HS寡糖将以不同的方式被磺基转移酶修饰，从而进一步增加它们的序列多样性。此外，糖基转移酶将被用来延长功能化的HP/HS寡糖，提供进入由不同结构域组成的寡糖的途径。在目标3中，精确设计的HP/HS寡糖将被检测其乙酰肝素酶抑制活性以及生长因子和血小板因子4的结合。将评估主干序列、氮取代和O-硫化的影响，以开发出一种高度特异的肝素酶抑制剂，具有较低的不良生物相互作用。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的新型治疗药物带来令人兴奋的机会。 公共卫生相关性：肝素(HP)和硫酸肝素(HS)与许多参与重要生物学过程的蛋白质相互作用，如细胞分化、病原体感染、癌症转移和凝血。该项目的长期目标是制备结构明确的HP/HS寡糖，并使用这些精确设计的化合物来建立HP/HS的结构和活性关系。这可能会导致开发抗凝血、癌症和病毒感染的新型治疗剂。

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.