IGF::OT::IGF TITLE: CHEMO-ENZYMATIC SYNTHESIS OF STRUCTURALLY DEFINED COMPLEX-TYPE N-GLYCANS

IGF::OT::IGF 标题：结构明确的复合型 N-聚糖的化学酶合成

• 批准号: 9154682
• 负责人: LIU YUNPENG, PH.D.
• 金额: $ 99.22万
• 依托单位: CHEMILY, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2017-09-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9154682
• 关键词: Chemicals; Complex; Contractor; Development; Diagnostic; Disease; Enzymes; Glycobiology; Glycoside Hydrolases; High Pressure Liquid Chromatography; Libraries; Malignant Neoplasms; Methodology; Methods; Natural Resources; Phase; Polysaccharides; Process; Research; Structure; Therapeutic; Vendor; base; carbohydrate binding protein; chemical synthesis; glycosyltransferase; improved

Access to structurally defined N-glycans is an urgent scientific need for glycan arrays for rapid analysis of carbohydrate binding proteins (CBPs), development of diagnostics and therapeutics for many diseases, as well as glycoanalysis. However, only a small number of N-glycans are currently commercially available, most of which are symmetric ones. The lack of structurally defined glycans has greatly hampered research in Glycobiology. Due to complexity and low abundance of N-glycans, it is extremely difficult to separate and purify homogenous N-glycans from natural resources. Thus chemical synthesis of N-glycans has been pursued instead. However, very limited N-glycans have been synthesized due to challenges in the chemical methodology. In the preceding Phase I proposal, the contractor developed a facile and efficient chemical core synthesis with an enzymatic extension (CCSEE) strategy and a rapid HPLC-based purification approach for N-glycan synthesis. The contractor successfully prepared 75 structurally defined N-glycans with a purity of 98%. In this Phase II project, the Contractor shall further improve the CCSEE strategy for synthesis of cancer-related N-glycans, including Neu5Gc-terminated, core-fucosylated, bisected, and 1,6 branched N-glycans. These glycans cover the chemical space of bi-, tri- and tetra-antennary complex-type N-glycans. This process involves several well-characterized glycosyltransferases, glycoside hydrolases, and other enzymes. All the proposed N-glycans shall be purified to 98%, and fully characterized by HPLC, MS, and partially by NMR.

获得结构上确定的N-聚糖是对用于快速分析碳水化合物结合蛋白（CBP）、开发许多疾病的诊断和治疗以及糖分析的聚糖阵列的迫切科学需求。然而，目前只有少量的N-聚糖是市售的，其中大多数是对称的。缺乏结构上确定的聚糖极大地阻碍了糖生物学的研究。由于N-聚糖的复杂性和低丰度，从天然资源中分离和纯化同质N-聚糖是极其困难的。因此，N-聚糖的化学合成已被替代。然而，由于化学方法学的挑战，已经合成了非常有限的N-聚糖。在之前的第一阶段提案中，承包商开发了一种简单有效的化学核心合成，采用酶促延伸（CCSEE）策略和基于HPLC的快速纯化方法进行N-聚糖合成。承包者成功制备了75种结构明确的N-聚糖，纯度为98%。在该二期项目中，承包商应进一步改进CCSEE的癌症相关N-聚糖合成策略，包括Neu 5Gc末端、核心岩藻糖基化、二等分和1，6分支N-聚糖。这些聚糖覆盖了双触角、三触角和四触角复合型N-聚糖的化学空间。该过程涉及几种充分表征的糖基转移酶、糖苷水解酶和其他酶。所有拟定N-聚糖均应纯化至98%，并通过HPLC、MS和部分NMR进行充分表征。