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

获得结构明确的n -聚糖是糖阵列快速分析碳水化合物结合蛋白（CBPs）、开发许多疾病的诊断和治疗以及糖分析的迫切科学需求。然而，目前市面上只有少量的n -聚糖，而且大多数是对称的n -聚糖。缺乏结构明确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，从天然资源中分离纯化均相n -聚糖是极其困难的。因此，n -聚糖的化学合成已被追求代替。然而，由于化学方法上的挑战，合成的n -聚糖非常有限。在之前的第一阶段提案中，承包商开发了一种使用酶延伸（CCSEE）策略的简单高效的化学核心合成和基于高效液相色谱的n -聚糖合成快速纯化方法。承包商成功制备了75个结构明确的n -聚糖，纯度为98%。在本二期项目中，承包商将进一步完善CCSEE合成癌症相关n -聚糖的策略，包括neu5gc端化、核心聚焦、分切和1,6支链的n -聚糖。这些聚糖覆盖了双、三、四天线络合型n -聚糖的化学空间。这个过程涉及到几种特征明确的糖基转移酶、糖苷水解酶和其他酶。所有提议的n -聚糖必须纯化至98%，并通过HPLC， MS和部分NMR进行充分表征。

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.