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

获得结构定义的N-糖链是糖链阵列的迫切科学需要，用于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方法，以及糖分析。然而，目前只有一小部分N-葡聚糖可以在商业上获得，其中大多数是对称的。缺乏结构明确的多糖极大地阻碍了糖生物学的研究。由于N-葡聚糖的复杂性和低丰度，从自然资源中分离纯化均一的N-葡聚糖是非常困难的。因此，N-葡聚糖的化学合成一直是人们追求的目标。然而，由于化学方法学上的挑战，N-葡聚糖的合成非常有限。在前一阶段的提案中，承包商开发了一种简单而高效的化学核心合成方法，其中包括酶延伸(CCSEE)策略和基于高效液相的N-糖聚糖合成的快速纯化方法。承包商成功地制备了75个结构定义的N-葡聚糖，纯度为98%。在该第二阶段项目中，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-末端、核心-岩藻糖基化、对分和1，6-支链N-聚糖。这些多聚糖覆盖双、三和四触角复合型N-多聚糖的化学空间。这一过程涉及几种特性良好的糖基转移酶、糖苷水解酶和其他酶。所有拟合成的N-葡聚糖的纯度均应达到98%，并通过高效液相色谱、质谱仪和核磁共振对其进行了充分的表征。

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.