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.
