IGF::OT::IGF SYNTHESIS OF HIGH-MANNOSE N-GLYCANS

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Cell-surface oligosaccharides appended to proteins and lipids are key binding epitopes in many critical biological processes, including bacterial infection, cell development and the immune response. Understanding these processes at the molecular level requires access to a wide range of oligosaccharides of known structure to support investigations of enzyme substrate binding and specificity, to screen for carbohydrate binding proteins, and to develop assays for enzymic activity. These oligosaccharides are currently unavailable in the commercial sector, thus impeding progress in this field. To address this deficiency, the proposed project focuses on the synthesis of oligosaccharides derived from the parent high-mannose oligosaccharide (14-mer) that is transferred en-bloc to polypeptides during translation. During Phase I of this• project, a "bottom-up" synthetic approach instead of a "top-down" approach to overcome the limitations of the latter was pursued. All five 0-glycosidic linkages found in naturally occurring high-mannose N-glycans of human glycoproteins were constructed, leading to the preparation of thirty-two (32) oligosaccharides (3- to 6-mers) using a generalized chemical approach. The Phase I project developed robust synthetic protocols, and the knowledge, experience and technical know-how gained during Phase I will help extend the chemical space to over 108 compounds in pending Phase II work. The entire chemical space of nested oligosaccharide fragments containing six or fewer monosaccharides (GlcNAc and Man residues), and nested fragments containing up to nine Man residues, will be covered. Methods will be developed to array this extensive library to support anticipated screening efforts by investigators in the glycoscience community.The Contractor shall provide services for the following: Seventy-six (76) high mannose oligosaccharides ranging in size from tri- to nona-saccharides shall be prepared by chemical synthesis. Each oligosaccharide shall be prepared in 0.2 mg or greater quantities and in high purity, the latter determined by NMR, high resolution mass spectrometry, high-pressure liquid chromatography and/or capillary electrophoresis. The 24-month Phase II project period shall provide the opportunity to significantly expand the high-mannose N-glycan product line and to position the contractor for commercialization of these new glycoscience products. The Phase II synthetic work shall be divided into four (4) project periods, each with specific outcomes in terms of final oligosaccharide products synthesized, characterized, and shipped to the NCI.