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

附着在蛋白质和脂质上的细胞表面寡糖是许多关键生物过程中的关键结合表位，包括细菌感染、细胞发育和免疫应答。在分子水平上理解这些过程需要获得广泛的已知结构的寡糖，以支持酶底物结合和特异性的研究，筛选碳水化合物结合蛋白，并开发酶活性测定。这些低聚糖目前在商业部门无法获得，因此阻碍了这一领域的进展。为了解决这一缺陷，拟议项目的重点是合成低聚糖衍生自母体高甘露糖低聚糖（14聚体），在翻译过程中整体转移到多肽。在该项目的第一阶段，采用了“自下而上”的综合方法，而不是“自上而下”的方法，以克服后者的局限性。构建了在人糖蛋白的天然存在的高甘露糖N-聚糖中发现的所有五个O-糖苷键，从而使用通用化学方法制备了三十二（32）种寡糖（3-至6-聚体）。第一阶段项目制定了强有力的合成方案，在第一阶段获得的知识、经验和技术诀窍将有助于将化学空间扩大到108个以上。 正在进行的第二阶段工作中的化合物。包含6个或更少单糖（GlcNAc和Man残基）的嵌套寡糖片段的整个化学空间，以及包含多达9个Man残基的嵌套片段将被涵盖。承包商应提供以下服务：应通过化学合成制备七十六（76）种高甘露糖寡糖，其大小从三到九不等。每种低聚糖的制备量应大于或等于0.2 mg，且应具有高纯度，后者可通过NMR、高分辨率质谱、高压液相色谱和/或毛细管电泳法测定。为期24个月的第二阶段项目期将提供机会，以显着扩大高甘露糖N-聚糖产品线，并定位这些新的糖科学产品的商业化承包商。第二阶段合成工作应分为四（4）个项目期，每个项目期都有最终低聚糖产品合成、表征和运输方面的具体成果 国家癌症研究所

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.