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

蛋白质和脂类上的细胞表面寡糖是许多关键生物学过程中的关键结合表位，包括细菌感染、细胞发育和免疫反应。在分子水平上了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，并开发酶活性的分析方法。这些低聚糖目前无法在商业部门获得，因此阻碍了这一领域的进展。为了解决这一不足，拟议的项目侧重于从母体高甘露糖低聚糖(14-聚体)衍生的低聚糖的合成，在翻译过程中将其整体转移为多肽。在这个·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。在天然存在的人糖蛋白的高甘露糖N-糖链中发现的所有五个0-糖苷键被构建，导致使用通用的化学方法制备三十二(32)个寡糖(3-到6-MERS)。第一阶段项目开发了强大的合成方案，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多个 正在等待第二阶段工作的化合物。包含6个或更少单糖(GlcNAc和Man残基)的嵌套低聚糖片段以及包含最多9个Man残基的嵌套片段的整个化学空间将被覆盖。将开发方法来排列这个庞大的文库，以支持糖科学界研究人员预期的筛选工作。承包商应提供以下服务：76(76)个大小从三糖到非那糖的高甘露低聚糖应通过化学合成来制备。每个低聚糖应以0.2毫克或更大的量制备，并以高纯度制备，后者通过核磁共振、高分辨率质谱仪、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将提供机会，大幅扩大高甘露糖N-葡聚糖产品线，并使承包商能够将这些新的糖科学产品商业化。第二阶段的合成工作应分为四(4)个项目期，每个项目期根据低聚糖产品的最终合成、表征和装运而有特定的结果 给国家情报局。

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.