SYNTHESIS OF CARBOHYDRATE LIGANDS FOR ADHESION MOLECULE L SELECTIN

粘附分子 L 选择素碳水化合物配体的合成

• 批准号: 6120244
• 负责人: STEVEN D ROSEN
• 金额: $ 0.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120244
• 关键词: biological products; biomedical resource; radiology; spectrometry; structural biology

Note There is a continution page for this abstract in Microwoft Word, and there are figures to be pasted in. Introduction. The selectins are a family of three adhesion molecules (L-, E- and P-selectin) that are involved in the initial attachment of blood-borne leukocytes to endothelial cells during the process of emigration from the bloodstream into the surrounding tissue. All three selectins bind to carbohydrate-based ligands on opposing cells in a calcium-dependent manner. Lselectin is unique among the selectins by virtue of its constitutive expression on all classes of circulating leukocytes. In addition, L-selectin plays a key role in leukocyte recruitment during a number of acute and chronic inflammatory conditions, focusing a tremendous amount of interest on the nature of the carbohydrate ligands on opposing endothelial cells. We have initiated a program aimed at the structural identification of carbohydrate ligands for L-selectin. Our approach involves analysis of the oligosaccharide structures on biological selectin ligands and the chemical synthesis of identified structures to direct ly demonstrate functional activity. Mass spectrometry is central to the characterization of our synthetic products, and will be the principal analytical tool in the direct structural identification of the carbohydrate epitopes on biological Lselectin ligands. Results and Discussion. Previous work in this laboratory has led to the molecular identification of two biological glycoprotein ligands for L-selectin, termed GlyCAM-l and CD34. The oligosaccharides on these glycoproteins are sulfated and sialylated, two modifications which were shown to be essential for L-selectin recognition. Preliminary characterization of the oligosaccharides on GlyCAM-1 using metabolic radiolabeling techniques has revelealed the presence of a novel capping group, 61-sulfo sialyl Lewis x [NeuAca2,3(SO4-6)Galbl,4(Fucal,3)GlcNAc, 1]. Thus, it is hypothesized that sulfation of the sialyl Lewis x tetrasaccharide on the 6'-position imparts high affinity binding activity to L-selectin. To test this hypothesis, we have designed a chemical/enzymatic synthesis for sulfated oligosaccharides related to structure 1. Our first target is compound 6 (scheme 1), in which the sialic acid residue of structure 1 has been replaced with a synthetically more accessible sulfate ester. The synthetic route begins with selective protection of the readily available disaccharide lactose (2) to afford derivative 3 in three steps. The 3'-, 4'- and 6-positions are then selectively liberated with acid to afford compound 4. Chemical sulfation proceeds selectively at the 3'- and 6'-positions yielding, after deprotection, disulfated intermediate 5. The structures of intermediates 2-5 have been assigned in part using mass spectrometry. Finally, enzymatic fucosylation using a recombinant fucosyltransferase (FucT V) and GDP-fucose will afford target moledule 6. Currently, we have completed the synthesis of 5 and tested this intermediate for L-selectin binding activity. Preliminary results indicate that compound 5 binds to L-selectin more potently than similar derivatives lacking the sulfate ester at the 61position. Thus, this key sulfate ester appears to contribute significantly to L-selecting binding activity. We anticipate that synthetic oligosaccharides such as compound 6 will be even more potent as L-selectin antagonists, and may demonstrate antiinflammatory activity in vivo. Finally, we plan to complement our metabolic radiolabeling analysis of the GlyCAM-1 oligosaccharides with direct characterizat ion by mass spectrometry.

请注意，在Microwoft上有此摘要的续订页面 Word，并且有要粘贴的图形。导言。这个 选择素是一个由三个黏附分子(L、E和E)组成的家族 P-选择素)参与了血传播的初始附着 外周血白细胞向内皮细胞迁移过程中的变化 血液流入周围组织。所有三种选择素都与 以碳水化合物为基础的配体在钙依赖的相对细胞上 举止。L选择素在选择素中是独一无二的，因为它 在所有类别的循环白细胞上都有组成性表达。在……里面 此外，L-选择素在白细胞募集中发挥关键作用。 一些急性和慢性炎症性疾病，集中在 人们对碳水化合物的性质非常感兴趣 对峙内皮细胞上的配体。我们已经启动了一个项目 目的是对碳水化合物配体进行结构鉴定 L-选择素。我们的方法包括分析低聚糖 生物选择素配体的结构及其化学合成 直接显示功能活性的已鉴定结构。 质谱学是表征我们合成的 产品，并将成为直接的分析工具 生物碳水化合物表位的结构鉴定 L-选择素配体。结果和讨论。以前在这方面的工作 实验室已经对两种生物进行了分子鉴定 L-选择素的糖蛋白配体，命名为GlyCAM-L和CD34.这个 这些糖蛋白上的寡糖被硫酸盐化和唾液酸化， 对L来说必不可少的两个修饰--选择素 承认。大豆低聚糖的初步表征 使用代谢放射性标记技术的GlyCAM-1揭示了 新型封端基团61-磺基唾液酸基Lewis x的存在 [NeuAca2，3(SO4-6)Galbl，4(Fucal，3)GlcNAc，1]。因此，它是假设的 唾液酸基Lewis x四糖在 6‘-位赋予L-选择素高亲和力结合活性。至 验证这一假设，我们设计了一种化学/酶合成 对于与结构1相关的硫化低聚糖。我们的第一个 目标是化合物6(方案1)，其中 结构1已被综合起来更易访问的 硫酸酯。合成路线从选择性保护开始 容易获得的二糖乳糖(2)来提供衍生物 3分三步走。然后选择性地将3‘-、4’-和6-位 用酸释放得到化合物4.化学硫化反应 选择性地在3‘和6’位置屈服，在解除保护之后， 二硫化中间体5.中间体2-5的结构为 部分用质谱仪进行了指认。最后，酶制剂 使用重组岩藻糖基转移酶(Fuct V)的岩藻糖基化和 GDP-FUSTOSE将承担目标模块6。目前，我们有 完成了5的合成，并对该中间体进行了测试 L-选择素结合活性。初步结果表明， 化合物5与L-选择素的结合比类似的衍生物更有效 61位缺少硫酸酯。因此，这个关键的硫酸盐 酯似乎对L的选择绑定有很大贡献 活动。我们预计合成的低聚糖，如 化合物6作为L-选择素拮抗剂将更加有效，并可能 在体内显示抗炎活性。最后，我们计划 补充我们对GlyCAM-1的代谢放射性标记分析 低聚糖的直接质谱学表征。