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.

注：在Microwoft中有此摘要的续页 字，还有数字要贴进去。 导论. 的 选择素是三种粘附分子（L-、E-和 P-选择素），参与血液传播的初始附着。 白细胞在移出过程中向内皮细胞迁移 血液流入周围组织 所有三种选择素 与相对细胞上的碳水化合物配体以钙依赖性 方式 L选择素是选择素中独特的，因为其 在所有类型的循环白细胞上的组成型表达。 在 此外，L-选择素在白细胞募集过程中起着关键作用， 一些急性和慢性炎症性疾病，重点是 人们对碳水化合物的性质产生了极大的兴趣 配体上的相对内皮细胞。 我们启动了一个项目 目的是鉴定碳水化合物配体的结构， 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化学合成 以直接证明功能活性。 质谱分析是表征我们的合成 产品，并将成为直接的主要分析工具， 生物活性肽糖表位的结构鉴定 L选择素配体。 结果和讨论。 以前的工作在此 实验室已经导致了两种生物的分子鉴定 L-选择素的糖蛋白配体，称为GlyCAM-1和CD 34。 的 这些糖蛋白上的寡糖被硫酸化和唾液酸化， 两种修饰被证明是L-选择素所必需的， 识别. 寡糖的初步表征 使用代谢放射性标记技术的GlyCAM-1揭示了 存在一个新的封端基团，61-磺基唾液酸刘易斯x [中性粒细胞2，3（SO 4 -6）Galbl，4（Fucal，3）GlcNAc，1]。 因此，假设 唾液酸基刘易斯x四糖的硫酸化 6 ′-位赋予L-选择素高亲和力结合活性。 到 为了验证这一假设，我们设计了一种化学/酶促合成方法， 对于与结构1相关的硫酸化低聚糖。 我们的第一 目标是化合物6（方案1），其中化合物6的唾液酸残基 结构1已被替换为合成上更容易获得的 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），得到衍生物 3分三步。 然后选择性地将3 '-、4'-和6-位 用酸释放，得到化合物4。 化学硫酸化收益 选择性地在3 ′-和6 ′-位，脱保护后， 二硫酸化中间体5。 中间体2-5的结构具有： 部分是用质谱法确定的。 最后，酶 使用重组岩藻糖基转移酶（FucT V）进行岩藻糖基化，以及 GDP-岩藻糖将提供目标分子量6。 目前已经 完成了5的合成，并测试了该中间体的 L-选择素结合活性。 初步结果表明 化合物5比类似衍生物更有效地与L-选择素结合 61位缺少硫酸酯。 因此，这种关键的硫酸盐 酯似乎对L-选择性结合有显著贡献 活动 我们预计，合成低聚糖，如 化合物6作为L-选择素拮抗剂甚至更有效，并且可以 在体内表现出抗肿瘤活性。 最后，我们计划 补充我们对GlyCAM-1的代谢放射性标记分析 通过质谱法直接表征低聚糖。