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: 6308892
• 负责人: STEVEN D ROSEN
• 金额: $ 0.99万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6308892
• 关键词: biological products; biomedical resource; carbohydrates; immunology; inflammation; lymphatic system; 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) thatare 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 bindto carbohydrate-based ligands on opposing cells in a calcium-dependent manner. L-selectin 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 directly 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 L-selectin 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-1 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, 6'-sulfo sialyl Lewis x [NeuAca2,3(SO4-6)Galb1,4(Fuca1,3)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 thansimilar derivatives lacking the sulfate ester at the 6'-position. 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 anti-inflammatory 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：Microwoft 中有此摘要的续页 文字，还有数字要贴进去。简介。 这 选择素是三个粘附分子（L-、E-和 P-选择素）参与血源性物质的初始附着 在迁移过程中白细胞向内皮细胞迁移 血液进入周围组织。 所有三个选择因子 与相对细胞上基于碳水化合物的配体结合 钙依赖性方式。 L-选择素在选择素中是独一无二的 凭借其对所有类别的流通的构成性表达 白细胞。 此外，L-选择素在白细胞中起着关键作用 一些急性和慢性炎症期间的募集 条件，将极大的兴趣集中在事物的本质上 相对的内皮细胞上的碳水化合物配体。 我们有 启动了一项旨在识别结构的计划 L-选择素的碳水化合物配体。 我们的方法涉及分析 生物选择素配体上的寡糖结构和 已识别结构的化学合成直接 展示功能活动。 质谱分析是该领域的核心 我们的合成产品的表征，并将成为主要 直接结构识别的分析工具 生物 L-选择素配体上的碳水化合物表位。 结果和 讨论。 该实验室之前的工作导致了分子 鉴定 L-选择素的两种生物糖蛋白配体， 称为 GlyCAM-1 和 CD34。 这些糖蛋白上的寡糖 被硫酸化和唾液酸化，这两种修饰被证明是 L-选择素识别所必需的。 初步表征 使用代谢放射性标记分析 GlyCAM-1 上的寡糖 技术揭示了一种新型封端基团的存在， 6'-磺基唾液酸路易斯 x [NeuAca2,3(SO4-6)Galb1,4(Fuca1,3)GlcNAc, 1]。 因此，假设唾液酸路易斯 x 的硫酸化 6'位上的四糖赋予高亲和力结合 L-选择素的活性。 为了检验这个假设，我们设计了一个 化学/酶法合成与相关的硫酸化寡糖 结构1。我们的第一个目标是化合物6（方案1），其中 结构1的唾液酸残基已被替换为 合成上更容易获得的硫酸酯。 合成路线 从选择性保护容易获得的二糖开始 乳糖(2)分三步得到衍生物3。 3'-、4'- 和 然后用酸选择性地释放 6'-位，以提供 化合物4。化学硫酸化选择性地在3'-和 6'-位置脱保护后产生二硫酸化中间体5。 中间体 2-5 的结构已部分指定为 质谱分析。 最后，使用酶促岩藻糖基化 重组岩藻糖基转移酶 (FucT V) 和 GDP-岩藻糖将提供 目标分子6。目前，我们已经完成了5个的合成 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与L-选择素的结合更强 比缺乏硫酸酯的类似衍生物有效 6'位。 因此，这种关键的硫酸酯似乎有助于 对L-选择结合活性有显着影响。 我们预计 合成寡糖如化合物 6 的功效会更强大 作为 L-选择素拮抗剂，并可能具有抗炎作用 体内活性。 最后，我们计划补充我们的新陈代谢 直接对 GlyCAM-1 寡糖进行放射性标记分析 通过质谱法表征。