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.

注意：Microft中的此摘要有一个连续页面 单词，有数字可以粘贴在介绍中。 这 选择素是一个三个粘附分子的家族（l-，e-和e-和 p-选择蛋白）与血液初始附着有关 在移民过程中，白细胞向内皮细胞 血液进入周围组织。 这三个选择 在A中绑定基于碳水化合物的配体 钙依赖性方式。 L-选择素在Selectins中是独一无二的 在所有循环类别上的构成表达的优点 白细胞。 此外，L-选择素在白细胞中起关键作用 多种急性和慢性炎症期间的招募 条件，将巨大的兴趣集中在 相反的内皮细胞上的碳水化合物配体。 我们有 启动了旨在结构识别的计划 L-选择素的碳水化合物配体。 我们的方法涉及分析 生物学选择配体的寡糖结构和 鉴定结构直接合成的化学合成 展示功能活性。 质谱是 我们的合成产品的表征，将成为主要 在直接结构识别的分析工具 生物L-选择蛋白配体的碳水化合物表位。 结果和 讨论。 该实验室的先前工作导致了分子 鉴定两个生物糖蛋白配体l-选择素， 称为GlyCAM-1和CD34。 这些糖蛋白上的寡糖 被硫化和溶解，两种修饰被证明为 L-选择素识别至关重要。 初步表征 使用代谢放射性标记在Glycam-1上的寡糖 技术使一个新颖的封盖组的存在， 6'-Sulfo siAlyl lewis X [neuaca2,3（SO4-6）Galb1,4（fuca1,3）GlcNAC，1]。 因此，假设siallyl刘易斯x的硫酸化 6'位置上的四糖赋予高亲和力结合 L-选择素的活性。 为了检验这一假设，我们设计了 与硫酸化的寡糖相关的化学/酶合成 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A取代 合成更容易获得的硫酸盐酯。 合成路线 从选择性保护随时可用的二糖开始 乳糖（2）在三个步骤中获得衍生物3。 3'-，4'-和 然后用酸选择性地解放6'位置以负担 化合物4。化学硫酸化在3'-和 脱落后的6'位置释放了中间体5。 中间体2-5的结构已部分使用 质谱法。 最后，使用A 重组岩藻糖基转移酶（FUCTV）和GDP凝血量将负担得起 目标moledule 6。目前，我们已经完成了5 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-选择素结合更多 有力地在泰米尔衍生物处缺乏硫酸酯的衍生物 6'位置。 因此，该关键硫酸酯似乎有助于 明显地对L-eletect的结合活性。 我们预料到这一点 合成寡糖（例如化合物6）将更有效 作为L-选择蛋白拮抗剂，可能表现出抗炎 体内活动。 最后，我们计划补充我们的代谢 与直接的糖基-1寡糖进行放射标记的分析 质谱法的特征化离子。