SYNTHESIS OF CARBOHYDRATE LIGANDS FOR ADHESION MOLECULE L SELECTIN

注：在Microwoft上有此摘要的续篇页面 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 细菌中相对细胞上的碳水化合物配体的结合 钙依赖方式。L-选择素在选择素中是独一无二的 论其在各类流通领域的本构表达 白细胞。此外，L-选择素在白细胞中起着关键作用 招募期间发生多起急、慢性炎症 条件，将极大的兴趣集中在 相反的内皮细胞上的碳水化合物配体。我们有 启动了一项旨在确定结构的计划 糖类配体为L-选择素。我们的方法包括分析 生物选择素配体上的寡糖结构和 已鉴定结构的化学合成直接 演示功能活动。质谱学是最重要的 我们的合成产品的特性，并将成为主要 直接结构鉴定中的分析工具 生物L-选择素配体上的碳水化合物表位。结果和 讨论。这个实验室之前的工作已经导致了分子 L-选择素两种生物糖蛋白配体的鉴定， 命名为GlyCAM-1和CD34。这些糖蛋白上的寡糖 是硫酸盐化和唾液酸化的，这两种修饰被证明是 对L来说是必不可少的--选择素识别。初步表征 利用代谢放射性标记技术研究GlyCAM-1上的寡糖 TECHICS发现了一个新的封顶团体， 6‘-磺基唾液酸基Lewis x[NeuAca2，3(SO4-6)Galb1，4(Fuca1，3)GlcNAc，1]。 因此，假设唾液酸基Lewis x的硫酸盐化 6‘-位上的四糖提供高亲和力结合 活动到L--选择素。为了验证这一假设，我们设计了一个 硫代低聚糖的化学/酶法合成 结构1。我们的第一个目标是化合物6(方案1)，其中 结构1的唾液酸残基已被替换为 合成更易获得的硫酸酯。合成路线 首先是对容易获得的双糖的选择性保护 乳糖(2)经三步反应得到衍生物3。3‘-、4’-和 然后用酸选择性地释放6‘-位以获得 化合物4.化学硫化选择性地在3‘-和 6‘-位，脱保护后生成二硫化中间体5。 中间体2-5的结构被部分地指定为 质谱学。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个目标分子的合成 并检测了该中间体与L-选择素的结合活性。 初步结果表明，化合物5与L-选择素结合较多 比类似的不含硫酸酯的衍生物更有效 6‘位。因此，这种关键的硫酸酯似乎对 对L的选择性结合活性有显著影响。我们预料到 人工合成的低聚糖，如化合物6，将更加有效 作为L-选择素拮抗剂，并可能表现出抗炎作用 活体内活动。最后，我们计划补充我们的新陈代谢 GlyCAM-1低聚糖的直接放射性标记分析 用质谱仪进行表征。

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.