Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands
Solid-phase synthesis for the identification of high-affinity bivalent lectin ligands
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DOI：
10.1021/jo0207271
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发表时间：
2003-07-25
影响因子：
3.6
通讯作者：
Toone, EJ
中科院分区：
文献类型：
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作者：
Debenham, SD;Snyder, PW;Toone, EJ
以碳水化合物为基础的治疗方法的发展一直受到蛋白质-碳水化合物络合的低亲和力的阻碍。由于大多数凝集素的低聚性质，多价的使用可能为高亲和力配体的创建提供了一种成功的策略。肽连接的多价配体库的固相评价有助于快速检查大部分连接体结构空间。如果这样的固相分析要复制溶液结合行为，则必须消除珠表面的分子间二价结合的可能性。在这里，我们报道了豆科植物凝集素concanavalin a的肽链、空间分离的单价和二价配体的固相合成和分析。同样的配体也在PEGA树脂上合成，以确定配体呈现对固相结合的影响。当我们开始确定测定灵敏度的下限时，意想不到的观察结果是，相对于单价配体，二价配体的分子间二价配体结合增强，这使得我们可以直接观察到防止分子间二价配体结合所需的表面阻断水平。对于结合位点相隔65 a的蛋白质，必须阻断约99.9%的Tentagel(1)表面位点和99.99%的PEGA头部总位点，以防止分子间二价结合。我们还报道了一价和二价肽连接配体的凝集和热液相结合研究。
The development of carbohydrate-based therapeutics has been frustrated by the low affinities that characterize protein-carbohydrate complexation. Because of the oligomeric nature of most lectins, the use of multivalency may offer a successful strategy for the creation of high-affinity ligands. The solid-phase evaluation of libraries of peptide-linked multivalent ligands facilitates rapid examination of a large fraction of linker structure space. If such solid-phase assays are to replicate solution binding behavior, the potential for intermolecular bivalent binding on bead surfaces must be eliminated. Here we report the solid-phase synthesis and analysis of peptide-linked, spatially segregated mono- and bivalent ligands for the legume lectin concanavalin A. Bead shaving protocols were used for the creation of beads displaying spatially segregated binding sequences on the surface of Tentagel resins. The same ligands were also synthesized on PEGA resin to determine the effect of ligand presentation on solid-phase binding. While we set out to determine the lower limit of assay sensitivity, the unexpected observation that intermolecular bivalent ligand binding is enhanced for bivalent ligands relative to monovalent ligands allowed direct observation of the level of surface blocking required to prevent intermolecular bivalent ligand binding. For a protein with binding sites separated by 65 A, approximately 99.9% of Tentagel(1) surface sites and 99.99% of the total sites on a PEGA bead must be blocked to prevent intermolecular bivalent binding. We also report agglutination and calorimetric solution-phase binding studies of mono- and bivalent peptide-linked ligands.