Mead Griffin, Hiley Megan, Ng Taryn, Fihn Conrad, Hong Kevin, Groner Myles, Miner Walker, Drugan Daniel, Hollingsworth William, Udit Andrew K
Department of Chemistry, Occidental College , Los Angeles, California 90041, United States.
Bioconjug Chem. 2014 Aug 20;25(8):1444-52. doi: 10.1021/bc500200t. Epub 2014 Jul 10.
Heparin is a sulfated glycosaminoglycan that is widely used as an anticoagulant. It is typically extracted from porcine or bovine sources to yield a heterogeneous mixture that varies both in molecular weight and in degree of sulfation. This heterogeneity, coupled with concern for contamination, has led to widespread interest in developing safer alternatives. Described herein are sulfated bacteriophage Qβ virus-like particles (VLPs) that elicit heparin-like anticoagulant activity. Sulfate groups were appended to the VLP by synthesis of single- and triple-sulfated ligands that also contained azide groups. Following conversion of VLP surface lysine groups to alkynes, the sulfated ligands were attached to the VLP via copper-catalyzed azide-alkyne cycloaddition (CuAAC). MALDI-MS analysis of the intermediate alkyne VLP indicated that the majority of the coat proteins contained 5-7 of the alkyne linkers; similar analysis of the intermediate alkyne particles conjugated to a fluorescein azide suggest that nearly the same number of attachment points (3-6) are modified via CuAAC. Analysis by SDS-PAGE with fluorescent staining indicated altered migration patterns for the various constructs: compared to the wild-type nanoparticle, the modified coat proteins appeared to migrate farther toward the positive pole in the gel, with coat proteins displaying the triple-sulfated ligand migrating significantly farther. Clotting activity analyzed by activated partial thrombin time (APTT) assay showed that the sulfated particles were able to perturb coagulation, with VLPs displaying the triple-sulfated ligand approximately as effective as heparin on a per mole basis; this activity could be partially reversed by protamine. ELISA experiments to assess the response of the complement system to the VLPs indicate that sulfating the particles may reduce complement activation.
肝素是一种硫酸化糖胺聚糖，被广泛用作抗凝剂。它通常从猪或牛的来源中提取，得到一种分子量和硫酸化程度都不同的异质混合物。这种异质性，加上对污染的担忧，引发了人们对开发更安全替代品的广泛兴趣。本文描述了具有类肝素抗凝活性的硫酸化噬菌体Qβ病毒样颗粒（VLP）。通过合成同时含有叠氮基团的单硫酸化和三硫酸化配体，将硫酸基团连接到VLP上。在将VLP表面的赖氨酸基团转化为炔烃后，通过铜催化的叠氮-炔环加成反应（CuAAC）将硫酸化配体连接到VLP上。对中间炔烃VLP的基质辅助激光解吸电离质谱（MALDI-MS）分析表明，大多数衣壳蛋白含有5-7个炔烃连接子；对与荧光素叠氮化物偶联的中间炔烃颗粒的类似分析表明，通过CuAAC修饰的连接点数量几乎相同（3-6个）。用荧光染色的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，各种构建体的迁移模式发生了改变：与野生型纳米颗粒相比，修饰后的衣壳蛋白在凝胶中似乎向正极迁移得更远，显示三硫酸化配体的衣壳蛋白迁移得更远。通过活化部分凝血活酶时间（APTT）测定分析的凝血活性表明，硫酸化颗粒能够干扰凝血，每摩尔VLP显示三硫酸化配体的抗凝效果与肝素大致相同；这种活性可以被鱼精蛋白部分逆转。酶联免疫吸附测定（ELISA）实验评估补体系统对VLP的反应表明，对颗粒进行硫酸化可能会减少补体激活。
