Chemical and enzymatic synthesis of multi-domain heparin mimetics

描述（由申请人提供）：肝素是一种抗凝剂，自20世纪30年代以来一直在使用，目前的年市场价值约为30 - 40亿美元。它是硫酸乙酰肝素的重度硫酸化形式，硫酸乙酰肝素是一种在几乎所有类型的动物细胞表面发现的多糖。两者都是基于连接到<$-N-乙酰葡糖胺的<$-糖醛酸的重复单元。在细胞中，这些多糖被一系列酶修饰，产生异质的、高度阴离子的链，这些链结合各种蛋白质。肝素的抗凝活性主要来自其对丝氨酸蛋白酶抑制剂抗凝血酶（AT）的作用。与AT结合的肝素抑制凝血级联酶因子Xa和因子IIa（凝血酶）。Xa因子的抑制只需要一个五糖序列，但凝血酶的抑制需要一个十六单糖序列，以同时结合AT和凝血酶。在这个尺寸范围内合成肝素基本上是不可能的，需要 来源于动物。这导致污染的问题，以及难以获得纯样品的结构活性研究。为了克服这些困难，刘健博士在美国查佩尔山的实验室开发了一种化学酶法，能够从5-10个残基合成肝素寡糖，其大小可结合单个蛋白质。我们将通过连接化学酶法合成的寡糖来构建能够结合多种蛋白质的肝素模拟物。Huisgen“点击”反应从叠氮基和末端炔产生1，2，3-三唑键，并将用于连接寡糖组分。我们通过开发一种将N-叠氮基乙酰葡糖胺添加到寡糖的非还原末端的酶促方法开始了该项目。还原末端将在柔性接头上显示末端炔。我们将使用这种方法组装三个肝素模拟物的AT结合和凝血酶结合肝素序列的组合组成。我们将通过亲和共电泳评价抗凝血酶的亲和力，并通过显色底物法评价抗IIa活性。将在鼠模型中检测药效学和称为肝素诱导的血小板减少症（HIT）的不良反应。

DESCRIPTION (provided by applicant): Heparin is an anticoagulant that has been in use since the 1930's and has a current annual market value of about $3-4 billion. It is a heavily sulfated form of heparan sulfate, a polysaccharide found on the surfaces of almost types of animal cells. Both are based on a repeating unit of ¿-uronic acid linked to ¿-N-acetylglucosamine. In the cell these polysaccharides are modified by a series of enzymes to yield heterogeneous, highly anionic chains that bind a large variety of proteins. The anticoagulant activity of heparin is largely derived from its action on the serine protease inhibitr antithrombin (AT). Heparin bound to AT inhibits the coagulation cascade enzymes factor Xa and factor IIa (thrombin). Inhibition of factor Xa only requires a pentasaccharide sequence, but inhibition of thrombin requires a sixteen-monosaccharide sequence to bind AT and thrombin simultaneously. The synthesis of heparin in this size range is essentially impossible, requiring it to be derived from animal sources. This leads to the problem of contamination, as well as difficulties in obtaining pure samples for structure-activity studies. To overcome these difficulties, Dr. Jian Liu's laboratory at UNC Chapel Hill has developed a chemoenzymatic method capable of synthesizing heparin oligosaccharides from 5-10 residues, the size for binding a single protein. We will build heparin mimetic capable of binding multiple proteins by linking chemoenzymatically synthesized oligosaccharides. The Huisgen "click" reaction yields a 1,2,3-triazole linkage from an azido group and a terminal alkyne and will be used to link the oligosaccharide components. We began this project by developed an enzymatic method for adding an N-azidoacetyl glucosamine to the nonreducing end of an oligosaccharide. The reducing end will display a terminal alkyne on a flexible linker. We will use this method to assemble three heparin mimetics consisting of combinations of the AT-binding and thrombin-binding heparin sequences. We will evaluate the affinity to antithrombin by affinity coelectrophoresis and assess anti-IIa activity with a chromogenic substrate method. Pharmocodynamics and the adverse effect known as heparin-induced thrombocytopenia (HIT) will be tested in a murine model.