Chemical and enzymatic synthesis of multi-domain heparin mimetics

多域肝素模拟物的化学和酶法合成

• 批准号: 8597816
• 负责人: Timothy Robert OLeary
• 金额: $ 3.2万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8597816
• 关键词: Acetylglucosamine; Adverse effects; Adverse reactions; Affinity; Alkynes; Amines; Animal Sources; Animals; Antibody Formation; Anticoagulants; Antithrombins; Arixtra; Azides; Binding; Cardiovascular Surgical Procedures; Cells; Cessation of life; Chemicals; Chemistry; Chromogenic Substrates; Clinical; Coagulation Process; Complex; Deep Vein Thrombosis; Disease Outbreaks; Dose; Drug Kinetics; Enzymes; Esters; Factor IIa; Factor Xa; Glucosamine; Hematologist; Heparin; Heparin Binding; Heparitin Sulfate; In Vitro; Incidence; Inorganic Sulfates; Label; Laboratories; Length; Libraries; Link; Marketing; Measures; Methods; Modeling; Monosaccharides; Mus; Oligosaccharides; Pathology; Patients; Pharmaceutical Preparations; Physicians; Platelet Factor 4; Polysaccharides; Production; Protein Binding Domain; Proteins; Pulmonary Embolism; Reaction; Sampling; Series; Serine Protease; Source; Structure; Surface; Testing; Thrombin; Thrombocytopenia; Toxic effect; Triazoles; Universities; Unspecified or Sulfate Ion Sulfates; Uronic Acids; Work; base; cycloaddition; design; flexibility; inhibitor/antagonist; link protein; mimetics; mouse model; public health relevance; sulfation; synthetic construct

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.

说明(申请人提供)：肝素是一种抗凝血剂，自1930年S以来一直在使用，目前年市场价值约为30-40亿美元。它是硫酸乙酰肝素的一种严重硫酸盐化形式，是一种在几乎各种动物细胞表面都能找到的多糖。两者都是基于与N-乙酰氨基葡萄糖相连的糖醛酸的重复单位。在细胞中，这些多糖被一系列酶修饰，产生多相的、高度负离子的链，这些链结合了大量的蛋白质。肝素的抗凝血活性主要来源于它对丝氨酸蛋白酶抑制剂抗凝血酶(AT)的作用。肝素与AT结合可抑制凝血级联酶FXa和FIIa(凝血酶)。抑制凝血因子Xa只需要一个五糖序列，而抑制凝血酶需要一个16个单糖序列来同时结合AT和凝血酶。在这种大小范围内合成肝素基本上是不可能的，需要它 从动物来源中提取。这导致了污染问题，以及难以获得用于结构活性研究的纯样品。为了克服这些困难，刘健博士在北卡罗来纳大学教堂山的实验室开发了一种化学酶法，能够从5-10个残基合成肝素低聚糖，5-10个残基的大小与单个蛋白质结合。我们将通过连接化学酶合成的寡糖来构建能够与多种蛋白质结合的肝素模拟物。惠斯根“点击”反应产生来自叠氮基团和末端炔的1，2，3-三唑键，并将用于连接低聚糖组分。我们首先开发了一种酶促方法，将N-叠氮乙酰氨基葡萄糖添加到低聚糖的非还原末端。减速端将末端炔显示在柔性连接件上。我们将使用这种方法组装由AT结合和凝血酶结合的肝素序列组成的三个肝素模拟物。我们将通过亲和共电泳法评价其与抗凝血酶的亲和力，并用发色底物法评价其抗IIa活性。药物动力学和被称为肝素诱导的血小板减少症(HIT)的副作用将在小鼠模型中进行测试。