第一部分:以細胞篩選合成五圓Iminocyclitols作為抗C型肝炎的研究 第二部分: 利用螢光標記醣類之方法定量金奈米粒子上醣分子濃度

Abstract

第一部分 C型肝炎病毒仰賴寄主系統進行複製。在寄主的系統中, N-型醣蛋白會被醣基轉移酶和醣解酶於內質網內修飾上不同的醣基.如果抑制這些酶,將可能嚴重影像醣蛋白成熟,轉運及分泌,甚至可改變細胞和病毒之間的辨識。iminocyclitol 2,5-dideoxy-2,5-imino-D- mannitol是已知有效的 α- and β-葡萄糖水解酶之抑制劑。以此構型為基礎,加上增加細胞穿透性的長碳鏈,利用醯胺鍵結方式與一系列的酸基於微量盤上合成約一百多個五圓Iminocyclitols分子。這些分子並未經過純化便直接進行細胞篩選抗C型肝炎有效結構以及細胞外α- and β-葡萄糖水解酶之抑制劑與已知的抗C型肝炎病毒抑制劑NB-DNJ相比許多更有效的分子結構被篩選出來。其中，含有苯環,硫吡啶及三氟甲基苯環結構之分子,最具有成為新藥的潛力。因刺這些分子,以及相對應之無常碳鏈的分子重新被合成及純化以進行對醣解酶IC50和Ki測試以及更詳細的抗c型肝炎病毒試驗.結果顯示長碳鏈分子雖然對醣解酶的抑制效果不是很理想,但是在細胞層級的測是當中卻展現較佳的抗病毒性。因此，利用簡單的組合式策略所合成的分子可以快速有效地利用細胞篩選方式得知具有潛力的抑制結構。 第二部分 醣基奈米材料是帶有許多醣基分子於奈米結構上的材料，這種材料是一種良好的蛋白質辨識平台。其中，圓形的材料常被應用於模擬細胞,例如醣奈米顆粒即可模仿細胞表面的醣基分子。這種獨特性質使的醣奈米顆粒可以被應用於解讀蛋白質及醣分子之間的效價性互動情形。然而關鍵性奈米粒子上醣分子的密度,卻很少被直接定量出來。在本篇研究當中,原本是想要值接利用直接測量奈米粒子上螢光醣分子的亮度來決定醣分子的密度,然而因為奈米粒子的本質而影響螢光醣分子的螢光強度,所以無法進行直接的測定。將檢量線改為螢光醣分子與定量的奈米粒子一起測定,希望可以得到更準確的螢光強度-醣分子濃度的檢量線。另外，原本所設計的雙螢光系統無法被利用於蛋白質及醣分子之間的親合性測試。未來將改成以酵素連結免疫吸附實驗及競爭性抑制的實驗以進行親合性測試。

Part I Replication of Hepatitis C virus (HCV) depends on host protein synthesis system. In this system, glycosyltransferases and glycosidases are involved in the processing of N-linked glycoproteins in the endoplasmic reticulum. Inhibition of these enzymes may have profound effects on maturation, transport, secretion of glycoproteins, and even alter cell-virus recognition processes. The iminocyclitol 2,5-dideoxy-2,5-imino-D- mannitol is a powerful inhibitor of a large range of α- and β-glucosidase. Based on this structure, compound with a cell-permeable alkyl chain, was synthesized and coupled with acid library in the microtiter plate. Without purification, >100 analogues were directly subjected to ex vivo anti-HCV infection cell based assay and showed good inhibitive ability despite most of them were of low inhibitive ability inα-glucosidase in vitro screening. Compared to the proven anti-HCV inhibitor NB-DNJ, several compounds were identified as better inhibitors. Five compounds with fused aromatic ring, thiopyridine or trifluoromethylbenzne showed good anti-HCV ability and were potent to become new drugs. These compounds were re-synthesized and purified to get their IC50 and Ki values for glucosidase and anti-HCV inhibition assay. Although the alkylated iminocyclitols showed only mildα-glucosidase inhibitory compared to corresponding non alkylated iminocyclitols, in cell-based assay they indeed showed good inhibition effect on anti-HCV inhibition. In brief, this work demonstrates the effectiveness of the simple combinatorial strategy for rapidly and efficiently discovering potent inhibitors by using cell-based assay system.

Part II Glyconanomaterials, nanomaterials carrying multiple carbohydrate ligands, provide a great platform for protein recognition research. Among the nanomaterials, spheral nanoparticle has been widely used as a model of cell, in which glyconanoparticle mimics the glycans on the cell surface. This property makes glyconanoparticle as an excellent material to study the multivalent interactions between glycan and protein. However, to study such interaction, the glycan density on nanoparticle surface is an important issue and a straightforward quantitative analysis of the interaction is rarely reported. In the study, direct measurement of the amount of fluorescent glycans on nanoparticles is affected by the property of nanoparticle. The standard curve of fluorescent glycans concentration to the fluorescence intensity should be modified with the existing of nanoparticles. Co-existence of two fluorescent compounds, coumarin and texas red, may influence the fluorescence of each other and is not ideal for study. Two modified experiments, enzyme - linked immunosorbent assay (ELISA) and fluorescence competition binding assay, are proposed here to determine the binding affinity of fluorescently labeled glyconanoparticles with corresponding lectins.