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研究生：	王人弘 Wang, Jen-Hon
論文名稱：	設計及合成五圓環亞胺醣作為法布瑞氏症疾病之藥理助疊小分子 Design and synthesis of five-membered iminosugurs as pharmacalogical chaperones for Fabry disease
指導教授：	鄭偉杰 Cheng, Wei-Chieh
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	103
中文關鍵詞：	亞胺醣、助疊小分子、法布瑞氏症
外文關鍵詞：	iminosugurs, pharmacological chaperones, Fabry disease
相關次數：	點閱：84 下載：3
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法布瑞氏症為一與X染色體相關的溶小體儲積症候群,因為缺乏溶小體內部的半乳醣水解酶而導致該水解酶的天然受質酰基鞘鞍醇三己糖(GL-3)在身體內不斷堆積在血管內皮細胞,使病人出現慢性神經疼痛,腸胃不適,眼睛混濁,血管角質瘤,擴張型心肌病等症狀.
目前正在發展中的化學助疊小分子療法為一對法布瑞氏症及其他溶小體儲積症候群的潛力療法,利用經由設計而成的小分子在內質網內對不正常摺疊蛋白質的活性作用區域進行選擇性結合並幫助它恢復其正常的3D結構使其可以順利通過內質網蛋白質品質控管系統(ER quality control)成功地被送到溶小體內去水解已儲積過多的受質,進一步減緩病人的症狀.
目前對法布瑞氏症助疊小分子的研究主要是以六圓環亞胺醣為主,以五圓環亞胺醣作為基底去設計助疊小分子去做系統性的研究並未被完整的建立及探討. 亞胺醣質子化之後的構形可以模擬水解酶在水解醣的過程中產生的過渡態(oxocarbonium ion).因此,我們以此做為出發點經由設計去合成不同構型的1-aminodeoxy-2,5-dihydroxymethyl-3,4-di-hydroxypyrrolidine作為骨架, 再經由快速的組合化學合成方法做不同取代基的修飾，產生出以吡咯烷為骨架的分子庫, 並檢測其對於recombinant α-galacotosidase A 及細胞內α-galacotosidase A的抑制能力. 經過初步的生物檢測後可以挑選出對酵素及細胞活性都具有較強生物活性的候選分子，當重新合成這些化合物後再做進一步的生物測試。在候選分子中,以骨架3,E6,H6,及H9等化合物對轉染α-galacotosidase A的COS7細胞突變及法布瑞氏症病人細胞具有不錯的助疊治療效果.
經由這一系列的過程，我們成功的用快速的化學方法從大量的分子庫中找到對α-galacotosidase A有助疊效果的新穎化合物.

Fabry disease is a X-linked lysosomal storage disorder caused by deficiency in lysosomal a-galactosidase A (α-Gal A) activity, resulting in progressive accumulation of neutral glycosphingolipids with terminal a-galactosyl residue, globotriaosylceramide (GL-3), in vascular endothelial cells. Patient with Fabry disease suffered from symptoms like chronic neuronopathic pain, gastrointestinal disturbances, angiokeratoma, cardiomyopathy, premature myocardial infarctions
Chemical chaperone therapy was as a potent strategy to treat Fabry disease and other LSDs using a specific-active-site small molecule selectively binding to α-Gal A as chaperone for mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation, increasing lysosomal trafficking and restoring cellular activity further. Currently, systematic study, design, and analysis of five-membered iminosugars-based chaperones toward α-galactosidases have not been extensively explored. Here, we synthesized galactose-like ADMDP (1-aminodeoxy-2,5-dihydroxy methyl-3,4-di-
hydroxypyrrolidine) from cyclic nitrones as scaffolds and adopted a combinatorial approach to rapidly generate various pyrrolidine-based library with substituent diversity by amide bond formation, followed by in vitro enzyme-based inhibition screening and in vivo cell-based inhibition to develop novel five-membered pharmacological chaperones for Fabry disease instead of only enzyme-based inhibition screening to avoid false positives that lacks relevant chaperone activity. The inhibitory ability of these libraries toward α-galacotosidase A were evaluated and the screening results showed some interesting and potent hits were found. After resynthesis of these hits, scaffold 3, E6, H6 and H9 showed well-responded to mutant α-Gal-A expressing in COS7 and Fabry patient cell line with N215S mutation.
Through our design of scaffolds and assistance of rapid diversification, and a series of bioevaluation, potent and new α-galacotosidase A chaperones could be efficiently discovered.

摘要	I
Abstract	II
Acknowledgement	IV
Table of Contents	V
Index of Figures	VIII
Index of Tables	XI
Index of Schemes	XII
Abbreviations	XIII
Chapter 1. Introduction	I
1-1 Introduction of iminosugars	I
1-1-1 Mechanism of enzymatic hydrolysis and the transition state mimics	I
1-2 Applications of iminosugars	2
1-3 Introduction of lysosomal storage disease	3
1-3-1 Background of fabry disease	5
1-3-2Therapeutic approach for fabry disease	6
1-4 Motivation	8
1-4-1 Design and strategy	10
Chapter 2. Results and discussion	13
2-1 Preparation of scaffolds 1, 2 from D-xylose	13
2-1-1 Preparation of scaffolds 3, 4 from L-ribose and D-lyxose	14
2-2 Library synthesis	18
2-3 Enzyme-based inhibition screening of recombinant lysosomal α-galactosidase	20
2-4 Cell-based inhibition of lysosomal α-galactosidase	22
2-5 General procedure of resynthesis compounds	25
2-6 Cloning of lysosomal α-galactosidase A in an cloning vector	26
2-7 Mutagenesis of lysosomal α-galactosidase A	27
2-8 Cell-based chaperone assay for COS7 transfected with hGLA	30
2-9 Cell-based chaperone assay for patient lymphocyte with N215S mutant	36
2-10 Enzyme-based characterization of potential hits	38
2-11 Conclusion	41
Chapter 3. Experimental section	42
3-1 General experimental procedure	42
3-2 General procedure for preparation of libraries 1-4	42
3-3 Procedure of preliminary enzyme-based screening	42
3-4 Procedure of cell-based screening	43
3-5 Total extraction of RNA	43
3-6 RT-PCR of total RNA	44
3-7 Cloning of lysosomal α-galactosidase	45
3-8 Mutagenesis of lysosomal α-galactosidase	46
3-9 Cell culture	46
3-10 Transient transfected of COS-7 and chaperone assay	47
3-11 Procedure of intracellular activity enhancement assay for N215S patient lymphocyte	48
3-12 Procedure and experimental data	49
Chapter 4. References	60
Appendix	64


                                    

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校內：2017-09-11公開
校外：2018-09-11公開

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