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研究生：	黃俊諺 Huang, Chun-Yen
論文名稱：	以組合化學方法合成α-L-鼠李醣水解酶的新穎抑制劑 Combinatorial approach toward synthesis of novel α-L-rhamnosidase inhibitors
指導教授：	鄭偉杰 Cheng, Wei-Chieh
共同指導教授:	黃福永 Huang, Fu-Yung
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：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	124
中文關鍵詞：	亞胺醣、環硝、鼠李醣、鼠李醣水解酶抑制劑
外文關鍵詞：	azasugar, cyclic nitrone, rhamnose, rhamnosidase inhibitors
相關次數：	點閱：64 下載：2
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亞胺醣衍生物對於抑制醣之水解酶和轉移酶所衍生的治療效果近年來越來越受到重視。從化學結構上分析，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，進而達到抑制水解酶的能力。論文中我們經由關鍵的中間產物-五員環硝，合成出不同構形的五員環多羥基吡咯烷分子，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α-L-rhamnosidase的抑制能力。首先藉由初步的生物檢測可以挑選出具有較強抑制效果的候選分子，當重新合成這些化合物後再做進一步的生物測試。在具有較強抑制效果的化合物中，亞胺醣衍生物39顯示出對於α-L-rhamnosidase 具有最強的抑制效果，其IC50為0.29 μM， Ki為0.24 μM，是一種競爭型的抑制劑。
經由這一系列的過程，我們成功地用快速的化學方法從大量的分子庫中找到對α-L-鼠李醣水解酶有抑制效果的化合物，而此方法也可以應用於其他水解醣酵素抑制的研究。

Therapeutic application of iminosugar derivatives for inhibiting sugar processing enzymes and transferases has received a lot of attention recently. From structure point of view, protonated conformation of iminosugars could mimic the transition state oxocarbenium ion, which are generated during the hydrolysis of sugar substrates by glycosidases, and exhibit inhibitory ability toward glycosidases. In this thesis, five-membered iminosugars-based scaffolds were prepared using five-membered chiral cyclic nitrones as key intermediates, followed by rapid conjugation with diverse acids through an amide coupling to generate five polyhydroxylated pyrrolidine-based libraries (5 x 127 = 635). The inhibitory ability of these libraries toward α-L-rhamnosidase were evaluated determine and the preliminary screening results showed some interesting and potent hits. After re-synthesis of these hits, the inhibitor 39 showed a Ki value of 0.24 μM against α-L-rhamnosidase with a competitive inhibition.
Through the design of scaffolds and assistance of rapid diversification, and in situ bioevaluation, potent and new α-L-rhamnosidase inhibitors could be efficiently discovered. Importantly, this approach can be applied to discovery of inhibitors against various sugar processing enzymes.

                 Table of Contents
摘要	                                                        I
Abstract	                                        II
謝誌                                                           III
Table of Contents	                                IV
Index of Figures	                                VI
Index of Tables	                                      VIII
Index of Schemes	                                IX
Abbreviations	                                         X
Chapter 1. Introduction                	                 1
1-1 Introduction of iminosugars	                         1
1-1.1 Mechanism of enzymatic hydrolysis and the transition state mimics	                                         1
1-2 Applications of iminosugars	                         2
1-3 Background of α-L-rhamnosidase	                 3
1-3.1 Relationship between α-L-rhamnosidase and rhamnosyl transferase	                                         4
1-3.2 Current known α-L-rhamnosidase inhibitors	         5
1-4 Design and strategy	                                 6
Chapter 2. Results and discussion	                 9
2-1 Preparation of scaffolds 1, 2, 3 from ribose	 9
2-1.1 Preparation of scaffolds 4, 5 from xylose	        13
2-1.2 1H NMR NOESY spectrums of 12, 14, 16 and 23	14
2-1.3 Preparation of reference compound 30	        17
2-2 Library synthesis	                                18
2-3 Bioassay	                                        23
2-4 Bioassay of re-synthesis compounds	                29
2-5 Conclusion	                                        32
Chapter 3. Experimental section	                        33
3-1 General experimental procedure	                33
3-2 General procedure for preparation of model 10	33
3-3 General procedure for preparation of libraries 31-35	                                                35
3-4 General procedure A for preparation of amide products	                                                35
3-5 Procedure of preliminary bio-screening	        35
3-6 Procedure of bioassay	                        36
3-6.1 Modified procedure of bioassay	                37
3-7 Procedure and experimental data	                38
3-7.1 Procedure and experimental data for reference compound 30	                                        47
3-7.2 Procedure and experimental data for re-synthesis amide products	                                        50
References	                                        54
Appendix	                                        60






                    Index of Figures
Figure 1.1 Mechanism of enzymatic hydrolysis and transition state mimics.	                                          2
Figure 1.2 Examples of iminosugars for the treatment of diseases.	                                          3
Figure 1.3 Mycobacterium tuberculosis cell wall glycan and the linker region.	                                  4
Figure 1.4 Currently potent α-L-rhamnosidase inhibitors (µM). (a, b and c represented compounds from different papers).	                                          6
Figure 1.5 Structure design and five proposed pyrrolidine-based scaffolds.	                                  7
Figure 1.6 Flowchart for the development of α-L-Rhamnosidase inhibitors.	                          8
Figure 2.1 1H-1H NOESY spectrum of 12.	                 15
Figure 2.2 1H-1H NOESY spectrum of 14.	                 15
Figure 2.3 1H-1H NOESY spectrum of 16.	                 16
Figure 2.4 1H-1H NOESY spectrum of 23.	                 17
Figure 2.5 Substituent diversify by amide bond formation.	                                                 19
Figure 2.6 Acid libraries A-E (1-10).	                 20
Figure 2.7 Acid libraries F-J (1-10).	                 21
Figure 2.8 Acid libraries K-L (1-10) and M (1-7).	 22
Figure 2.9 Primary inhibitory screening of library 31 at 100μM toward α-L-rhamnosidase.	                         24
Figure 2.10 Primary inhibitory screening of library 32 at 100μM toward α-L-rhamnosidase.	                         25
Figure 2.11 Primary inhibitory screening of library 33 at 100μM toward α-L-rhamnosidase.	                         25
Figure 2.12 Primary inhibitory screening of library 34 at 100μM toward α-L-rhamnosidase.	                         26
Figure 2.13 Primary inhibitory screening of library 35 at 100μM toward α-L-rhamnosidase.	                         26
Figure 2.14 Inhibition potential of scaffolds 1-5 at 100 μM against enzyme.	                                         27
Figure 2.15 Primary inhibitory screening of selected potent hits from library 33 at 1 μM.	                         27
Figure 2.16 Chemical structures of potent α-L-rhamnosidase inhibitor.	                                         28
Figure 2.17 The Lineweaver-Burk plots of (a) 36, (b) 37, (c) 38, (d) 39 (e) 40 and (f) scaffold 5 for the inhibition of α-L-rhamnosidase. The increasing concentrations of substrate were used to determine the Ki values and the data were plotted as 1/v versus 1/[S].	                 31
Figure 2.18 The Lineweaver-Burk plots of reference inhibitor 30 for the inhibition of α-L-rhamnosidase. The increasing concentrations of substrate were used to determine the Ki values and the data were plotted as 1/v versus 1/[S].	                                         31
Figure I Km plot of naringinase with substrate para-nitrophenyl α-L-rhamnopyranoside.	                 62
Figure II IC50 plots of (a) 36, (b) 37, (c) 38, (d) 39, (e) 40, (f) scaffold 5 and (g) 30.	                         64




                      Index of Tables
Table 2.1 Preparation of dehydroxylated 8.	         10
Table 2.2 Preparation of scaffold 1.	                 11
Table 2.3 Inhibition potential of iminosugars 36-40, 5 and 30 against α-L-rhamnosidase.	                         29





                      Index of Schemes
Scheme 2.1 Preparation of polyhydroxylated pyrrolidine-based scaffold 1.	                                  9
Scheme 2.2 Preparation of scaffold 2.	                 12
Scheme 2.3 Preparation of scaffold 3.	                 12
Scheme 2.4 Preparation of scaffold 4.	                 13
Scheme 2.5 Preparation of scaffold 5.	                 14
Scheme 2.6 Preparation of reference compound 30.	 18
Scheme I Resynthesis of potential structures showed strong inhibitory ability.	                                 61


                                    

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