• 學位論文

Guidelines for Drug Design and Drug target Discovery: Case studies on Zinc-containing Proteins and ADP-ribosyltransferases

In the era of post-genomics, computer is a powerful tool of aiding drug design and exploring useful information in databases. Due to the high cost of developing new drugs, computer aided drug design have been widely used in drug discovery and lead compound optimization. In this thesis we try to deduce some guidelines of drug design and drug target discovery for zinc-containing proteins and ADP-ribosyltransferases, as these enzymes are important drug targets for various diseases such as cancer, bacteria infections, bacterial resistance to antibiotics, rheumatoid arthritis, diabetes, and endotoxic shock. Chapter 2. We reveal the physical basis for the observed differences between structural and catalytic Znsites: In most catalytic sites, water is found bound to Zn2+ as it transfers the least charge to Zn2+ and is less bulky compared to the protein ligands, enabling Zn2+ to serve as a Lewis acid in catalysis. In most structural sites, however, ≥2 Cys are found bound to Zn2+, as Cys transfers the most charge to Zn2+ and reduces the Zn charge to such an extent that Zn2+ can no longer act as a Lewis acid; furthermore, steric repulsion among the bulky Cys(S) prevents Zn2+ from accommodating another ligand. Chapter 3. We discuss the differential effects of commonly observed Zn-His-Bkb vs. Zn-His-[Asp/Glu] triad on Zn-core stability and reactivity. We also reveal the advantage of a second-shell Asp/Glu carboxylate in catalytic Zn-cores: relative to a Bkb carbonyl group, it increases (i) the HOMO energy of the cationic/neutral zinc core, (ii) the reactivity of the attacking Zn-bound OH, (iii) electron transfer to the substrate, and (iv) the stability of the metal complex upon electron transfer. Chapter 4. We discover a conserved structural motif for recognizing nicotinamide adenine dinucleotide in poly(ADP-ribose) polymerases and ADP-ribosylating toxins and discuss the implications for structurebased drug design. This locally conserved structure binds the nicotinamide mononucleotide moiety in a structurally conserved ringlike conformation. The biological implications/applications of locally conserved structures for toxins/PARPs and the nicotinamide mononucleotide are discussed.

• 學位論文

天然物Ottelione A 和B 及Ingenol 之全合成研究

本論文共分為兩個部分，第一部分是天然物ottelione A和B之全合成研究。以(-)-quinic acid（111）為起始物經數步反應獲得化合物(-)-92後，經由1,4-加成及碘化反應得到α-碘基酮化合物(-)-94並且利用了分子內自由基環化反應為關鍵步驟建構順式-六,五-駢環，經過二十三個步驟完成(+)-ottelione A（1）的全合成，總產率為1.7%。最後將(+)-ottelione A（1）於鹼性性條件進行反應就可以得到 (-)-ottelione B（2）。 本論文第二部分是天然物ingenol之合成研究。以(+)-3-carene (20)為起始物經數步反應獲得化合物90後，經由1,4-加成及碘化反應得到具有烯丙基苯硫基支鏈的α-碘基酮化合物172，以自由基環化反應為關鍵步驟建立ingenol的A、C環部分，更避免在環化時產生開三員環的副產物之產生。

• 學位論文

7,7-二甲基-1-(嗎福啉基-4-基)-雙環[2.2.1]庚烷-2-醇催化烷基鋅不對稱加成亞胺反應之研究

本論文利用由樟腦磺酸衍生之β-胺基醇30為掌性配位基，應用在烷基鋅對亞胺之不對稱加成反應，建立具有光學活性的二級胺。 以0.6當量的β-胺基醇於室溫（25°C）下，在甲苯和正己烷之混合溶劑中，促進烷基鋅試劑對亞胺進行不對稱加成反應，得到二級胺產物之產率為70-97%，鏡像選擇性最高可達98% ee。

• 學位論文

反式-4-羥基-L-脯胺醯胺/樟腦磺醯胺衍生之有機催化劑應用於醛類加成至反式-β-硝基苯乙烯之不對稱Michael反應研究

本篇論文旨在研究樟腦架構結合反式-4-羥基-L-脯胺酸設計合成出新穎的有機催化劑，催化異丁醛與反式-β-硝基苯乙烯進行不對稱有機催化Michael加成反應。篩選最佳有機催化劑，探討溶劑種類、催化劑及添加劑的當量數對產物產率以及鏡像選擇性的影響；篩選出化合物42作為最佳的有機催化劑並且應用在催化異丁醛加成至一系列不同取代基的反式-β-硝基苯乙烯的不對稱Michael反應，可以得到91-99%的產率與84-98%的鏡像超越值；也將催化劑42應用在催化不同的α-單取代醛類加成至反式-β-硝基苯乙烯的不對稱Michael反應，可以得到94-96%的產率與80/20-95/5的非鏡像選擇性，其中syn產物具有93-97%的鏡像超越值。

• 學位論文

硼酸修飾表面製備醣微陣列晶片的發展及應用

醣微陣列晶片是一種可同時篩選多種醣體、使用較少的樣品，快速且高通量的分析方式。對於醣生物學的研究領域，醣晶片是一種快速且有效分析醣體與生物分子交互作用的方法。在本研究中，根據硼酸能與醣體上的二元醇形成環狀硼酸酯的特性，發展一種新穎且簡便的醣晶片製程，可提供穩定、共價鍵固化的醣晶片。雖然硼酸酯化反應在水溶液中是一種可逆的反應，但由於醣體上許多羥基的多價體效應，以及硼酸酯共價鍵的特性，醣體固化在玻片表面後不容易被洗掉。除了單醣之外，這樣的固化方式適用於各種大小的醣體，且不影響晶片表面上的醣體與蛋白質的結合能力。此外，市售的醣體或是由生物樣品純化的醣體，可直接固化在硼酸修飾的表面，不需要額外的化學修飾。在篩選分析醣體與蛋白質交互作用與定量分析交互作用的親和力強弱的應用中，硼酸修飾的醣晶片提供了一種便利且快速的平台。

• 學位論文

樟腦衍生物為掌性輔助基之亞胺在不對稱反應及天然物合成上的應用

本論文含三個部份：第一部份是以樟腦磺醯胺為掌性輔助基進行AG7088類似物15的合成；第二部份則是利用樟腦醯胺為掌性輔助基進行甘胺酸酯之不對稱1,4-加成反應生成具光學活性之焦谷胺酯；第三部份則是進行生物鹼Isoretronecanol 119與Trihydroxyheliotridane 121 的合成。 ㄧ、AG7088類似物15的合成：利用樟腦磺醯胺為掌性輔助基之亞胺18為起始物與-bromoketone 30進行反應，共需6項步驟合成出AG7088類似物15，總產率為35%。 二、不對稱1,4-加成反應：利用樟腦醯胺為掌性輔助基之亞胺48與-不飽和酸酯進行不對稱1,4-加成反應，得到單一立體異構物（>98% de），並有71-90%產率。經由X-ray單晶繞射分析得知91之碳2及碳3立體化學為(R,S)；次要產物108為(S,S) 。利用亞胺48與methyl tiglate進行不對稱1,4-加成反應，得到化合物99及100，產率為93%及19:1的非鏡像選擇性。經由X-ray單晶繞射分析得知99之碳2、碳3及碳4之立體化學為(R,S,R)。亞胺48經不對稱1,4-加成反應、水解並合環2個步驟可合成出焦谷胺酯49（2步產率81%）。 三、生物鹼Isoretronecanol 119 與 Trihydroxyheliotridane 121的合成：利用亞胺48與-不飽和酸酯133進行不對稱1,4-加成反應，並應用於生物鹼Isoretronecanol 119與 Trihydroxyheliotridane 121的合成上。合成Isoretronecanol 119共花8個步驟，總產率為23%。合成Trihydroxyheliotridane 121共花9個步驟，總產率為16%。

• 學位論文

針對岩藻糖水解酶及乙醯基胺葡萄糖水解酶建立具有強效且選擇性的抑制劑

Glycosidases are enzymes that catalyze the cleavage of glycosidic bonds. Because of their roles in metabolism, protein post-translational modifications, cell-cell interactions, as well as in viral and bacterial infections, glycosidases have been the target of various therapeutic interventions. The development of glycosidase inhibitors has been useful for increasing knowledge of mechanistic details of their corresponding target enzymes and for probing the functions of specific glycoconjugates. This thesis describes a rapid method for discovering potent, selective inhibitors for - fucosidase (Fuc) and N-acetyl--hexosaminidase (Hex). These inhibitors were either investigated for their binding interactions using X-ray crystallography, isothermal titration calorimetry, and pH profile analysis, or were evaluated at the cellular level to explore their potential for use in future applications. Chapter 1 describes the significance of, and presents background information on, glycosidases and their inhibitors. We developed an efficient method for examining the activity and selectivity of various inhibitors on two -fucosidases - one (TmF) from Thermotoga maritima and the other (HuF) from human in this study. A variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were prepared in microplates and then screened without purification to assess their inhibitory activity levels on the two -fucosidases. Among the FNJ derivatives tested, the majority of the C1-substituted FNJs were slow, tight-binding inhibitors of TmF, but acted as reversible inhibitors of HuF. The best C1-substituted inhibitor exhibited a 13,700-fold difference in affinity between the two -fucosidases (Chapter 2). We applied C1-substituted FNJ derivatives to distinguish between TmF and Corynebacterium fucosidase. The flexibility of the loop (TmF sequence 44-65) was found to be critically associated with inhibition potency (Chapter 3). Subsequently, to determine the dynamic motion of the Fuc/inhibitor that interact from low to high binding affinities, we obtained nine X-ray structures, consisting of TmF and FNJ derivative complexes. The structures had dissociation constant (Ki) values in the M to pM range. The analysis of these complex structures identified several factors important in improving binding affinity. The low M Ki level structures provided sufficient electrostatic and H-bond interactions for stabilization of loops 1 and 2, in the main control of Y64, D224 and E266. Further improvement of Ki from the nM to pM is attributed to the increase of hydrophobic interactions and entropy. The flexibility of the aglycone portion and the resulting hydrophobic and H-bond interactions likely contributed to further fine-tuning of affinities in the Ki pM range (Chapter 4). Previous works have provided evidence of the uptake of L-fucose from gastric cancer cells to H. pylori. In that study, fucosidase activity was detected in the culture medium of H. pylori-infected gastric cancer cells. Here, for the purpose of rapid and efficient purification, the previously developed 1-aminomethyl-1-deoxy-FNJ was immobilized to agarose beads. The resulting affinity chromatography and subsequent liquid chromatography-tandem mass spectrometric analysis identified human -L-fucosidases 2 as the key enzyme involved in the aforementioned L-fucose transfer (Chapter 5). Finally, human Hex isozymes are considered important glycosidase targets for drug discovery because of their connection to osteoarthritis and lysosomal storage disorders. We developed GlcNAc-type iminocyclitols as potent and selective Hex inhibitors. The most potent inhibitor had a Ki of 0.69 nM against human Hex B and was 2.5105 times more selective for Hex B than for a similar human enzyme, O-GlcNAcase. These glycosidase inhibitors were shown to modulate intracellular levels of glycolipids, including ganglioside-GM2 and asialoganglioside-GM2 (Chapter 6).

• 學位論文

桿狀病毒早期基因對內源及外源啟動子表現之影響

Baculovirus viral DNA replication and transcriptional regulation are essential components to the life cycle of the virus and also important issues for expression of the engineered protein using this useful viral tool. The polyhedrin upstream sequence (pu) has been previously shown to function as an enhancer in an infection dependent manner. It is also the second known enhancer identified in baculovirus in addition to the homologous regions (hrs). Differing from hrs, the enhancer’s function of pu requires the presence of viral infection, suggesting that virus-encoded factors are required for pu’s enhancer function. To identify these factor(s), a cosmid library of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genomic DNA was constructed. Sequential fragmentation/analysis reveals 3 virus-encoded factors affecting pu’s enhancer function. These factors include IE1, IE2, and PE38 with IE1 being essential and IE2/PE38 being stimulatory. Northern blot analysis result further showed that pu and IE1 function together to stimulate the transcription activity of target promoter, and pu is therefore defined as a transcriptional enhancer. Of the three orfs that constitute pu, orf6 encodes the late expression factor 2 (lef-2) of AcMNPV and is the best studied one. It is one of the 20 late expression factors which are essential for baculoviral late gene expression. It’s been implicated to involve in both origin-dependent DNA replication and late gene expression. To better study the role of lef-2 in the life cycle of the virus, a lef-2 deletion bacmid was constructed and analyzed. The results show that lef-2 is indeed required for efficient viral DNA replication and late/very late gene expression. However, low-level of viral DNA replication could be detected in the absence of lef-2, suggesting that the function of this gene may be stimulatory rather than essential, as previously suggested, for viral DNA replication. The temporal/spatial distribution of LEF-2 in the infected cells was also analyzed. In addition, it was found that LEF-2 was associated with the nucleocapsids of both budded viruses and occluded-derived viruses, suggesting that its function may be required immediately after viral entry into host cells. In addition to arthropods, baculovirus can also enter a variety of mammalian cells with varied efficiencies and has therefore been proposed as an alternative gene delivery tool. To optimize baculovirus transduced gene expression in target mammalian cells, we found that the known baculovirus trans-activator, IE1, could stimulate the expression of cytomegalovirus immediate early (CMVie) promoter. This IE1-mediated stimulation of CMVie promoter activity can be further enhanced by the presence of hr, the known baculovirus enhancer for better gene expression.