http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/58 2026-04-14T22:27:28Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18192 標題: ﰑﰎﰙﰚﰛﰜﰝﰞﰟﰠﰡﰢﰣﰤﰑﰥﰦﰧﰨﰑﰎﰙﰚﰛﰜﰝﰞﰟﰠﰡﰢﰣﰤﰑﰥﰦﰧﰨﰩ化學探針的設計與合成及其在生化方面之應用; Design, synthesis and evaluation of chemical probes for biochemical applications 作者: Yu-Ling Hsu; 許玉羚 摘要: 化學探針能選擇性地標示目標蛋白質，為近代蛋白質體學上其中一種強力不可或缺的工具。本論文合成和設計幾種重要目標蛋白的化學探針，並介紹其發展及在生化方面的應用。第一部分針對蛋白激酶的結構特性設計的二個探針分子庫，皆以平行合成的組合式化學建構：第一個探針分子庫具有5'對苯磺醯氟腺苷和生物素標籤；第二個分子庫以3'疊氮去氧胸苷和腺苷為架構，接上三種親電子基。主架構的疊氮結構使其為一天然標籤基團。二個探針分子庫針對蛋白激酶的標示效能也在本論文進行驗証和比較。 穿心蓮內酯為一多功能的抗發炎和抗癌藥物，但相關的抑制機制卻還未完全明暸，因此建構具螢光團的穿心蓮內酯探針有助於進行生理中抑制和抗癌機制的追蹤和研究。第二部分合成和設計天然物穿心蓮內酯的可穿透細胞螢光探針，第一個可穿透細胞穿心蓮內酯螢光探針的合成經驗也有助於未來此類探針或抑制劑的開發和改良。本論文也合成α-L-岩藻糖苷酶醌甲基生成機制活性探針，以鹼催化置換β變旋異構物成α結構的關鍵中間體，並進而合成帶有螢光團的可穿透細胞; Development of small-molecular tools which selectively react with designated protein families has been found powerful in modern functional proteomics. In this dissertation, we evaluated two kinase probe libraries according to the structure and functions of protein kinases. The first library adopted a 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSBA) skeleton and a biotin reporter. The second library explored a novel molecular framework by attaching three kind of electrophilic warheads on 3'-azido 2', 3'-dideoxy base (AZT and AZA) recognition unit. The azido group at 3' position is a native clickable tag without further modification. The labeling performances of these two probe libraries toward kinases were compared. Despite the therapeutic importance of this multifunctional herbal compound Andrographolide, the inhibition and regulation mechanism are still ambiguious. Herein, we designed a novel andrographolide-based, cell permeable fluorescent probe for in vivo identification of target proteins participating in related cancer or diseases. This first-time synthesized Andrographolide-based, cell permeable fluorescent probe provide valuable information for the synthesis method in the future. In addition, cell-permeable activity-based probes for α-L-fucosidase were evaluated by the key base-promoted epimerization step. These two probes carrying mono or difluoromethylphenyl group would generate reactive quinone methide as a trapping device after hydrolysis. In the last part, quantitative isotope-coded azido tags were synthesized and applied to detect the level of S-nitrosylation proteome with drug treatment. 2015-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/101069 標題: 點擊化學策略在磁珠上引入醣基的研究; Click Chemistry for Glycan Functionalization on Magnetic Beads 作者: 巫梓璿; Tzu-Hsuan Wu 摘要: 腫瘤相關醣抗原（tumor-associated carbohydrate antigens, TACAs） 在人類癌細胞表面有過度表現的現象。因此，如何辨識這些 TACAs 已成為生物分析與藥物開發中極需解決的問題。適配體（aptamer），即短鏈 DNA 或 RNA 寡核苷酸，近年來已成為一種常用的生物感測工具。其獨特的核酸序列可藉由 體外篩選技術（in vitro selection） 產生，並透過特殊的三維複雜摺疊結構，對特定標的物（如小型生物分子與胜肽）展現高度的專一性與親和力。 本實驗室欲取得特定適配體序列以辨認以下三種特定腫瘤相關抗原：Tn、sTn 與 Tf。，以利後續應用。為了進行體外篩選，需要一種固相載體來在多輪篩選過程中捕捉序列。磁珠（magnetic beads, MBs）對大多數化學物質具有正交性，且操作便利，因此是一種強而有力的固相載體。在本研究以兩步驟的方式將 sTn 抗原導入磁珠。第一步引入使帶有 BCN官能基的spacer修飾於氨基磁珠（NH2-MBs）上，第二步引入使帶有疊氮尾部的抗原與經過 BCN 活化的磁珠以環張力促進叢集炔-疊氮點擊反應（SPAAC）的方式進行偶聯。 為了驗證磁珠上成功引入的化學修飾，本研究使用茚三酮（ninhydrin）測試得知NH2-MB氨基含量。BCN spacer 的完全轉換則透過茚三酮測試中吸光度下降的反推來驗證，並藉由紅外光譜（IR spectra）的比較來確認 BCN spacer 的成功導入。最後，利用 Shukla’s assay 測得磁珠上唾液酸（sialic acid）的負載量。; Tumor-associated carbohydrate antigens (TACAs) are overexpressed on the surface of human carcinoma cells. Therefore, recognizing these TACAs is a prompt issue for bioanalysis and drug development. Aptamer, a short DNA or RNA oligonucleotide have become a popular biosensor in recent years. The unique sequence of the nucleic acid can be generated by a technique called in vitro selection to exhibit high affinity and selectivity toward the particular target such as small biomolecules and peptides by the unique three-dimensional complex folding. Our laboratory aimed to obtain the specific aptamer sequences that could recognize three tumor-associated antigens: Tn, sTn, and Tf, for subsequent applications. To perform in vitro selection, a solid support is requested to capture the sequences through multiple rounds. The magnetic beads (MBs) can be the powerful solid support in this job for its orthogonality to most of the chemicals and the convenience to operate. In this work, we demonstrated a two-step process of introducing the sTn onto the MBs. The first step involved the introduction of the BCN-functionalized spacer to modify the amino magnetic beads (NH2-MBs). In the second step, the azide-modified antigen would be coupled to the BCN-activated beads through strain-promoted alkyne–azide cycloaddition (SPAAC). To verify the successful chemical modifications introduced onto the MBs, the NH2 loading of the starting MBs was determined by the ninhydrin test. The complete conversion of the BCN spacer on the MBs would be verified by the reverse engineering of the absorbance loss of the ninhydrin test. We had also verified the successful introduction of BCN spacer by the comparison of the IR spectra. Finally, the sialic acid loading of the final target beads would be measured by the Shukla’s assay. 2025-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/37773 標題: 麩胺酸側鏈長短對於螺旋內離子對作用力以及精胺酸側鏈電荷對於辨識核糖核酸與穿透細胞膜的影響; Effect of Glutamate Side Chain Length on Intrahelical Ion Pairing Interaction and Arginine Side Chain Charge on RNA Recognition and Cellular Uptake 作者: Ming-Huei Weng; 翁明暉 摘要: 靜電作用力在穩定蛋白質結構與蛋白質辨識中，是一個重要的作用力。帶電荷胺基酸能夠參與螺旋內離子對作用力，且蛋白質內超過三成的殘基符合α螺旋結構。令人不解的是，自然界中帶電荷胺基酸的側鏈長度皆不一樣。為了探討帶電荷胺基酸側鏈長度對螺旋內離子對作用力的影響，對於可能具有螺旋內麩胺酸-精胺酸和精胺酸-麩胺酸作用力的胜肽做不同側鏈長度及不同殘基間距的研究。胜肽的螺旋性藉由圓偏光二色性光譜儀測得。根據pH 7的數據顯示，胜肽中帶電荷胺基酸側鏈長度越長 (麩胺酸、天門冬胺酸)，具有越高的α螺旋結構。 在人類免疫缺陷病毒中的蛋白質Tat49-57中，具有兩個生物功能: 其一是細胞穿透能力，具有潛力運用到藥物傳遞，其二是可以和TAR核糖核酸結合，是引起後天免疫缺乏症候群關鍵性的一步。至今仍不清楚在Tat49-57中，哪個帶正電荷精胺酸對這兩個生物功能的活性較為重要。因此，將Tat49-57中帶正電荷的精胺酸改成不帶電的瓜胺酸，並對其進行辦識核糖核酸以及細胞穿透的研究。利用膠體電泳位移和螢光各向異性度分析對TAR核糖核酸與Tat胜肽衍生物的解離常數進行研究。利用流式細胞儀評估Tat胜肽衍生物穿透Jurkat細胞膜且進入細胞內部的效率並用螢光顯微鏡觀察。序列中52、53、55號位置的精胺酸對於辦識TAR核糖核酸非常重要。精胺酸側鏈電荷對於細胞穿透的重要性取決於胜肽的濃度。在30微莫耳胜肽濃度下，序列中53、56、57號位置的精胺酸對於細胞穿透的重要性大於 其它位置的精胺酸。57號位置的精胺酸在120微莫耳濃度下對於胜肽細胞穿透的能力影響重大。; Electrostatic interactions are one of the dominant forces in stabilizing protein structure and in protein recognition. Charged amino acids participate in ion pairing interactions, and more than 30% protein residues adopt an α-helix conformation. It is unclear why the four charged amino acids have the different side chain lengths. Therefore, the effect of charged amino acids side chain lengths on intrahelical ion pairing interactions was investigated. Peptides with potential intrahelical Glu-Arg and Arg-Glu interactions were explored with various side chain lengths and spacings. Helicity for peptides was determined by circular dichroism (CD) spectroscopy. Based on CD spectra at pH 7, the longer side chain length (Glu vs Asp), the more the helicity. The human immunodeficiency virus (HIV) Tat 9-mer (Tat49-57) has two biological functions: cellular penetration, which may enable drug delivery, and binding to TAR RNA, which is a crucial step in HIV-1 virus proliferation to cause the acquired immune deficiency syndrome. However, it is unclear which positively charged Arg in Tat49-57 is important for the two biological functions. To examine the effect of Arg side chain charge on RNA recognition and on cellular uptake, each positively charged Arg in Tat49-57 was replaced with a neutral citrulline one at a time. The dissociation constant for the TAR RNA-Tat derived peptide complexes was studied by gel shift and fluorescence anisotropy assays. The cellular uptake efficiency for Jurkat cells was assessed by flow cytometry and the peptide-treated Jurkat cells were observed by an inverted fluorescence microscope. The arginines near the middle of the sequence (52, 53, and 55) play important roles for TAR RNA recognition. The importance for Arg side chain charge for cellular uptake depends on the peptide concentration. The positive charge on Arg53, Arg56, and Arg57 are more important compared to the other arginines for penetrating at 30 μM peptide concentration. The positive charge on Arg57 is crucial for penetrating at 120 μM peptide concentration. 2011-01-01T00:00:00Z http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33718 標題: 麩胺酸側鏈長度於螺旋內離子對作用力的影響以及離胺酸側鏈長度和胺基酸位置對螺旋程度的影響; Effect on Helical Content: Positional Effects, Lys and Arg Side Chain Length, and Glu Side Chain Length in Intrahelical Ion Pairs 作者: Hao-Chun Hsu; 許皓鈞 摘要: 蛋白質超過30%結構含有α螺旋結構。Lifson-Roig理論是一個用來描述胜肽的螺旋結構多寡的理論，其假設每個胺基酸形成螺旋的傾向不受胺基酸所在的位置影響。為了驗證這個假設，我們合成了一系列的胜肽，利用圓二色光譜儀測量此系列胜肽的螺旋程度，並且將胺基酸在6, 11和16號位置的w值求出。在6號和11號位置的w值很相近，但16號位置的w值卻非常的高。 為了探討胺基酸側鏈長短對w值的影響，我們利用固相胜肽合成技術設計並合成一系列含有精氨酸長度不同的非自然界胺基酸S-2-amino-6-guanidinohexanoic acid (Agh),S-2-amino-4-guanidino-butyric acid (Agb), S-2-amino-3-guanidinopropionic acid (Agp)和離氨酸長度不同的非自然界胺基酸Ornithine (Orn), S-2,4-diaminobutyric acid (Dab), S-2,3-diaminopropionic acid (Dap)並根據修飾過的 Lifson-Roig理論測量其w值。在精氨酸長度不同的非自然界胺基酸中，精氨酸的w值最高，顯示精氨酸形成螺旋的傾向最高。在離氨酸長度不同的非自然界胺基酸中，w值隨著側鏈長度增長而增加。 螺旋內離子對作用力可以穩定結構．側鏈長度與正負電荷相對間距會影響螺旋程度．為了研究側鏈長度對於影響程度的影響，利用固相胜肽合成技術合成並設計一系列含有合成出與精氨酸長度不同的非自然界胺基酸S-2-amino-6-guanidinohexanoic acid (Agh),S-2-amino-4-guanidino-butyric acid (Agb), S-2-amino-3-guanidinopropionic acid (Agp)以及與穀胺酸長度不同的天門冬胺酸的胜肽序列，包括AspAgh4, GluAgh4, AspAgb3, GluAgb3, AgpAsp5以及 AgpGlu5. 利用圓二色光譜儀測量不同胜肽在pH 2-12範圍下的螺旋程度。圓二色光譜儀測得訊號結果包括個別胺基酸本身對於螺旋喜好程度、胺基酸序列中側鏈之間與的作用力以及胺基酸側鏈與螺旋骨架N端C端作用力．本研究中，根據圓二色光譜儀訊號顯示pH 7情況下，螺旋程度大小依序為GluAgh4 > AspAgh4, GluAgb3 > AspAgb3, AgpGlu5 > AgpAsp5. 當負電荷胺基酸側鏈越長，其表現出的螺旋程度也相對較高。; One third of all protein residues adopt a helical conformation. Statistical mechanical models such as modified Lifson-Roig theory are used to describe the conformational ensemble of monomeric helical peptides. One basic assumption in these statistical models is that the helix propensity for a given amino acid is position independent. To test this assumption, the helix propensity for neutral non-Ala residues at various guest positions were derived from circular dichroism spectroscopy (CD) data. Helix propensities were similar for positions 6 and 11 for the same amino acid, but much higher at position 16. Helix propensity (w) for Arg and Lys analogs were derived based on modified Lifson-Roig theory to investigate the effect of Lys and Arg side chain length on helix formation. The helix propensity (w) for Arg analogs followed the trend: wArg > wAgh > wAgb > wAgp, indicating the uniqueness of the Arg side chain length in helix formation. In contrast, all three Lys analogs were energetically unfavorable for N-capping. Orn and Dap were energetically favorable for C-capping, whereas Dab was energetically unfavorable for C-capping. All three Lys analogs were energetically unfavorable at internal helix positions. Electrostatic ion pairing interactions between oppositely charged amino acids can stabilize proteins and helical structures. To study the effect of Glu side chain length and relative spacing on intrahelical ion pairing interaction, peptides AspAgh4, GluAgh4, AspAgb3, GluAgb3, AgpAsp5, and AgpGlu5 were synthesized and studied by CD at pH 2-12. Based on CD data at pH 7, the helical content of the peptides followed the trend GluAgh4 > AspAgh4, GluAgb3 > AspAgb3 and AgpGlu5 > AgpAsp5. The results showed that helicity increases with increasing side chain length of the negatively charged residue (Glu vs Asp). 2011-01-01T00:00:00Z