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研究生:陳昱竑
研究生(外文):Yu-Hung Chen
論文名稱:合成兩種光親和性探針用於研究植物之防禦機制以及乳癌用藥泰莫西芬的副作用來源
論文名稱(外文):Synthesis of Two Photoaffinity Probes to Investigate Defensive Response in Plants and Adverse Side Effects of Tamoxifen
指導教授:陳昭岑
指導教授(外文):Chao-Tsen Chen
口試委員:陳逸然冀宏源
口試委員(外文):Yet-Ran ChenHung-Yuan Chi
口試日期:2017-03-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:181
中文關鍵詞:蛋白質體學光親和性探針光反應基團CAPE1泰莫西芬矽羅丹明同位素編碼
外文關鍵詞:proteomicsphotoaffinity probephotoreactive groupCAPE1tamoxifenSi rhodamineisotope code
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 本論文主要分為兩部分,第一部分為針對植物的防禦機制的研究。系統素(Systemin)等胜肽類植物激素主導了植物的抗蟲害防禦反應,2014年陳逸然博士團隊發現了新的一段肽類植物激素CAPE1,具有類似系統素啟動防禦機制的功能,但其結合蛋白以及訊息傳遞的細節尚未被完全了解。本研究希望進一步了解CAPE1的結合蛋白與其訊號傳遞機制,進一步可以將此胜肽應用為生物可降解與環境友善的農藥,在提升農業產率的同時不對環境造成汙染。因此本研究設計並合成了以CAPE1多肽序列為辨識端的光親和性探針PhoLeu-CAPE1用於研究CAPE1之結合蛋白,如圖A所示,將此探針作用於植物葉片的蛋白質體後照光進行共價性鍵結,希望能夠標示並鑑定未知的CAPE1結合蛋白。探針設計上引入光親和性胺基酸photo-leucine作為光反應基團,並以生物素作為親和性標籤,活性測試結果顯示設計的探針仍舊保有CAPE1的活性,並正在進行結合蛋白的標示。
 第二部分為針對乳癌治療藥物泰莫西芬的研究。在乳癌的治療上泰莫西芬(Tamoxifen)是針對雌性激素受體陽性反應ER-(+)病患的一線用藥,但是在使用上泰莫西芬仍具有許多的副作用,包括熱潮紅、噁心、嘔吐、陰道不適、陰道出血等生理不適,同時也增加罹患子宮內膜癌的風險,這些副作用降低了泰莫西芬的專一性治療效果。此外文獻也指出泰莫西芬針對非ER-(+)的細胞具有引發計畫性細胞凋亡(Apoptosis)的作用,但詳細的作用機制仍未被完全了解。因此,本研究另一個研究目標即是希望發展光親和性探針探索這些副作用的來源。因此,設計並合成了一個以泰莫西芬的生理代謝物4-羥基泰莫西芬(4-hydroxytamoxifen, 4-OHT)為辨識基團的光親和性探針4-OHT-diaz-alkyne,如圖B (a)所示。此探針引入兩階段標示的概念,希望利用點擊化學(Click reaction)在蛋白質標示完成後再接上標籤基團。與探針對應的標籤基團部分也設計了多重功能的標籤SiRho-N3-Biotin,如圖B (b)所示,可以同時引入螢光團以及生物素標籤,在螢光團的選擇上使用放光波段在近紅外光且量子產率更高的矽羅丹明,而生物素標籤部分則額外引入同位素編碼鑑定的功能。希望這些探針能夠標示到尚未被發現的結合蛋白,並且利用設計的標籤達到更佳的訊號輸出功能。
This thesis is mainly divided into two parts. The first part is to study defensive response in plants. Systemin regulates the expression of defensive genes in tomato leaves in response to insect attacks or other severe mechanical wounding. In 2014, Chen et al discover a novel polypeptide plant hormone named CAPE1. CAPE1 shares similar defensive function with systemin, but its binding protein and the detail signal transduction pathway haven’t been fully understood. These peptidic plant hormones have great potential to become biodegradable and environment friendly pesticide. Thus, a photoaffinity probe PhoLeu-CAPE1 with CAPE1 peptide sequence as the recognition part to investigate CAPE1 binding protein was designed and synthesized. After incubation with plant leaves proteome and photoactivation with UV light, this probe can covalently link to the binding protein for further analysis (Figure A). Despite he probe equips photo-leucine as a photoreactive group and biotin as an affinity tag, it can still trigger defensive response as the native CAPE1. The binding protein labeling is still under progress.
The second part is to study the origin of tamoxifen-induced side effect. Tamoxifen is the first-line drug to treat breast cancer patient with estrogen receptor positive. However, the treatment accompanies with many adverse side effects, including hot flashes, vaginal bleeding or discharge, as well as increasing the risk of endometrial cancer. Furthermore, tamoxifen induces apoptosis in estrogen receptor negative cell lines, but the detail mechanism hasn’t been fully understood. The origin of these adverse effects needs to be sought. Herein, a photoaffinity probe 4-OHT-diaz-alkyne with 4-hydroxytamoxifen (4-OHT) as the recognition unit was designed and synthesized (Figure B-a). A two-step labeling concept was introduced in the probe design. To utilize azide-alkyne cycloaddition to attach tag after protein labeling. A multiple functional tag SiRho-N3-Biotin featuring near infarred dye Si-rhodamine with higher quantum yield as fluorophore and the isotope-coded affinity tag for mass spectrometry detection was designed and synthesized (Figure B-b). These photoaffinity probes were expected to find the unknown binding proteins, followed by coupling with the tag for further purification and identification.
目錄 i
圖目錄 iv
Scheme目錄 vi
表目錄 vii
中文摘要 viii
英文摘要 x

第一章、蛋白質體學與光親和性探針之介紹 1
1.1 蛋白質體學之介紹 1
1.2 Activity-based probe 2
1.3 Affinity-based probe 4
1.4 光親和性基團 7
1.4.1 芳香族疊氮 8
1.4.2 二苯甲酮 9
1.4.3 偶氮環丙烯 10
1.5 光親和性胺基酸 12
1.6 標籤報導基團 13
1.6.1 親和性標籤 14
1.6.2 螢光團 14
1.6.3 同位素標籤 15
1.7 兩階段標示 17

第二章、胜肽激素啟動之植物防禦機制介紹及其光親和性探針之設計 21
2.1 系統素Systemin簡介 22
2.2 新穎植物激素CAPE1介紹 25
2.3 研究動機 28
2.4 探針CPRG-CAPE1之分子設計以及逆合成分析 28
2.5 探針CPRG-CAPE1之合成 31
2.6 探針PhoLeu-CAPE1之分子設計以及逆合成分析 39
2.7 探針PhoLeu-CAPE1之合成 42
2.8 實驗結果與討論 48

第三章、乳癌用藥泰莫西芬介紹及其光親和性探針之設計 50
3.1 類固醇激素簡介 50
3.2 雌性激素簡介 51
3.3 雌性激素受體結構與其作用機制 52
3.3.1 雌性激素受體之配位子結合區域分析 52
3.3.2 雌性激素受體之作用機制 55
3.4 乳癌的治療與選擇性雌激素受體調節物 56
3.5 研究動機 58
3.6 探針4-OHT-diaz-alkyne之分子設計以及逆合成分析 59
3.7 探針4-OHT-diaz-alkyne之合成 61
3.8 多重功能標籤基團Rho-N3-Biotin之分子設計以及逆合成分析 67
3.9 多重功能標籤基團Rho-N3-Biotin之合成 73
3.10 同位素編碼親和性標籤N3-Biotin-D8之合成 80
3.11 預期結果 83

實驗部分 84
一、一般敘述 84
二、合成步驟及光譜數據 85
參考文獻 122
附錄 128
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