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研究生:曾子銘
研究生(外文):Tzu-Ming Tseng
論文名稱:在固相多肽合成中引入同型半胱胺酸硫內酯的方法開發
論文名稱(外文):Methodology Development Towards the Introduction of γ-thiolactone in SPPS
指導教授:林渝亞
指導教授(外文):Yu-ya Lin
學位類別:碩士
校院名稱:國立中山大學
系所名稱:化學系研究所
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:2020
畢業學年度:108
語文別:中文
論文頁數:94
中文關鍵詞:固相胜肽合成高半胱胺酸同型半胱胺酸硫內酯胜肽組織蛋白去乙醯基酶CHAP 31H3K27AsuNHOHN-酰基苯並三唑
外文關鍵詞:histone deacetylaseshomocysteine thiolactonepeptideN-acyl benzotriazsolid-phase peptide synthesisH3K27AsuNHOHCHAP 31
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胜肽藥物已成為新型的治療藥劑,並且具有良好的藥理特性,例如高選擇性、低毒性、代謝穩定性等優勢。而硫醇具有很強的親核性,可以在溫和條件下進行各種化學轉換,並且已被用於生物分子例如脫氧核糖核酸、胜肽和蛋白質的化學修飾中。但由於巰基官能基團的反應性,使得以往巰基的保護基團的去保護步驟是對環境不利且原子經濟不佳的。而透過硫代內酯的胺解便是解決此問題有效方法之一。因此本論文希望以固相胜肽合成的方法將同型半胱胺酸硫內酯建立在胜肽序列中。
本論文嘗試合成兩種包含同型半胱胺酸硫內酯之胜肽序列 (CHAP 31 含硫衍生物與 H3K27AsuNHOH 含硫衍生物) 之前驅物。CHAP 31 與 H3K27AsuNHOH 皆為選擇特異性的組織蛋白去乙醯基酶抑制劑,因此先以這兩種胜肽序列做為參考。
本研究透過微波加熱合成儀進行固相胜肽合成程序,在胜肽序列中構建的鄰氨基苯胺基於酸性條件下經亞硝酸鹽形成 N-酰基苯並三唑 (N-acyl benzotriazole) 結構,爾後嘗試用同型半胱胺酸硫內酯 (Homocysteine thiolactone, HCT) 作為切除樹酯的試劑,並利用實驗室中已開發的方法──經同型半胱胺酸硫內酯醯基置換進行開環而合成含高半胱胺酸 (Homocysteine, HCys) 之胜肽。
雖然尚未完成最終產物的合成,但已逐步檢視各個流程,並將其改善。待此方法成熟後再進行硫的衍生化反應,並確認其生物活性,後續再改變胜肽序列,比較其生物活性。
Peptide drugs have become a new type of therapeutic agent, and have good pharmacological properties, such as high selectivity, low toxicity, metabolic stability and other advantages. Thiols have strong nucleophilicity and can perform various chemical transformations under mild conditions, and have been used in the chemical modification of biological molecules such as deoxyribonucleic acid, peptides and proteins. However, due to the reactivity of the sulfhydryl functional group, the deprotection steps of the sulfhydryl protecting group is unfavorable to the environment and poor atom economy. Aminolysis through the reaction between thiolactone and amine is one of the effective methods to solve this problem. Therefore, this thesis hopes to establish homocysteine thiolactone in the peptide sequence.
This thesis attempts to synthesize two peptide sequences containing homocysteine thiolactone (The precursor of thioether analogues of CHAP 31 and H3K27AsuNHOH). Both CHAP 31 and H3K27AsuNHOH are selective HDIs, so were chosen as the target peptide sequences.
In this study, a solid-phase peptide synthesis procedure was carried out using a microwave heating synthesizer. The o-aminoaniline constructed in the peptide sequence allows the formation of the N-acyl benzotriazole structure via reaction with nitrite under acidic conditions. Next, homocysteine thiolactone (HCT) was used as a reagent to cleave the resin resulting in HCT residue at the C-terminus. The HCT-containing peptide could then be subjected to the method developed in the laboratory to yield a homocysteine-containing peptide by aminolysis of homocysteine thiolactone.
Although the synthesis of the final product has not been completed, each step has been gradually reviewed and optimized. After this method matures, the sulfur derivatization reaction is performed to confirm its biological activity, and then the peptide sequence is changed to compare its biological activity.
The investigation done in thesis would enable future work regarding the synthesis of thiolactone peptide by SPPS in the laboratory to be performed more efficiently.
論文審定書 i
致謝 ii
中文摘要 iv
Abstract v
目次 vii
圖目錄 x
流程目錄 xi
表目錄 xii
光譜目錄 xiii
縮寫表 xiv
第一章 緒論 1
1.1 胜肽合成 3
1.1.1. 固相胜肽合成之載體 3
1.1.2. 固相胜肽合成之步驟 4
1.1.3. 微波輔助胜肽合成 5
1.1.4. 偶聯試劑 6
1.2 同型半胱胺酸硫內酯 (γ-thiolactone) 8
1.3 利用鄰氨基苯胺修飾固相多肽合成之碳端 10
1.4 組織蛋白去乙醯基酶 (Histone deacetylases, HDACs) 12
1.4.1. 組織蛋白乙醯化與去乙醯化 12
1.4.2. HDACs 的種類介紹 13
1.4.3. HDACs 的作用機制 15
1.4.4. 組織蛋白去乙醯基酶抑制劑 (Histone deacetylase inhibitor, HDI) 17
第二章 文獻回顧 19
2.1 選擇性組織蛋白去乙醯基酶抑制劑 19
2.1.1. 環狀胜肽之合成 21
2.1.2. 線性組織蛋白去乙醯基酶抑制劑 22
第三章 研究動機 24
第四章 結果與討論 27
4.1 胜肽片段之製備 28
4.1.1. 各類胺基酸胺端保護之合成 28
4.1.2. 3’-Fmoc-Dbz-OH之合成 29
4.1.3. Fmoc-Tyr(Me)-OH之合成 29
4.1.4. HCT 之旋光對拆分方法 30
4.2 固相多肽合成 (Solid phase peptide synthesis, SPPS) 32
4.2.1. 微波固相多肽合成使用試劑與反應條件 32
4.2.2. Kaiser 試劑檢測 33
4.3 CHAP 31 含硫衍生物與 H3K27AsuNHOH 含硫衍生物之合成 34
4.3.1. CHAP 31 含硫衍生物之逆合成分析 34
4.3.2. H3K27AsuNHOH 含硫衍生物之逆合成分析 36
4.3.3. CHAP 31 含硫衍生物與 H3K27AsuNHOH 含硫衍生物之微波固相多肽合成 38
4.3.4. 二氨基苯甲酸環化反應 39
4.3.5. 去除樹酯之結果與討論 39
4.4 固相胜肽合成合成步驟之探討 42
4.4.1. NH2-Gly-Gly-Gly-Dbz-NH2 之合成 42
4.4.2. NH2-Lys-Ala-Ala-Arg-Dbz-NH2 之合成 44
4.5 結論 47
第五章 參考資料 48
第六章 實驗步驟與光譜數據 54
6.1 儀器設備與藥品材料 54
6.2 合成步驟與光譜數據 56
6.2.1 胺基酸衍生物合成基本步驟 56
6.2.2 化合物 8 的合成步驟 59
6.2.3 化合物 10 的合成步驟 63
6.2.4 固相胜肽合成 65
6.2.5 化合物 27-2 的合成步驟 67
光譜資料 69
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