臺灣博碩士論文加值系統

天然化學連接法（Native chemical ligation，NCL）是高效實用的主流多肽/蛋白質化學合成方法，彌補了固相多肽合成法在合成多肽鏈長度上的局限。但天然化學連接法依賴半胱氨酸（Cysteine）以進行多肽鏈間的連接，而半胱氨酸在天然蛋白質中含量極少（約1%），極大地限制了NCL 的運用。
本實驗室先前開發出一種光解輔助基以修飾天門冬氨酸，並成功將其引入寡肽之間的天然化學連接。本研究目標是將類似光解基團修飾在天門冬醯胺上，使其成為可直接應用於固相多肽合成（Solidphase-peptide-synthesis, SPPS）的氨基酸組件。使用被修飾的天門冬醯胺代替半胱氨酸在水相緩衝溶液中進行天然化學連接。完成連接後施加紫外線光照，在溫和的反應條件下除去完成使命的輔助基團，得到“天然”的目標多肽。

Native chemical ligation(NCL) is a practical and efficient methodology for peptide/proteins synthesis. However, the rare presence of the cysteine residue (1.4% content in proteins) has limited the utility of NCL (most peptide pharmaceuticals rarely contain an internal cysteine).
The previously work in our lab already developed a photocleavable auxiliary to modify Asp. Such building block has been used in NCL between dipeptides. The method of this job relied on Photocleavable-auxiliary-modified Asn/Gln to achieve NCL. Modified Asn/Gln can take place the role of Cys in NCL. Using solid-phase peptide synthesis (SPPS) to synthesis peptide sequences, then ligate peptide sequences by NCL. We use UV irradiation after NCL to obtain target peptide mild and rapidly.
The objective of this job is use such method to achieve NCL between
short-sequence peptides ligation and produce a valuable peptide/protein.

目錄

目錄......................................................i
圖目錄...................................................iv
表目錄...................................................vi
流程目錄................................................vii
氨基酸總表.........................................viii
縮寫對照表...............................................ix
第一章 緒論............................................1
1-1 前言..............................................1
1-2 傳統多肽合成......................................2
1-3 固相多肽合成法....................................4
1-4 天然化學連接法....................................6
1-4-1 天然化學連接法的起源........................6
1-4-2 天然化學連接法的反應機構....................6
1-5 不需要半胱氨酸的天然化學連接法....................8
1-5-1以自由基反應的方式移除硫醇輔助基.............8
1-5-2 多肽主鏈修飾光解輔助基.....................12
1-5-3 醣輔助連接法...............................14
1-5-4 側鏈輔助法.................................18

1-6其他新穎的多肽連接法..............................20
1-6-1 絲氨酸/蘇氨酸連接法........................20
1-6-2 酮酸-羥胺連接法............................22
1-6-3輔助基介導天然化學連接法....................22
1-7 固相多肽合成法合成C端為硫酯的多肽...............23
1-7-1 樹脂的選擇.................................24
1-7-2 硫醇離去基的選擇...........................26
1-7-3 固相多肽合成法合成被修飾成硫酯的多肽.......27
1-7-4 硫醇離去基的替代基團.......................29
1-7-5 硫醇催化劑.................................30
1-8 天然化學連接法在蛋白質合成的實際運用.............31
第二章 研究動機與構想.................................33
2-1 於天門冬醯胺側鏈引入光解基團.....................33
2-2 天門冬醯胺組件的實際運用.........................35
第三章 結果與討論.....................................36
3-1 合成氨基酸衍生物物...............................36
3-1-1 光解輔助基團...............................36
3-1-2 合成氨基酸骨架及氨基酸組件.................43
3-1-3 氨基酸組件的光解測試.......................47
3-2 合成天然化學連接法所需的多肽.....................52
3-2-1 合成C端多肽...............................53
3-2-2 合成N端多肽...............................54
3-3 未來展望.........................................59
3-3-1 將輔助基團應用於其他氨基酸.................59
3-3-2 探討分子內醯基遷移環數影響及側鏈立障之影響.60
第四章 實驗部分........................................62
4-1 一般實驗方法.....................................62
4-1-1 核磁共振光譜...............................62
4-1-2 質譜.......................................62
4-1-3 薄層色譜層析法.............................63
4-1-4 製備及薄層色層分析法.......................63
4-1-5 管柱層析法.................................64
4-1-6 化學藥品...................................64
4-2 固相多肽合成法...................................65
4-2-2 樹脂前處理及引入第一個氨基酸單元...........65
4-2-3 多肽序列建構與從樹脂上切除之方法...........66
4-3 使用高效液相層析法純化產物.......................67
4-3-1 沖提系統...................................67
4-3-2 儀器與管柱.................................67
4-3-3 分析與純化條件.............................67
4-4 實驗步驟及光譜資料...............................69
光譜附錄...............................................97
參考文獻................................................180
 
圖目錄

圖1 進行耦合反應的限制性-將能幹擾的官能基保護............2
圖2 偶合反應之副產物.....................................2
圖3 進行偶合反應的限制性-外消旋導致位向不專一............3
圖4 進行偶合反應的限制性-反應過程產生aspartimide.........4
圖5 固相多肽合成法.......................................5
圖6 分子內醯基遷移路徑...................................6
圖7 纈氨酸與半胱氨酸之間的肽鍵合成.......................6
圖8 天然化學連接法的反應機構.............................7
圖9 各天然氨基酸在天然蛋白質中的含量.....................8
圖10 以除硫反應得到不含半胱氨酸的多肽....................9
圖11 自由基除硫反應的反應機構...........................10
圖12 含硫輔助基的氨基酸衍生物...........................11
圖13 除硫反應不具有選擇性...............................12
圖14 光解輔助基應用於天然化學連接法.....................12
圖15 以光解輔助基團連接組蛋白的一段多肽和泛素...........13
圖16 醣輔助連接法.......................................15
圖17 用醣輔助連接法或側鏈輔助連接法合成多肽.............19
圖18 合成HIV-2 Tat......................................20
圖19 絲氨酸/蘇氨酸連接法................................21
圖20酮酸-羥胺連接法.....................................22
圖21輔助基介導天然化學連接法............................23
圖22 組裝C端為硫酯的多肽...............................26
圖23 Danishefsky團隊所開發的硫酯鍵多肽合成方法..........27
圖24 固相多肽合成法建構低活性硫酯基的多肽................28
圖25 以固相多肽合成法建構含硫酯基的多肽..................29
圖26 以固相多肽合成與親和醯基取代反應建構C端多肽........30
圖27 硫醇催化劑的發展過程................................31
圖28 紅血球生成素衍生物的合成............................32
圖29 光解輔助基團建立於天門冬醯胺的側鏈上................33
圖30 光解反應機構........................................34
圖31天門冬醯胺組件的合成................................35
圖32 將穀胺醯胺組件引入固相多肽合成及天然化學連接........35
圖33化合物1和化合物9之差異.............................41
圖34輔助基團運用在建構在多肽上的兩種方案................45
圖35 兩種氨基酸組件的逆合成分析..........................46
圖36 Schofield教授提出的光解反應測試.....................51
圖37 目標多肽片段之合成方案..............................53
圖38 粗產物的HPLC圖譜...................................56
圖39 質譜上已有的產物....................................57
圖40 由質譜推測可能有的產物..............................58
圖41 光解輔助基運用於其他氨基酸..........................59
表目錄

表1 Diederichsen團隊的輔助基及其效率的探討...............14
表2 醣輔助連接法效率之探討...............................16
表3 不同中間體環數對醣輔助連接法效率的探討...............18
表4氨基酸側鏈立障對絲氨酸/蘇氨酸連接法效率的影響........20
表5 固相多肽合成法常用的樹脂.............................24
表6 氨基酸組件化合物21的光解反應能力探究................49
表7 探討化合物21、25和27的光解能力.......................52
表8 延伸N端多肽長度.....................................60
表9 改變N端氨基酸側鏈...................................61
表10 顯色劑配方..........................................63表11 HPLC系統條件與梯度洗脫條件..........................68
 
流程目錄

流程一 光解輔助基的逆合成分析............................37
流程二 化合物1的合成路徑................................38
流程三 改良化合物5的合成路徑............................39
流程四 對取代反應的探討與優化............................40
流程五 改良後的化合物1合成方案..........................41
流程六 化合物9的合成計劃................................42
流程七 化合物9的合成路線................................43
流程八 兩種氨基酸組件的合成路徑..........................47
流程九 化合物27之合成路徑與光解.........................51
流程十 C端多肽的合成流程.................................55
流程十一 固相多肽合成法合成N端多肽之流程................56

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