利用氟標記輔助化學及酵素合成寡醣

Fluorous-tag Assisted Chemoenzymatic Synthesis of Oligosaccharides

侯凱齡(Kai-Ling Hou)

指導教授 ： 林俊成

國立清華大學/理學院/化學系/碩士(2013年)

摘要

本論文中利用IMPACTTM-CN system (Intein Mediated Purification with Affinity Chitin-binding Tag) 系統，成功表達出岩藻糖激酶/鳥苷二磷酸岩藻糖焦磷酸化酶 (FKP)、α-1,3-岩藻糖轉移酶 (α-1,3-FucT) 與 -1,3-乙醯半乳糖胺基轉移酶/半乳醣轉移酶 (LgtD)。利用FKP合可製得百毫克的GDP-Fuc，結合α-1,3-FucT與實驗室已發表之唾液酸轉移酶 (PmST)，成功合成Lewis X和sialyl Lewis X。
為了進一步合成不同的多醣體，表達及結合實驗室已有的半乳糖激酶 (GalK)、α-1,4-半乳糖轉移酶 (LgtC)、β-1,3-N-乙醯葡萄糖胺轉移酶 (HpGnT)，並針對醣基轉移酶進行非專一受質的忍受度探討，成功合成出P1抗原。此外，利用氟標記之物質，可快速純化目標產物的特性，進一步將氟標記修飾在雙醣的還原端連結鏈上，結合酵素反應進行目標物合成，並經由氟的固相萃取 (Fluorous Solid-phase Extraction，F-SPE) 可大幅縮短純化時間為三十分鐘，是一種有效率的合成方法。
另外，研究發現α-1,3-FucT可以接受含氟標記之N-乙醯基乳糖胺 (LacNAc)、sialyl LAcNAc、LacdiNAc (N,N-diacetyl-lactosamine) 當作受質，進行醣基化反應 (fucosylation) 後，生成含氟標記之Lewis X、sialyl Lewis X和dimeric Lewis X。

關鍵字

酵素合成 ； 氟 ； 抗原 ； 寡醣

並列摘要

In this thesis, we employed IMPACTTM-CN system to overexpress L-fucokinase/GDP-fucose pyrophosphorylase (FKP), α-1,3-fucosyl- transferase (FucT), and β-1,3-N-acetylgalactosaminyltransferase/ β-1,3-galactosyltransferase (LgtD). FKP was used to synthesize GDP-Fucose. The synthesis was performed on preparative scales (230 mg) and the reaction was monitor by high performance liquid chromatography. After quenching the reaction with ethanol, the crude products were purified by gel filtration using BioGel P-2 resin (Bio-Rad). In combination with α-1,3-fucosyltransferase and other glycosyltransferases, Lewis X and sialyl Lewis X have been synthesized successfully.
To synthesize P1 antigen, other enzymes such as galactokinase (GalK), β-1,3-N-acetylglucosaminyltransferase (HpGnT) and α-1,4-galactosyltransferase (LgtC) were expressed. After optimization of enzymatic reaction conditions, P1 antigen was synthesized successfully. Furthermore, an fluorous assisted separation was developed to facilitate the purification of enzymatic reaction products. By using a fluorous tag at the reducing end of saccharide, optimized conditions of the enzyme, and fluorous solide phase extraction, the separation time was remarkably reduced to 30 min for each seperation.
In addition, α-1,3-fucosyltransferase was found to tolerate a wide range of substrates appended with a fluorous tag such as LacNAc, sialyl LAcNAc, and LacdiNAc in this study. After fucosylation, these substrates were transformed to fluorous tagged Lewis X, sialyl Lewis X and dimeric Lewis X.

並列關鍵字

enzymatic synthesis ； fluorous ； antigen ； oligosaccharides

參考文獻

112. 游景晴，國立清華大學化學研究所，博士論文，奈米粒子系統應用: 酵素固化與高通量酵素篩選平台開發，民國101年。

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