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研究生(外文):Jui-Chi Lee
論文名稱(外文):Chemical Synthesis of GDP-Alkynyl Fucose and its Application as a Substrate for Different Kinds of Fucosyltransferases
指導教授(外文):Chi-Huey WongChung-Yi Wu
外文關鍵詞:fucosealkynyl fucosefucosyltransferasefluorousaluminum-coated glass slide
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岩藻醣 (fucose) 常見於細胞表面的各種醣化合物 (glycoconjugates) 中,對於細胞的性質與功能具有相當重要的影響。為了了解岩藻醣在生物體內的分布與表現,翁院長研究團隊發展了以岩藻醣類似物-炔基岩藻醣 (alkynyl fucose) 作為生物探針的技術1, 2,並對人類肝癌細胞及乳癌細胞進行測試,確定此種合成物質可以被細胞所接受,並可藉由螢光影像反映出被標記的岩藻醣在細胞的表現情形。為了進一步探討此一岩藻醣類似物應用在生物個體的情形,我也和陳鈴津老師實驗室合作,以老鼠為測試對象進行生物測試。
在另一方面,目前已知有至少十三種不同類型的岩藻醣轉移酵素(fucosyltransferase, FucT)在人類細胞中被發現,但其中何者可接受前述的岩藻醣類似物,則是尚待解決的問題。為了進行酵素活性測試,我也合成出GDP-alkynyl fucose作為岩藻醣予體(fucosyl donor)及N-acetyl-lactosamine (LacNAc)類似物作為岩藻醣受體(fucosyl acceptor)。我們與林俊宏老師實驗室合作,針對現有的幾種岩藻醣轉移酵素(FucT-Hp, FucT-9, FucT-4)進行測試;另外,我們也分別以GDP-fucose及lactose作為岩藻醣予體及岩藻醣受體,進行對照組的酵素活性測試。
傳統上,岩藻醣轉移酵素活性的測試方法各有其限制與缺點,如同位素標定法(radio-labeling),同位素標定化合物的合成不甚方便且具有風險;利用NADH消耗量進行測試固然可行,但因其過程中包含三種酵素(FucT, pyruvate kinase, lactate dehydrogenase),使結果具有較大的不確定性;螢光檢測(fluorescence imaging)靈敏且快速,但無法同時比較兩種以上同時作用的酵素之間的關係。為此,我們實驗室發展出一套以氟化材料(fluorinated materials)、鍍鋁玻璃晶片(aluminum-coated glass slide, ACG slide)及雷射脫附離子化質譜分析(laser desorption/ionization mass spectrometry)為基礎的酵素活性分析方法。分析過程快速而方便,靈敏度高,可同時比較多種酵素。在本研究中,我們也運用此一分析方法進行岩藻醣轉移酵素的活性測試。
為了進行酵素活性測試,我也將LacNAc接上一段氟碳鏈(fluorous alkyl chain)以應用於上述分析方法。結果顯示GDP-alkynyl fucose雖然可為上述幾種岩藻醣轉移酵素(FucT-9, FucT-4)所接受,但效果不若GDP-fucose佳。以此初步研究為基礎,未來需要針對其他種類的岩藻醣轉移酵素及不同受質進行測試,以確定炔基岩藻醣在細胞內被接受的程度為何,並以此評估其作為岩藻醣探針的效果。
Fucose appears in many glycoconjugates on cell surface and plays critical roles in the biological features and physiological functions of cells. In order to study the distribution and expression of fucose in living organisms, a fucose analog, namely alkynyl-fucose, was designed to label the fucosyl glycoconjugates.1, 2 In these studies, alkynyl fucose can be accepted in human hepatocellular carcinoma cells and breast adenocarcinoma cells. In the help of Dr. Alice Lin-Tsing Yu’s laboratory, we administrated mice with alkynyl fucose to explore its application in living organisms.
In addition, there were at least thirteen different fucosyltransferases (FucT) found in human cells. However, it is not sure that which FucT can accept alkynyl fucose. In order to solve this problem, we designed GDP-alkynyl fucose and N-acetyl-lactosamine as the fucosyl donor and acceptor respectively. Cooperating with Dr. Chun-Hung Lin’s laboratory, the activities of several fucosyltransferases, including FucT-Hp, FucT-9 and FucT-4, were tested.
Traditionally, determination assays of fucosyltransferase activity have their limits and drawbacks. For example, it is difficult and danger to synthesize isotope-tagged compounds by radio-labeling technique; assaying by determination of NADH consumption is feasible, but the included enzymes (FucT, pyruvate kinase, lactate dehydrogenase) make the detection result indefinite; fluorescence imaging is sensitive and quick, but it is not applied to comparison between more than two enzymes. Therefore, an enzyme activity determination assay based on fluorinated materials, aluminum-coated glass (ACG) slide and laser desorption/ionization time-of-flight mass spectrometry was developed. It is sensitive, convenient and able to compare multiple enzymes at a single experiment. In this study, fucosyltransferase activity was determined by this assay.
LacNAc was linked to a fluorous alkyl chain and used in the assay described above. The results revealed that GDP-alkynyl fucose can be tolerated by FucT-9 and FucT-4 but not as well as GDP-fucose. Based on the preliminary test, different fucosyltransferases and substrates will be assayed to assess the feasibility of alkynyl fucose probe in cells in the future.
Acknowledgement i
Abstract (Chinese) ii
Abstract (English) iv
Abbreviation vi

1. Introduction 1
1.1 Glycobiology 1
1.2 The significance of fucose and fucosylation 3
1.3 Using alkynyl fucose as a probe to profile fucose expression patterns in cells and organelles 5
1.4 Development of Matrix-Free Laser Desorption/Ionization-Time of Flight Mass Spectroscopy Using Aluminum-Coated Glass Slide 10
2. Result and Discussion 14
2.1 Chemical Synthesis of Alkynyl-Fucose Analog 14
2.2 Chemical Synthesis of the Fucosyl Donor 17
2.3 Chemical Synthesis of the Fucosyl Acceptor 18
2.4 Monitoring fucosylation by laser desorption/ionization TOF MS on fluorinated ACG slide 21
3. Conclusion 30
4. Experimental 31
4.1 Materials 31
4.2 General 32
4.3 Instrumentation 32
4.4 List of known compounds 33
4.5 Synthesis of 1,2,3,4-Tetraacetyl Alkynyl Fucose Probe 33
4.6 Synthesis of GDP-Fucose and GDP-Alkynyl-Fucose 38
4.7 Synthesis of LacNAc-linker 43
4.8 Analysis of fucosylation by laser-desorption/ionization time-of-flight mass spectroscopy on aluminum-coated glass slide 49
5. Reference 52

Appendix 57
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