Molecular Mechanism of Symplectoteuthis Bioluminescence Using Coelenterazine Analogs

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研究生：	周峻名
論文名稱：	Molecular Mechanism of Symplectoteuthis Bioluminescence Using Coelenterazine Analogs
指導教授：	磯部稔 Minoru Isobe 汪炳鈞 Uang, Biing-Jiun
口試委員:	洪上程 Hung, Shu-Chen 陳淑慧 Chen, Shu-Hui 廖俊臣 Liao, Chun-Chen
學位類別：	博士 Doctor
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	103
中文關鍵詞：	腔腸素、脫氫腔腸素、生物冷光、發光蛋白
外文關鍵詞：	coelenterazine, photoprotein, symplectin
相關次數：	點閱：2 下載：0
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在發光蛋白(photoprotein)中，symplectin是少數在結合位置與活化位置皆用共價鍵連接脫氫腔腸素(DCL)，此種鍵結是半胱氨酸的SH基團進行共軛加成所產生的。此發光蛋白在產生冷光前，其發色團(chromophore)分子會先從結合位置移至活化部位Cys-390。
本研究的第一部分是開發高效率的合成方式以獲得烯醇酮(enol-aldehyde)的等同物：酮縮醛(keto-acetal 2-6a, 2-20b, 2-20c)與酮碸(keto-sulfone 2-24b, 2-24c)，因為烯醇酮是獲得腔腸素與其類同物等此類螢光素的重要片段。再將較佳的縮合反應應用在氨基吡嗪與各式酮縮醛或酮碸上，相較於以往氨基吡嗪與烯醇酮的反應，此種改良後的合成法可獲得較多量的腔腸素與其類同物。
第二部份描述在使用具有8-(4'-甲氧苯基)-與8-(2'-萘基)-的發色團與含有天然發色團的symplectin作用後，分析螢光的變化來研究發色團與發光蛋白的動態結合。此實驗中，我們在微酸性與可產生冷光的條件下(pH=7.8)觀察螢光的變化，並透過液相層析的方法分析冷光發生後的萃出物，證實無法產生螢光的萘基類同物在pH=6.0時可取代發光蛋白中原有的腔腸素，並與螢光蛋白形成鍵結。
第三部份，利用半胱氨酸加成物2-43b與水母素(aequorin)反應以模擬脫氫腔腸素與symplectin的反應機構。在結果中發現，分子內的過氧基的氧化反應會削減冷光的發生，透過此反應並佐以先前的研究，確立了半胱氨酸加成物2-43b中的2'碳上的立體位向。

Symplectin, which is one of the few photoproteins, forms covalent bonds with the dehydrocoelenterazine (DCL) at the binding sites and the active site. This binding takes place through the SH’s of the cysteine residues via conjugate addition reaction. This photoprotein contains the chromophore molecules at the binding cite first, and then moves to the active cite Cys-390 for the luminescence.
In the first part of this dissertation, we developed an efficient synthetic route to yield keto-acetal (2-6a, 2-20b, and 2-20c) and keto-sulfone (2-24b and 2-24c) as the equivalent of enol-aldehyde, which was the key segment toward coelenterazine (CL) and its analogs as luminescent molecules. The improved synthesis of coelenterazine and its analogs employed an advanced condensation with the aminopyrazines using various keto-acetal and keto-sulfone segments, which resulted in much higher yields to give the final imidazopyrazinone heterocycles than the previous method using enol-aldehyde.
In the second part, we focus on these dynamic aspects of the chromophore using the natural photoprotein by analyzing the fluorescence changing of the DCL chromophores analogs with 8-(4'-methoxyphenyl)- or 8-(2'-naphthyl)-group and 2-(2’,4’-difluorophenyl)-group. Exchanges of these chromophores were monitored the fluorescence at slightly acidic media and also from the luminescence function observed at the optimum pH 7.8. The non-fluorescent naphthyl analogs was even proven to make the covalent bond formation at pH 6.0 and evidently to obtain the corresponding luminescent product amide by liquid chromatographic detection from the spent solutions.
In the third part, the cysteine adducts 2-43b were treated to aequorin as the model of DCL in symplectin. This result showed us that the intramolecular peroxide oxidation happened on cysteine residue and lead decrease of bioluminescence. The intramolecular peroxide oxidation combined with the previous study lead us to determinate the absolute configuration of C2' position in cysteine adducts.

中文摘要    i
Abstract    ii
Acknowledgement    iii
Abbreviation    iv
Index    vii
List of Figures    x
List of Schemes    xiii
List of Tables    xiv

1. Introduction    1
1-1. Background of bioluminescence    1
1-1-1. Firefly luciferin    2
1-1-2. Bacterial luciferin    4
1-1-3. Dinoflagellate luciferin    5
1-1-4. Imidazopyrazinone    7
1-2. Review of symplectin    10
1-3. Review of synthetic routes toward coelenterazine and dehydrocoelenterazine    14
1-4. Proposal of the study    17
2. Results and Discussion    22
2-1. Synthesis of coelenterazine and its analogs    22
2-1-1. Retrosynthesis study of coelenterazine and dehydrocoelenterazine    22
2-1-2. Novel synthetic route toward the equivalent of keto-acetal    22
2-1-2. A precursor synthesis of coelenteramine analogs    27
2-1-3. Synthesis of coelenterazine and dehydrocoelenterazine    29
2-2. Chromophore exchange in symplectin    32
2-2-1. Preparation of symplectin solution    32
2-2-2. Bioluminescence activity    33
2-2-3.  Dynamic Profile of the Chromophore Exchange by using 2,4-diF-DCL-naphtyl    35
2-3. Study of oxidation of cysteine residue.    45
2-3-1. Synthesis and analysis of sulfoxide products from cysteine adducts.    45
3. Conclusions and Summary    54
 
4. Experimental Section    57
4-1. General Methods    57
4-2. Synthetic method and analytical data    59
4-2-1. 1-(1,3-Dioxolan-2-yl)-2-(4-methoxyphenyl)ethanone (2-6a)    59
4-2-2. 2-(4-Methoxybenzyl)-1,3-dithiane (2-13a)    60
4-2-3. 2-(4-Methoxybenzyl)-1,3-dithiane-2-carbaldehyde (2-14a)    61
4-2-4. 2-(2-(4-Methoxybenzyl)-1,3-dithian-2-yl)-1,3-dioxolane (2-15a)    62
4-2-5. 2-(2,4-Difluorophenyl)-N-methoxy-N-methylacetamide (2-16b)    63
4-2-6. 2-(2,6-Difluorophenyl)-N-methoxy-N-methylacetamide (2-16c)    64
4-2-7. 3-(2,4-Difluorophenyl)-1-methoxy-1-(phenylthio)propan-2-one (2-19b)    65
4-2-8. 3-(2,6-Difluorophenyl)-1-methoxy-1-(phenylthio)propan-2-one (2-19c)    66
4-2-9. 3-(2,4-Difluorophenyl)-1,1-dimethoxypropan-2-one (2-20b)    67
4-2-10. 3-(2,6-Difluorophenyl)-1-methoxy-1-(phenylsulfonyl)propan-2-one (2-24b)    68
4-2-11. 3-(2,6-Difluorophenyl)-1-methoxy-1-(phenylsulfonyl)propan-2-one (2-24c)    69
4-2-12. 5-Iodopyrazin-2-amine (2-28)    70
4-2-13. 3-Bromo-5-iodopyrazin-2-amine (2-29)    70
4-2-13. 3-Bromo-5-(4-methoxyphenyl)pyrazin-2-amine (2-30)    71
4-2-14. 4-(5-Amino-6-bromopyrazin-2-yl)phenol (2-31)    72
4-2-14. General procedures of synthesis of coelenterazine    72
4-2-15. General procedures of synthesis of coelenteramide    75
4-3. Bioluminescence experimental section    77
4-3-1. Preparation of apo-symplectin solution    77
4-3-2. Measurements of fluorescence spectra    78
4-3-3. Measurements of integrated light amount from bioluminescence    80
4-3-4. Chemiluminescence measurement    80
4-3-5. Analysis of bioluminescence product    81
4-3-6. Fluorescence measurement of synthetic compound    81
4-4. Analysis of oxidation on cysteine residue    82
4-4-1. Preparation of Cysteine adduct 2-43b    82
4-4-2. Analysis of synthetic standard    83
4-4-3. Bioluminescence measurement of Renilla luciferase    83
4-4-4. Bioluminescence measurement of aequorin    84
4-4-5. The simulation of cysteine adducts    84
4-5. Supporting information    85
4-6. Publications    103
                                

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