鳶烏賊生物螢光的合成與生物有機研究｜國立清華大學博碩士論文庫

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研究生：	童郁雯 Tung, Yu-Wen
論文名稱：	鳶烏賊生物螢光的合成與生物有機研究 Synthesis and Bioorganic Studies on Symplectoteuthis Bioluminescence
指導教授：	磯部稔 Minoru Isobe
口試委員:	汪炳鈞莊敬
學位類別：	碩士 Master
系所名稱：	理學院 - 化學系 Department of Chemistry
論文出版年：	2012
畢業學年度：	100
語文別：	英文
論文頁數：	166
中文關鍵詞：	去氫腔腸螢光素、鳶烏賊、生物螢光
外文關鍵詞：	dehydrocoelenterazine, Symplectoteuthis oualaniensis, bioluminescence
相關次數：	點閱：3 下載：0
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鳶烏賊(Symplectoteuthis oualaniensis)是一個會發藍光的螢光烏賊，牠的螢光
蛋白叫做symplectin; 而去氫腔腸螢光素(DCL)是symplectin 的發光基質，為一個
天然的發色團。之前的研究發現合成的發色團2,4-二氟-去氫腔腸螢光素
(2,4-diF-DCL)可發出比天然的發色團(DCL)高兩倍的生物螢光量。所以我們推測
當symplectin 在pH 6 時(孵育期incubation)，2,4-二氟-去氫腔腸螢光素
(2,4-diF-DCL)與天然發色團去氫腔腸螢光素(DCL)交換。
為了證明發色團的交換，各種八號位置改變成甲氧苯基(methoxyphenyl)或噻
吩(thiophene)的二氟-去氫腔腸螢光素的類似物被合成出來。這些類似物在八號位
置多增加一個共振系統，所以其紫外光-可見光吸收、螢光發射、化學發光和生物
發光的波長產生紅位移。在本篇研究中，二氟-去氫腔腸螢光素的類似物與其他可
能會在symplectin 生物發光過程中產生的物質的特徵性質被詳細檢驗。有這些合
成物質的資料，在symplectin 內的發色團交換可以被追蹤並估算出交換的比例。

A luminous squid, Symplectoteuthis oualaniensis, emits blue light, and its
photoprotein has been named as symplectin. The luminous substrate of symplectin is
dehydrocoelenterazine (DCL) as natural chromophore. A synthetic chromophore,
2,4-difluoro-dehydrocoelenterazine (2,4-diF-DCL), was found to emits 2 times
bioluminescence light amount comparing to natural form chromophore (DCL). The
exchange of chromophore between 2,4-diF-DCL and DCL in symplectin might happen
during incubation at pH 6.
To prove the exchange of the chromophores, several diF-DCL analogs with
modification at 8-position (methoxyphenyl and thiophene) were synthesized. These
analogs have one more conjugated system at the 8-position resulting in red-shift in
UV-Vis absorbance, fluorescence emission, chemiluminescence and bioluminescence
wavelength. In this study, the characteristics of DCL analogs and other compounds
which are producible in symplectin luminescence process were examined. With these
data from authentic samples, chromophore exchange in symplectin could be monitored
and percentage of exchange could be estimated.

中文摘要    II
Abstract    III
Acknowledgement    IV
Index    V
Figure index    VIII
Table index    XIII
Scheme index    XIV
Abbreviation    XVIII
Chapter 1 Introduction    1
1 General introduction    1
2 Review on synthesis of coelenterazine    4
3 Our group synthesis of dehydrocoelenterazine and its analogs    6
4 Review on biological studies of symplectin luminescence system    9
4.1 Previous results    9
4.2 Postulated mechanism    14
5 Research purpose: chromophore exchange    17
Chapter 2 Synthesis of coelenterazine analogs, dehydrocoelenterazine analogs and luminescence product    21
1 Coelenteramine analogs    21
2 Coelenteramide analogs    24
3 Coelenterzine and dehydrocoelenterzine analogs    26
Chapter 3 Comparison of benzyl, methoxyphenyl and thiophenyl substituted analogs    30
1 Purity examination and hydrophobicity comparison    31
2 UV-Vis absorbance and FL emission comparison of coelenteramide and coelenteramine analogs    35
3 UV-Vis absorbance of DCL analogs and its cysteine adducts    42
3.1 DCL analogs’ affinity to cysteine by calculating association constant    44
3.2 DCL analog-Cys-adducts at different pH    57
4 Fluorescence emission of cysteine adducts    59
4.1 Cysteine adducts formation observed by fluorescence increasing    60
4.2 Cysteine-adducts stability under degassed condition    69
5 Estimation of bioluminescence wavelength    75
6 Summary    79
Chapter 4 Chromophore-exchanging and bioluminescence    82
1 Bioluminescence total light yield    83
2 Incubation (pH 6): reconstituted symplectin and chromophore-exchanging    87
3 Bioluminescence (pH 8)    90
3.1 Fluorescence emission change during bioluminescence    90
3.2 Bioluminescence wavelength    93
4 Bioluminescence product analysis    95
4.1 Alkaline shift of authentic coelenteramide and coelenteramine analogs    95
4.2 Differentiation of coelenteramide and coelenteramine in spent solution from bioluminescence    98
Chapter 5 Conclusion    101
1 Conclusion    101
2 Future plan    103
Chapter 6 Experimental section    104
1 General methods    104
2 Experimental procedures and analytical data    106
2.1 Compound 2-8    106
2.2 Compound 2-10    107
2.3 Compound 2-11    108
2.4 Compound 2-13    109
2.5 Compound 2-18    110
2.6 Compound 2-20    112
2.7 Compound 2-21    114
2.8 Compound 2-23    116
2.9 Compound 2-25    118
2.10 Compound 2-27    119
2.11 Compound 2-28    120
2.12 Compound 2-29    121
2.13 Compound 2-31    122
2.14 Compound 2-32    123
2.15 Compound 2-35    124
2.16 Compound 2-36    125
2.17 Compound 2-37    126
Chapter 7 References    128
Supporting information    130

                                

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