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論文中文名稱:開發長波長螢光探針利用quinone-methide rearrangment-elimination方式偵測具生理意義的thiols和DT-diaphoras [以論文名稱查詢館藏系統]
論文英文名稱:Development of a long-wavelength fluorescent probe for physiologically significant thiols and DT-diaphorase based on quinone-methide-type reaction [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生物科技研究所
出版年度:97
中文姓名:丁國能
英文姓名:Kuo-Neng Ting
研究生學號:95688019
學位類別:碩士
語文別:中文
口試日期:2008-06-18
論文頁數:72
指導教授中文名:黃聲東
口試委員中文名:郭憲壽;林俊茂;吳瑞裕
中文關鍵詞:硫氫衍生物脱氫還原酵素隱藏式螢光劑
英文關鍵詞:ThiolsDT-diaphoraseLatent Fluorophore
論文中文摘要:我們設計合成一種新型隱藏式螢光分子 BCC,它具有長波長螢光性質,可以用來檢測在生理學上具重要意義的thiols衍生物質或是觀察DTD活性表現。其BCC(7)螢光發射器設計原理是運用thiols或DTD於生理溫度水溶液下經過一連串自發性及不可逆性的反應後釋放出螢光劑。反應機制是由DTD或thiols還原螢光分子的benzoquinone後形成hydroquinone中間體,然後再進行quinone-methide-type rearrangement reaction,因此釋放出螢光劑。而螢光劑激發後會於趨近紅外光譜區域波長595nm散發出螢光訊號。我們證明利用BCC釋放螢光訊號來偵測,可以簡單方便檢測thiols的濃度和DTD活性表現。 NEM對BCC與thiol反應螢光釋放有競爭抑制現象。而且由反應後的螢光訊號差異得知BCC對其他氨基酸或生物性還原劑,螢光釋放的反應性很差。我們也證實這種新型的螢光指示劑對偵測不同濃度thiols (10~100uM)反應後,所產生的螢光訊號有線性關係,且希望它能成為生物檢測器應用於未來臨床疾病的觀察。
論文英文摘要:We synthesized a new long-wavelength latent fluorogenic probe BCC (7) to detect physiologically significant thiols or monitoring redox enzyme. The fluorogenic chemical transformation of BCC triggered by DTD or thiols is through a tandem reactions, thiol induced benzoquinone reduction, and quinone-methide-type rearrangement reaction, which are spontaneous and irreversible at physiological temperature in aqueous media. The fluorescence signal revealed by this process is specific and exhibited in the near red spectrum region with emission maxima at 595nm. BCC is simple to use for detecting biological thiols and real time monitoring DTD activity. It could be competitively inhibited by thiol scavenger, NEM. The fluorescent response of BCC is insensitive to various amino acids and biological reductants. This novel fluorimetric indicator demonstrates a good relationship in detecting thiols in 10 to 100uM range, which presents to the applicability for the construction of fiber-optic biosensors in the future clinical diagnostic.
論文目次:摘要............................................................................. I
ABSTRACT................................................................. II
誌謝.............................................................................III
目錄.............................................................................IV
圖目錄.......................................................................... VI
第一章 前言..................................................................1
第二章 文獻探討..........................................................2
2.1 麩胱甘肽.................................................................2
2.1.1 麩胱甘肽生理功能..............................................2
2.1.2 麩胱甘肽解毒機制..............................................3
2.2 同半胱胺酸.............................................................4
2.2.1 同半胱胺酸生理功能..........................................4
2.2.2 同半胱胺酸的代謝..............................................5
2.3 半胱胺酸.................................................................6
2.4 Thiol偵測概論..........................................................7
2.5 DT-Diaphorase生理功能........................................10
2.5.1 DT-Diaphorase測試概論.....................................11
2.6 螢光原理.................................................................12
2.7 隱藏式螢光分子.....................................................14
2.7.1 隱藏式螢光分子偵測thiol..................................14
2.7.2 隱藏式螢光分子偵測氧化還原狀態..................17
第三章 隱藏式螢光設計概念.......................................18
3.1 Quinone-thiol interactions..........................................18
3.2 The quinone-methide-rearrangment-elimination........20
3.3 選擇適當的螢光劑...................................................22
3.4 研究動機...................................................................24
第四章 實驗方法與設備................................................25
4.1 儀器與試藥..............................................................25
4.1.1 實驗儀器...............................................................25
4.1.2 實驗室藥品...........................................................26
4.2 實驗方法與討論.......................................................27
4.2.1 BCC對DTD/NADH的測試方法.........................27
4.2.2 DTD活性即時性觀察實驗步驟............................28
4.2.3 HPLC檢設方法......................................................29
4.2.4 BCC對thiols反應條件實驗方法.........................30
4.2.5 BCC對thiols即時性方法.....................................32
4.2.6 Thiols檢量線方法.................................................33
4.2.7 NEM共同競爭測試..............................................34
4.2.8 BCC對其他胺基酸和還原劑實驗方法...............35
4.2.9 Micheal addition機制測試方法...........................36
4.3合成化合物 (6).......................................................37
4.4合成Latent fluorophore BCC (7)............................38
第五章 結果與討論......................................................39
5.1 Latent fluorophore BCC (7)總合成方法..................39
5.2 BCC對DTD/NADH的測試...................................41
5.3 DTD活性即時性觀察..............................................42
5.4 HPLC 測試cumarin 5的釋放測試.........................43
5.5 DTD對BCC的親合性測試....................................44
5.6 BCC對thiols反應條件測試...................................46
5.7 BCC對thiols即時性觀察.......................................47
5.8 Thiols檢量線測試....................................................48
5.9 BCC對thiol反應後cumarin 5的釋放測試...........50
5.10 NEM共同競爭測試................................................51
5.11 螢光釋放機制釋放探討.........................................52
5.11.1 BCC對其他胺基酸和還原劑測試......................53
5.11.2 Micheal addition機制測試...................................54
第六章 結論....................................................................56
參考文獻..........................................................................57
附錄圖一化合物(6) 1HNMR.........................................64
附錄圖二化合物(6) 13CNMR........................................65
附錄圖三化合物(6) Mass....................................................66
附錄圖四 化合物(6)IR...................................67
附錄圖五 BCC(7) 1HNMR................................................68
附錄圖六 BCC(7) 13CNMR................................................69
附錄圖七 BCC(7) Mass..................................................70
附錄圖八 BCC(7) IR.......................................................71
附錄圖39 coumarin (5) 0.1~0.4uM減量線......................72
圖40 coumarin (5) 0.4~0.9uM減量線.......................72
表格一BCC對DTD/NADH動力學實驗據............72
論文參考文獻:[1] Soltaninassab, S. R., Sekhar, K. R.,Meredith, M. J. and Freeman, M. L., Multi-faceted regulation of γ–glutamylcysteine synthetase, J.Cell Physiol. 182, 2000, 163-170.
[2] Sugiyma, K. I., Izawa S. and Inoue, Y., The Yap1p-dependentinduction of glutathione synthesis in heat shock response of saccharomyces cerevisiae. J. Biol. Chem. 275, 2000, 15535–15540.
[3] Blaauboer, B. M., Niesink, J. M., Vries, J. and Hollinger, M. A., Biotransformation: detoxication and bioactivation. In Toxicology, 1996, pp 41-45. CRC Press, London.
[4] Griffith, O. W., Biologic and pharmacologic regulation of mammalian glutathione synthesis. Free Radic. Biol. Med, 27, 1999, 922-935.
[5] Lu, S. C., Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB J. 13, 2000, 1169-1183.
[6] Wu, D., Meydani, S. N., Sastre, J., Hayek, M. and Meydani, M., In vitro glutathione supplementation enhances interleukin-2 production and mitogenic response of peripheral blood mononuclear cells from young and old subjects. J. Nutr. 124, 1994, 655-663.
[7] Ibrahim, W., Lee, U.-S., Szabo, J., Bruckner, G. and Chow, C. K., Oxidative stress and antioxidant status in mouse kidney: effects of dietary lipid and vitamin E plus iron. J. Nutr. Biochem. 10, 1999, 674-678.
[8] Bleser, P. J. D., Xu, G., Rombouts, K., Rogiers, V. and Geerts, A., Glutathione levels discriminate between oxidative stress and transforming growth factor-β signaling in activated rat hepatic stellate cells. J. Biol. Chem. 274, 1999, 33881–33887.
[9] Kang S-S, Wang PWK, Malinow MR., Hyperhomocysteinemia as a risk factor for
occlusive vascular disease. Annual Review of Nutrition. 12, 1992, 279-298.
[10] Ueland PM, Refsum H, Brattstrom L., Plasma homocysteine and cardiovascular disease In :Francis RB Jr(ed) Atherosclerotic cardiovascular disease, hemostasis, and endothelial function. Marcel Dekker, New York, 1992, pp 183-236
[11] KS McCully., Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis .,Am J Pathol ., 56 , 1969, 111-128
[12] Finkelstein JD. Methionine metabolism in mammals. Journal of Nutrition Biochemistry.,1, 1995, 228-237.
[13] Ueland PM., Homocysteine species as components of plasma redox thiol status. Clinical Chemistry., 41, 1995, 340-342.
[14] Fiskerstrand T, Refsum H, Kvalheim G, Ueland PM. Homocysteine and other thiols in plasma and urine: automated determination and sample stability. Clinical Chemistry.,39, 1993, 263-271.
[15] Slivka, A. and Cohen. G., Brain ischemia markedly elevates levels of the neurotoxic amino acid, cysteine, Brain Res, 608, 1993, 33-37
[16] Karlsen. R. L., Grofova. I., Sorenssen. M. D. and Fonnum. F, Morphological changes in rat brain induced byl-cysteine injection in newborn animals, Brain. Res, 208, 1981, 167-180
[17] Klingman, J. G. and Choi, D. W, Toxicity of sulphurcontaining amino acids on cultured cortical neurones. Neurology, 39, 1989, 397–398.
[18] Olney. J. W, Zorumski. C., Price, M. T., and Labruyere. J, L-cysteine, a bicarbonate-sensitive endogenous excitotoxin J. Science, 248, 1990, 596-599
[19] John W. Oleny, OI-LAN HO., Brain Damage in Infant Mice following Oral Intake of Glutamate, Aspartate or Cysteine, Nature, 227, 1970, 609-611
[20] John W. Olney, Oi Lan Ho, Vesela Rhee, Bruce Schainker Cysteine-induced brain damage in infant and fetal rodents Brain Res, 45, 1972, 309-313
[21] Pedersen, O. O. and Karlsen, R. L., The toxic effect of L-cysteine on the rat retina. A morphological and biochemical study. Invest. Ophthalmol, Vis. Sci, 19, 1980, 886-892
[22] Misra, C. H., Is a certain amount of cysteine prerequisite to produce brain damage in neonatal rats? Neurochem. Res, 14, 1989, 253-257.
[23] Olney, J. W.. Role of excitotoxins in developmental neuropathology. APMIS 10, 1993 (Suppl. 40), 103–112.
[24] R. Janáky, V. Varga, A. Hermann, P. Saransaari, and S. S. Oja., Mechanisms of L-Cysteine Neurotoxicity , Neurochemical. Research, 25 , 2000, 1397-1405
[25] Heafield, M. T., Fearn, S., Steventon, G. B., Waring, R. H., Williams, A. C., and Sturman, S. G, Plasma cysteine an sulphate levels in patients with motor neuron, Parkinson’s and Alzheimer’s disease, Neurosci. Lett, 110, 1990, 216-220.
[26] Perry, T. L., Norman, M. G., Young, V. W., Whiting, S., Crichton, J. U., Hansen, S., and Kish, S. J. , Hallervorden-Spatz disease: cysteine accumulation and cysteine dioxygenase deficiency in the globus pallidums, J. Ann. Neurol, 18, 1985, 482-489.
[27] Lehmann, A., Hagberg, H., Orwar, O., and Sandberg, M., Cysteine sulphinate and cysteate: mediators of cysteine toxicity in the neonatal rat brain? Eur. J. Neurosci, 5, 1993, 1398-1412.
[28] Schurr, A., West, C. A., Heine, M. F., and Rigor, B. M. The neurotoxicity of sulfur-containing amino acids in energy-deprived rat hippocampal slices. Brain Res, 601, 1993, 317-320.
[29] Slivka, A. and Cohen, G. Brain ischemia markedly elevates levels of the neurotoxic amino acid, cysteine. Brain Res, 608, 1993, 33-37.
[30] Winters R. A., Zukowski J., Ercal N., Matthews R. H., Spitz D. R, Analysis of Glutathione, Glutathione Disulfide, Cysteine, Homocysteine, and Other Biological Thiols by High-Performance Liquid Chromatography Following Derivatization by N-(1-Pyrenyl)maleimide , Analytical, Biochemistry, 227, 1995, 14-21
[31] Kingo Itaya, Isamu Uchida, Vernon D. Neff, Electrochemistry of polynuclear transition metal cyanides: Prussian blue and its analogues , Acc. Chem. Res,19, 1986, 162-168
[32] I. M. Kolthoff, Cyrus Barnum, The Anodic Reaction and Waves of Cysteine at the Dropping Mercury Electrode and at the Platinum Micro Wire Electrode , J. Am. Chem. Soc, 62, 1940, 3061-3065
[33] Rabenstein. D. L, Saetre. R, Mercury-based electrochemical detector for liquid chromatography for the detection of glutathione and other sulfur-containing compounds Anal. Chem, 49, 1977, 1036
[34] G.. Ellman, Tissue sulfhydryl groups, Arch. Biochem. Biophy, 82, 1959, 70-77
[35] Maedo. H., Matsuno. H., Ushia. M., Katayame. K., Saeki. K., N. Ithoh., 2,4-Dinitrobenzenesulfonyl Fluoresceins as Fluorescent Alternatives to Ellman's Reagent in Thiol-Quantification Enzyme Assays, Angew. Chem. Int. Ed, 44, 2005, 2922-2923
[36] Navarro, F., Arroyo, A., Martin, S. F., Bello, R. I., de Cabo, R.Burgess, J. R., Navas, P., Villalba, J. M. Biofactors, 1999, pp:163-170.
[37] David Siegel, Emiko M. Bolton, Jeanne A. Burr, Daniel C. Liebler, and David Ross The Reduction of -Tocopherolquinone by Human NAD(P)H:Quinone Oxidoreductase: The Role of -Tocopherolhydroquinone as a Cellular Antioxidant, Mol. Pharmacol, 52, 1997, 300-305.
[38] Schlager JJ, Powis G, Cytosolic NAD(P)H:(quinone-acceptor)oxidoreductase in human normal and tumor tissue: effects of cigarette smoking and alcohol, Int. J. Cancer. 45, 1990, 403-409.
[39] Smitskamp-Wilms E, Giaccone G, Pinedo HM, van der Laan BF, Peters GJ., DT-diaphorase activity in normal and neoplastic human tissues; an indicator for sensitivity to bioreductive agents? Br. J. Cancer, 72, 1995, 917-921.
[40] Shutsung Liao, H. G. Williams-Ashman, Enzymatic oxidation of some non-phosphorylated derivatives of dihydronicotinamide, Biochem. Biophys. Ees. Commun, 4, 1961, 208-213
[41] Rabinowitz, J. D.; Vacchino, J. F.; Beeson, C.; McConnell, H. M., Potentiometric Measurement of Intracellular Redox Activity, J. Am. Chem. Soc, 120, 1998, 2464-2473
[42] 鍾嘉明,有機發光二極體成長條件與特性研究,碩士學位論文,中原大學,桃園中壢,92年
[43] Zlokarnik G, Negulescu PA, Knapp TE, Mere L, Burres N, Feng L, Whitney M, Roemer K, Tsien RY, Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter., Science, 279, 1998, 84-88.
[44] Chang, M. C. Y., Pralle, A., Isacoff, E. Y.; Chang, C. J, A Selective, Cell-Permeable Optical Probe for Hydrogen Peroxide in Living Cells, J. Am. Chem. Soc, 126, 2004, 15392-15393.
[45] Chandran, S. S.; Dickson, K. A., Raines, R. T., Latent Fluorophore Based on the Trimethyl Lock, R. T., J. Am. Chem. Soc, 127, 2005, 1652-1653.
[46] Wei Jiang, Qingquan Fu, Hongyou Fan, Joe Ho, Wei Wang, A Highly Selective Fluorescent Probe for Thiophenols, Angew. Chem. Int. Ed, 46, 2007, 1 – 5
[47] Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G., Guiwen., A Rhodamine-Based Fluorescent Probe Containing a Se-N Bond for Detecting Thiols and Its Application in Living Cells, J. Am. Chem. Soc, 129, 2007, 11666-11667
[48] Zhang, M.; Yu, M.; Li, F.; Zhu, M.; Li, M.; Gao, Y.; Li, L.; Liu, Z.; Zhang, J.; Zhang, D.; Yi, T.; Huang, C, A Highly Selective Fluorescence Turn-on Sensor for Cysteine/Homocysteine and Its Application in Bioimaging, J. Am. Chem. Soc, 129, 2007, 10322-10323.
[49] Tremblay, M. S.; Sames, D. A New Fluorogenic Transformation: Development of an Optical Probe for Coenzyme Q, Org. Lett., 7, 2005, 2417-2420
[50] Alkmini Digga, Svetlana Gracheva, Callum Livingstone, James Davis, Potentiometric detection of thiols: a mechanistic evaluation of quinone–thiol interactions, Electrochemistry Communications., 5, 2003, 732-736
[51] S. Gracheva., C. Livingstone., Davis. J. Anal. Chem., 76, 2004, 3833-3836
[52] Yadav. J. S, Swamy. T, Subba Reddy B.V., Krishna Rao. D, Organic synthesis in water: Green protocol for the conjugate addition of thiols to p-quinones, J. Mol. Catal, 274, 2007, 116-119
[53] Flader C, Liu J, Borch RF, Development of novel quinone phosphorodiamidate prodrugs targeted to DT-diaphorase, J. Med. Chem, 43, 2000, 3157-3167
[54] Chiang. L., Chu. C-Y, Chem. Comm, 6, 2003, 2728-2729
[55] Coquery. M., Morin. A., Bécue. A, Lepot. B, Priority substances of the European Water Framework Directive: analytical challenges in monitoring water quality, Trends Anal. Chem., 23, 2004, 127-136.
[56] Otto S. Wolfbeis, Ernst Koller and Petra Hoghmuth, The Unusually Strong Effect of a 4-Cyano Group upon Electronic Spectra and Dissociation Constants of 3-Substituted 7-Hydroxycoumarin. Chem. Soc. Jp., 58, 1985, 731-734.
[57] Bittner S, Gorohovsky S, Paz-Tal LO, Becker JY, Synthesis, electrochemical and spectral properties of some omega-N-quinonyl amino acids, Amino Acid, 22 , 2002, 71-79.
[58] Paz, M. M.; Tomasz, M., Reductive Activation of Mitomycin A by Thiols Org. Lett., 3 , 2001, 2789-2792.
[59] A. R. Menotti, J. Am. Chem. Soc, 65 ,1943, 1209-1211.
[60] Grace-Ann M. Lobo, Sneha A. Chitre, Spandan M. Rathod, Robert B. Smith, Ray Leslie, Callum Livingstone, James Davis, Determination of Total Reduced Thiol Levels in Plasma Using a Bromide Substituted Quinone, Electroanalysis, 19, 2007, 2523-2528.
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