臺灣博碩士論文加值系統

藉由設計及合成不同的錯合物，探討其熱力學性質及做為人工水解&;#37238;催化受質的效力是本實驗室感興趣的研究主題，對於鑭系金屬大環配位子的錯合物尤感興趣，由於鑭系金屬擁有高氧化態、路易士酸性強、高配位數及電子密度高等特性，且與大環配位子形成之錯合物穩定性高，使其非常適合應用在研究人工水解&;#37238;的領域上，並且希望能藉此研究更了解鑭系金屬在配位化學上的特性。
本實驗室先前已合成並研究一系列之錯合物，發現錯合物中金屬離子的配位水數目越多以及錯合物所帶之正電荷越高，其水解DNA的模型受質BNPP (bis(4-nitrophenyl)-phosphate)的效果越好。
在本論文中，我們合成了新的雙體大環錯合物，m-12N3O-dimer(1, 3-bis(1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl)benzene)及m-ODO2A-dimer(1, 3-bis[4, 10-bis(carboxymethyl)- 1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl]benzene)，分別與過渡金屬與鑭系金屬錯合後進行熱力學實驗決定穩定常數以及水解催化BNPP與RNA的模型受質HPNP(2-hydroxypropyl-4-nitrophenyl phosphate)的動力學實驗，並與前人已研究之錯合物及已發表之文獻比較其差異性。
由初步的水解實驗結果顯示雙核鑭系金屬大環錯合物水解BNPP及HPNP的速率都隨著環境中pH的上升而增加，直到pH10-10.5開始下降。另外其整體效率比單體錯合物LnODO2A+高上四至五個數量級；但是卻比EuDO2A+差了約一個數量級。

We have been interested in the design, synthesis and characterization of artificial nucleases and ribonucleases by employing macrocyclic lanthanide complexes. We use lanthanide ions to interact with ligands to form complexes. Because their high
coordination number, Lewis acidity, charge density and oxidation states, they could be suitable for hydrolysis of the model compound of DNA / RNA, BNPP / HPNP.
In our previous studies, we found that with more number of inner-sphere coordinated water molecules and a positive charge in the complexes, the more efficiency for hydrolysis. However, different ligands and lanthanide ions will influence the efficiency.
In this research, we synthesize two new ligands, m-12N3O-dimer(1, 3-bis(1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl)benzene) and m-ODO2A-dimer(1, 3-bis[4, 10-bis(carboxymethyl)- 1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl]benzene) complex with transition and lanthanide ions, respectively. The result of hydrolysis studies shows that the complexes in dimer form are more effective than in monomer form with the same moiety. But the m-Eu2(ODO2A-dimer) has still lower efficiency compare to EuDO2A+. Besides hydrolysis studies, the thermodynamic characteristics are also important. These could help us to understand the whole mechanism of hydrolysis and the coordination features with lanthanide ions more accurately, and develop more efficient complexes in the future.

中文摘要.....................................................................................................................i
英文摘要....................................................................................................................ii
誌謝...........................................................................................................................iii
目錄...........................................................................................................................iv
第一章 緒論............................................................................................................1
前言與研究背景................................................................................................1
文獻回顧............................................................................................................7
研究目的...........................................................................................................30
第二章 配位子的合成...........................................................................................31
N,N’,N’’-tritosyldiethylenetriamine(N3Ts3)的製備..........................................31
1,5-ditosyloxy-3-oxapentane(O3Ts2) 的製備...................................................31
4, 7, 10-tritosyl-1-oxa-4, 7, 10-triazacyclododecane(12N3OTs3) 的製備........32
1-oxa-4, 7, 10-triazacyclododecane(12N3O) 的製備.......................................33
Tricyclo[8, 2, 1, 04, 13]1-oxa-4,7,10-triazacyclotridecane(12N3O-tricyclic) 的製備...................................................................................................................... 34
1, 3-bis(1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl)benzene (12N3O-dimer) 的製備...................................................................................................................35
1, 3-bis(1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl)benzene hydrochloride salt (12N3O-dimer ‧ HCl salt) 的製備....................................................................36
1, 3-bis[4, 10-bis(carboxymethyl)- 1, 4, 10-triaza-7-oxacyclododec-1-ylmethyl]benzene hydrochloride salt (ODO2A-dimer ‧ HCl salt) 的製備.................................................................36
第三章 金屬離子溶液的配製與標定..................................................................38
金屬離子溶液的濃度標定方法......................................................................38
金屬離子溶液的濃度標定原理......................................................................40
金屬離子溶液的標定步驟..............................................................................41
溶液與藥品的配製..................................................................................41
標定金屬離子溶液..................................................................................43
第四章 配位子的質子化常數之研究..................................................................44
配位子溶液的濃度標定..................................................................................44
儀器的準備與藥品的配製......................................................................44
配位子溶液的標定步驟..........................................................................46 
配位子的質子化常數的滴定原理..................................................................50
配位子的質子化常數的計算結果..................................................................51
第五章 配位子與金屬離子穩定常數之研究..................................................... 54
配位子與金屬離子之間穩定常數的計算原理..............................................54
配位子與金屬離子之間穩定常數的研究......................................................55
儀器及滴定溶液的準備..........................................................................55
錯合物的滴定步驟..................................................................................56
穩定常數的計算......................................................................................56
實驗結果與分析討論..............................................................................57
第六章 金屬離子錯合物之動力學研究..............................................................65
動力學實驗之原理..........................................................................................65
動力學實驗之測定方法與準備......................................................................66
儀器及藥品的準備與溶液的配製..........................................................67
動力學反應溶液的配製..........................................................................69
動力學實驗之步驟..........................................................................................70
動力學實驗之計算方法..................................................................................70
動力學實驗之結果與討論..............................................................................71
加入NaOH(aq)水解BNPP / HPNP之control ........................................72
m-Eu2(ODO2A-dimer)在不同pH下水解BNPP / HPNP...................... 74
m-Eu2(ODO2A-dimer) vs. BNPP.....................................................74
m-Eu2(ODO2A-dimer) vs. HPNP.................................................... 78
m-Yb2(ODO2A-dimer)在不同pH下水解BNPP / HPNP..................... 81
m-Yb2(ODO2A-dimer) vs. BNPP....................................................81
m-Yb2(ODO2A-dimer) vs. HPNP....................................................85
m-Zn2(12N3O-dimer)在不同pH下水解HPNP.....................................86
m-Cu2(12N3O-dimer)在不同 pH下水解HPNP....................................89
第七章 結論與未來展望..................................................................................... 91
參考文獻................................................................................................................. 93
附錄..........................................................................................................................98

1. Kramer, R., Bioinorganic models for the catalytic cooperation of metal ions and functional groups in nuclease and peptidase enzymes. Coordin Chem Rev 1999, 182.
2. Oivanen, M.; Kuusela, S.; Lonnberg, H., Kinetics and mechanisms for the cleavage and isomerization of the phosphodiester bonds of RNA by Bronsted acids and bases. Chem Rev 1998, 98 (3), 961-990.
3. Hegg, E. L.; Burstyn, J. N., Toward the development of metal-based synthetic nucleases and peptidases: a rationale and progress report in applying the principles of coordination chemistry. Coordin Chem Rev 1998, 173, 133-165.
4. Thompson, J. E.; Kutateladze, T. G.; Schuster, M. C.; Venegas, F. D.; Messmore, J. M.; Raines, R. T., Limits to catalysis by ribonuclease A. Bioorg Chem 1995, 23 (4), 471-481.
5. Komiyama, M.; Takeda, N.; Shigekawa, H., Hydrolysis of DNA and RNA by lanthanide ions: mechanistic studies leading to new applications. Chemical Communications 1999, (16), 1443-1451.
6. Trawick, B. N.; Daniher, A. T.; Bashkin, J. K., Inorganic mimics of ribonucleases and ribozymes: From random cleavage to sequence-specific chemistry to catalytic antisense drugs. Chem Rev 1998, 98 (3), 939-960.
7. Komiyama, M., Sequence-Specific and Hydrolytic Scission of DNA and Rna by Lanthanide Complex-Oligodna Hybrids. J Biochem-Tokyo 1995, 118 (4), 665-670.
8. Ott, R.; Kramer, R., DNA hydrolysis by inorganic catalysts. Appl Microbiol Biot 1999, 52 (6), 761-767.
9. Kuimelis, R. G.; McLaughlin, L. W., Mechanisms of ribozyme-mediated RNA cleavage. Chem Rev 1998, 98 (3), 1027-1044.
10. Maldonado, A. L.; Yatsimirsky, A. K., Kinetics of phosphodiester cleavage by differently generated cerium(IV) hydroxo species in neutral solutions. Organic & Biomolecular Chemistry 2005, 3 (15), 2859-2867.
11. Jebasingh, B.; Alexander, V., Synthesis and relaxivity studies of a tetranuclear gadolinium(III) complex of DO3A as a contrast-enhancing agent for MRI. Inorganic Chemistry 2005, 44 (25), 9434-9443.
12. Coleman, J. E., Structure and Mechanism of Alkaline-Phosphatase. Annu Rev Bioph Biom 1992, 21, 441-483.
13. Gellman, S. H.; Petter, R.; Breslow, R., Catalytic Hydrolysis of a Phosphate Triester by Tetracoordinated Zinc-Complexes. Journal of the American Chemical Society 1986, 108 (9), 2388-2394.
14. Kovari, E.; Kramer, R., Rapid phosphodiester hydrolysis by an ammonium-functionalized copper(II) complex. A model for the cooperativity of metal ions and NH-acidic groups in phosphoryl transfer enzymes. Journal of the American Chemical Society 1996, 118 (50), 12704-12709.
15. Burstyn, J. N.; Deal, K. A., Selective catalytic hydrolysis of a simple phosphodiester by a macrocyclic copper(II) complex. Inorganic Chemistry 1993, 32 (17), 3585-3586.
16. Deck, K. M.; Tseng, T. A.; Burstyn, J. N., Triisopropyltriazacyclononane Copper(II): An Efficient Phosphodiester Hydrolysis Catalyst and DNA Cleavage Agent. Inorganic Chemistry 2002, 41 (4), 669-677.
17. Hegg, E. L.; Mortimore, S. H.; Cheung, C. L.; Huyett, J. E.; Powell, D. R.; Burstyn, J. N., Structure-reactivity studies in copper(II)-catalyzed phosphodiester hydrolysis. Inorganic Chemistry 1999, 38 (12), 2961-2968.
18. Deal, K. A.; Burstyn, J. N., Mechanistic Studies of Dichloro(1,4,7-triazacyclononane)copper(II)-Catalyzed Phosphate Diester Hydrolysis†. Inorganic Chemistry 1996, 35 (10), 2792-2798.
19. Morrow, J. R.; Buttrey, L. A.; Shelton, V. M.; Berback, K. A., Efficient catalytic cleavage of RNA by lanthanide(III) macrocyclic complexes: toward synthetic nucleases for in vivo applications. Journal of the American Chemical Society 1992, 114 (5), 1903-1905.
20. Morrow, J. R.; Buttrey, L. A.; Berback, K. A., Transesterification of a Phosphate Diester by Divalent and Trivalent Metal-Ions. Inorganic Chemistry 1992, 31 (1), 16-20.
21. Amin, S.; Voss, D. A.; Horrocks, W. D.; Morrow, J. R., Restoration of catalytic activity by replacement of a coordinated amide group: Synthesis and laser-induced luminescence studies of the phosphate diester transesterification catalyst [Eu(NBAC)](3+). Inorganic Chemistry 1996, 35 (26), 7466-+.
22. Baker, B. F.; Khalili, H.; Wei, N.; Morrow, J. R., Cleavage of the 5‘ Cap Structure of mRNA by a Europium(III) Macrocyclic Complex with Pendant Alcohol Groups. Journal of the American Chemical Society 1997, 119 (38), 8749-8755.
23. Chappell, L. L.; Voss, D. A.; Horrocks, W. D.; Morrow, J. R., Effect of mixed pendent groups on the solution and catalytic properties of europium(III) macrocyclic complexes: Bifunctional and monofunctional amide and alcohol pendents in septadentate or octadentate ligands. Inorganic Chemistry 1998, 37 (16), 3989-3998.
24. McCue, K. P.; Morrow, J. R., Hydrolysis of a Model for the 5'-Cap of mRNA by Dinuclear Copper(II) and Zinc(II) Complexes. Rapid Hydrolysis by Four Copper(II) Ions. Inorg Chem 1999, 38 (26), 6136-6142.
25. Iranzo, O.; Elmer, T.; Richard, J. P.; Morrow, J. R., Cooperativity between Metal Ions in the Cleavage of Phosphate Diesters and RNA by Dinuclear Zn(II) Catalysts. Inorganic Chemistry 2003, 42 (24), 7737-7746.
26. Iranzo, O.; Richard, J. P.; Morrow, J. R., Structure-activity studies on the cleavage of an RNA analogue by a potent dinuclear metal ion catalyst: Effect of changing the metal ion. Inorganic Chemistry 2004, 43 (5), 1743-1750.
27. Morrow, J. R.; Nwe, K.; Andolina, C. M., Tethered Dinuclear Europium(III) Macrocyclic Catalysts for the Cleavage of RNA. Journal of the American Chemical Society 2008, 130 (44), 14861-14871.
28. Rossiter, C.; Mathews, R.; Delmundo, I.; Morrow, J., Cleavage of a RNA analog containing uridine by a bifunctional dinuclear Zn(II) catalyst. Journal of Inorganic Biochemistry 2009, 103 (1), 64-71.
29. Subat, M.; Woinaroschy, K.; Anthofer, S.; Malterer, B.; Konig, B., 1,4,7,10-Tetraazacyclododecane Metal Complexes as Potent Promoters of Carboxyester Hydrolysis under Physiological Conditions. Inorganic Chemistry 2007, 46, 4336-4356.
30. Subat, M.; Woinaroschy, K.; Gerstl, C.; Sarkar, B.; Kaim, W.; Konig, B., 1,4,7,10-Tetraazacyclododecane Metal Complexes as Potent Promoters of Phosphodiester Hydrolysis under Physiological Conditions. Inorganic Chemistry 2008, 47, 4661-4668.
31. Gomez-Tagle, P.; Yatsimirsky, A. K., Kinetics of phosphodiester hydrolysis by lanthanide ions in weakly basic solutions. J Chem Soc Dalton 1998, (18), 2957-2959.
32. Gomez-Tagle, P.; Yatsimirsky, A. K., Phosphodiester hydrolysis by lanthanide complexes of bis-tris propane. Inorganic Chemistry 2001, 40 (15), 3786-3796.
33. Gunnlaugsson, T.; Davies, R. J. H.; Kruger, P. E.; Jensen, P.; McCabe, T.; Mulready, S.; O’Brien, J. E.; Stevenson, C. S.; Fanning, A.-M., Cyclen based lanthanide ion ribonuclease mimics: the effect of pyridine cofactors upon phosphodiester HPNP hydrolysis. Tetrahedron Letters 2005, 46 (21), 3761-3766.
34. Camargo, M. A.; Neves, A.; Bortoluzzi, A. J.; Szpoganicz, B.; Fischer, F. L.; Terenzi, H.; Serra, O. A.; Santos, V. G.; Vaz, B. G.; Eberlin, M. N., Efficient Phosphodiester Hydrolysis by Luminescent Terbium(III) and Europium(III) Complexes. Inorganic Chemistry 2010, 49.
35. Fenton, D. E.; Vigato, P. A., Macrocyclic Schiff-Base Complexes of Lanthanides and Actinides. Chem Soc Rev 1988, 17 (1), 69-90.
36. Huskens, J.; Torres, D. A.; Kovacs, Z.; Andre, J. P.; Geraldes, C. F. G. C.; Sherry, A. D., Alkaline earth metal and lanthanide(III) complexes of ligands based upon 1,4,7,10-tetraazacyclododecane-1,7-bis(acetic acid). Inorganic Chemistry 1997, 36 (7), 1495-1503.
37. 吳柏宏, 鑭系金屬離子錯合物水解磷酸酯鍵之動力學研究. 國立交通大學生物科技所 2003, 碩士論文.
38. Chang, C. A.; Wu, B. H.; Kuan, B. Y., Macrocyclic lanthanide complexes as artificial nucleases and ribonucleases: Effects of pH, metal ionic radii, number of coordinated water molecules, charge, and concentrations of the metal complexes. Inorganic Chemistry 2005, 44 (19), 6646-6654.
39. 林玉淳, 大環配位子結構與質子化系統及鑭系金屬離子錯合物結構之分子模擬. 國立交通大學生物科技所 2006, 碩士論文.
40. 蔡政憲, 鑭系金屬錯合物之熱力學與水解磷酸酯鍵之動力學研究. 國立交通大學生物科技所 2006, 碩士論文.
41. 陳郁頻, 大環配位子DO2PA之熱力學研究與HEDTA鑭系金屬錯合物水解磷酸酯鍵之動力學研究. 國立交通大學生物科技所 2007, 碩士論文.
42. 王翊帆, 大環配位子 ODO2A之穩定常數研究與其鑭系金屬錯合物之水解磷酸酯鍵效力. 國立交通大學生化工程所 2007, 碩士論文.
43. 吳幸怡, 大環配位子 ONO2A之穩定常數研究與其鑭系金屬錯合物之水解磷酸酯鍵效力. 國立交通大學生化工程所 2007, 碩士論文.
44. Cabral, M. F.; Costa, J.; Delgado, R.; Dasilva, J. J. R. F.; Vilhena, M. F., Protonation and Metal Complexation Studies on Some Oxa-Diaza-Macrocyclic Ligands. Polyhedron 1990, 9 (23), 2847-2857.
45. Chaves, S.; Cerva, A.; Delgado, R., Tris(carboxymethyl)oxatriazamacrocycles and their metal complexes †. Journal of the Chemical Society, Dalton Transactions 1997, (22), 4181-4190.
46. Amorim, M. T. S.; Chaves, S.; Delgado, R.; Dasilva, J. J. R. F., Oxatriaza Macrocyclic Ligands - Studies of Protonation and Metal Complexation. J Chem Soc Dalton 1991, (11), 3065-3072.
47. Gans, P.; Sabatini, A.; Vacca, A., Investigation of equilibria in solution. Determination of equilibrium constants with the HYPERQUAD suite of programs. Talanta 1996, 43 (10), 1739-1753.
48. Chang, C. A.; Chen, Y. H.; Chen, H. Y.; Shieh, F. K., Capillary electrophoresis, potentiometric and laser excited luminescence studies of lanthanide(III) complexes of 1,7-dicarboxymethyl-1,4,7,10-tetraazacyclododecane (DO2A). J Chem Soc Dalton 1998, (19), 3243-3248.
49. Fujioka, H.; Koike, T.; Yamada, N.; Kimura, E., A new bis(zinc(II)-cyclen) complex as a novel chelator for barbiturates and phosphates. Heterocycles 1996, 42 (2), 775-787.
50. Koike, T.; Takashige, M.; Kimura, E.; Fujioka, H.; Shiro, M., Bis(Zn-II-cyclen) complex as a novel receptor of barbiturates in aqueous solution. Chem-Eur J 1996, 2 (6), 617-623.
51. Kimura, E.; Aoki, S.; Koike, T.; Shiro, M., A tris(Zn-II-1,4,7,10-tetraazacyclododecane) complex as a new receptor for phosphate dianions in aqueous solution. Journal of the American Chemical Society 1997, 119 (13), 3068-3076.
52. Brown, D. M.; Usher, D. A., Hydrolysis of hydroxyalkyl phosphate esters: effect of changing ester group. Journal of the Chemical Society (Resumed) 1965, 6558.
53. Schiller, A.; Scopelliti, R.; Severin, K., Enhanced hydrolytic activity of Cu(ii) and Zn(ii) complexes in highly cross-linked polymers. Dalton Transactions 2006, (32), 3858.