臺灣博碩士論文加值系統

本論文主要以軟性的「電噴灑游離質譜法(Electrospray ionization mass spectrometry)」，提供一個方便、快速的方法，研究具有抗癌活性的鉑族金屬化合物與DNA之間的鍵結關係，做為將來雙股螺旋DNA非共價鍵結及藥物設計的基礎研究。
　　論文分成三大部分，其一，研究具有抗癌活性Ru金屬化合物對於單股DNA上鹼基G位置的選擇性，實驗結果認為: Ru金屬化合物對於單股DNA上「中間」位置的鹼基G，有較高的選擇性﹔其二，研究實驗的環境與DNA的雙股螺旋結構穩定度之間的關聯性，實驗結果認為:在負電的模式之下，使用isopropanol為揮發溶劑且與水的的比例達75%以上、毛細管溫度為120-140℃且加入的NH4OAc的量為DNA濃度的200-500倍時，可以得到最大量的雙股螺旋DNA﹔最後，研究具有抗癌活性雙核鉑金屬化合物與雙股螺旋DNA產生反應時，彼此之間的鍵結關係﹔實驗結果認為: 具有抗癌活性雙核鉑金屬化合物與雙股螺旋DNA產生反應時是先以ionic/electrostatic force形式相互吸引，最後形成Pt-N coordination bond。

The gentle nature of the ESI proess, however, means that ESI-MS is also finding application for the study of noncovalent and other fragile biomolecular complexes.
The thesis included three section:
First section: Determination of Binding Location Preference between Short Oligonucleotide and Ru-complexes Analyzed by Ion Trap Mass Spectrometry
The interaction of two ruthenium (II) complexes, [Ru(terpy)(bpy)Cl]Cl and cis-[Ru(bpy)]Cl2, with single-stranded DNA wereinvestigated by electrospry ionization mass spectrometry. The DNA binding sits preference of [Ru(terpy)(bpy)Cl]Cl and cis-[Ru(bpy)]Cl2 were located at central guanine, which was determined by tandem mass spectrometry technique. The Ru-complexes bounds to guanine also enhanced the loss of guanine-Ru, and gave a-B and w fragments.
Second section: Factors Influence on Gas-Phase Stability of Double Stranded Oligonucletoides Studied by Electrospray Mass Spectrometry
The topic studied in relativity between gas-phase stability of double stranded oligonucletides and experimental system. According to this experimental results: the experimental system was set up at capillary temperature at 120-140℃; adding concentration of NH4OAc is 200-500 times over concentration of oligonucletides; the volatile solvent- isopropanol have been used as and the compositions again water 75%.
Third stction: Kinetic of the DNA binding Azole-Bridged Dinuclear Platinum(II) compounds by Electrospray ionization Mass Spectrometry
The kinetics of the reaction between three new derivatives of azole-bridged dinuclear platinum (Ⅱ) complex , (1), [{cis-Pt(NH3)2}2(μ-OH)-(μ-pyrazolate)][NO3]2 (2), [{cis-Pt(NH3)2}2(μ-OH)-(μ-1,2,3-triazolate)][NO3]2 and (3), [{cis-Pt(NH3)2}2(μ-OH)-(μ-phenyltriazolate)][NO3]2 and complementary double-stranded DNA was investigated by electrospray ionization mass spectrometry. Products with different binding modes were observed and shows competition at different time intervals. Ionic/electrostatic binding products dominate at early stage of reaction and gradually transform into covalently modified adduct by removal of hydroxyl leaving group. We proposed that the binding of the dinuclear platinum complexes to the double-stranded nucleic acids may be considered as a three-step process: rapid initial electrostatic binding followed by covalent attachment of the drug by sequential displacement of the leaving group to the N7 position of guanosines, giving first monofunctional and subsequently bifunctional adducts. The binding affinity of three derivatives to complementary 12-mer oligonucleotides are in the order of 3>1»2, which are determined by the ratio of intensity of electrostatic adduct to that of unbound free DNA. On the hand, comparison of the relative intensities of mono- and difunctional platinum covalent adduct suggested that the reactivity for these Pt(Ⅱ) complexes are in the order 2 > 1 > 3, parallel to the trend of cytotoxicity.
Compared to other technique, the study shows that ESI is a sensitive and fast tool for confirmation of binding and determination of stoichometry for both covalent and noncovalent systems.

目錄
論文摘要
第一章 緒論
1-1 細胞內的遺傳物質 1
1-1.1 DNA的發現 1
1-1.2 DNA的化學組成 2
1-1.3 DNA的結構 5
1-2 DNA與惡性腫瘤之關聯性 6
1-3 癌症治療 7
1-4 抗癌藥物 8
1-4.1 鉑金屬化合物 9
1-4.2 鉑族金屬抗癌藥的抗癌作用機制 11
1-5 金屬抗癌藥物的未來展望 14
1-5.1 釕金屬化合物 15
1-5.2 多核鉑金屬化合物 16
1-6 藥物發展與質譜技術 17
第二章 質譜分析法
2-1 質譜儀的演進 19
2-2 電噴灑游離質譜法 (Electrospray ionization mass spectrometer, ESI/MS) 的演進 22
2-3 電噴灑游離質譜法 (Electrospray ionization mass spectrometer, ESI/MS) 的游離機制 23
2-4 質譜法 (mass spectrometry)的質量分析器 27
第三章 Determination of Binding Location Preference between Short Oligonucleotide and Ru-complexes Analyzed by Ion Trap Mass Spectrometry
3-1 研究背景 31
3-2 實驗藥品與方法 34
3-2.1 藥品來源 34
3-2.2 藥品配製 38
3-2.3 儀器設備 38
3-3 結果與討論 38
3-3.1 HPLC的純化 44
3-3.2 何為誘導碰撞解離(collisional-induced dissociation, CID) 53
3-3.3 MS/MS碎裂後之質量鑑定 54
3-3.4 MS/MS碎裂機制之探討 59
3-3.5 [s.s.DNA+Ru complex]的解離機制 65
3-3.6 利用串聯式質譜儀探討s.s.DNA與Ru化合物cis-[Ru(bpy)2]Cl2、[Ru(terpy)(bpy)Cl]Cl之反應性 78
3-3.7 79
3-3.8 結論 81
第四章 Factors Influence on Gas-Phase Stability of Double Stranded Oligonucletoides Studied by Electrospray Mass Spectrometry
4-1 研究背景 82
4-2 實驗藥品與方法 85
4-2.1 藥品來源 85
4-2.2 藥品配製 85
4-2.3 儀器設備 85
4-3 結果與討論 87
4-3.1 DNA濃度效應 89
4-3.2 溶劑效應 91
4-3.3 毛細管溫度(capillary temperture) 95
4-3.4 醋酸銨濃度效應 98
4-3.5 結論 101
第五章 Kinetic of the DNA binding Azole-Bridged Dinuclear Platinum(II) compounds by Electrospray ionization Mass Spectrometry
5-1 研究背景 102
5-2 實驗藥品與方法 106
5-2.1 藥品來源 106
5-2.2 藥品配製 106
5-2.3 儀器設備 107
5-3 結果與討論 111
5-3.1 DNA的質量鑑定 112
5-3.2 DNA與雙核鉑金屬鍵結化合物的質量鑑定 114
5-3.3 DNA與雙核鉑金屬鍵結化合物結構的安定度 134
5-3.4 DNA與雙核鉑金屬鍵結模式 140
5-3.5 DNA與雙核鉑金屬鍵結後雙股螺旋DNA的穩定性
144
5-3.6 三種雙核鉑金屬鍵結與DNA反應性的比較 145
5-3.7 在細胞體內和細胞體外雙核鉑金屬藥物與DNA鍵結反應性之比較 149
5-3.8 pH值與反應速率 152
第六章 結論及未來方向 153
參考文獻 155

參考文獻
1. 三浦謹一郎，劉文正，DNA與遺傳訊息，1996，台北市編譯館
2. Blackburn, G. J.; Gait, M. J. Nucleic Acids in Chemistry and Biology 1990, Oxford University press, Tokyo
3. Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry III 2000, Worth Press, New York
4. Waston, J. D.; Crick, F. H. C. Nature, 1953, 171, 737-738
5. 生物化學圖解，Peter N. Campbell Anthony D. Smith
6. Organic chemistry K.Peter C. Vollhardt and Nell E. Schore
7. K. J. Light-Wahl, D. L. Springer, B. E. Winger, C. G. Edmonds, D. G. Camp, II, B. D. Thrall, and R. D. Smith, J. Am. Chem. Soc. 1993, 115, 803-804
8. Antonio Triolo, Federico M. Arcamone, Andrea Raffaelli and Piero Salvadori, J. Mass Sperctrom. 1997, 32, 1186-1194
9. 圖解病理學，蔡東龍
10. Antonio Triolo, Federico M. Arcamone, Andrea Raffaelli and Piero Salvadori, J. Mass Sperctrom. 1997, 32, 1186-1194
11. Thomson, J. J. Ray of Positive Elctricity and their Application to Chemical Analysis, Green and Co. 1998
12. Nier, A. O. Rev. Sci. Instrum. 1947, 18, 415
13. Munsun, M. S. B.; Field, F. H. J. Am. Chem. Soc. 1960, 88, 2621
14. Beckey, H. D. Principles of Field Ionization and Desorption Mass Spectrometry, 1977
15. Barber, M.; Bordoil, R. S.; Ellott, G. J.; Sedgwick, R. D.; Tyler, A. N. Anal. Chem. 1982, 54, 645A
16. Benninghoven, A.; Rudenauer, F. G.; Werner, H. W. Second Ion Mass Spectrometry : Basic Concepts, Instrumental Aspects, Applications and Trends 1987
17. Cotter, R. J. Anal. Chem. 1988, 60, 781A
18. Unsold, E.; Hillenkamp, F.; Nitsche, R. Analysis 1990, 4, 115
19. Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984, 88, 1451
20. Karas, M.; Hillenkamp, F. Anal. Chem. 1998, 60, 2299
21. Mack, L. L.; Kralic, P.; Rheude, A.; Dole, M. J. Chem. Phys. 1970, 52, 4977
22. Dole, M.; Mack, L. L.; Hines, R. L. J. Chem. Phys. 1968, 49, 2240
23. Wong, S. F.; Meng, C. K.; Fenn, J. B. J. Chem. Phys. 1988, 92, 546
24. Fenn, J. B.; Meng, C. K.; Wong, S. F.; Meng Science 1989, 246, 64
25. AlekSendriv, M. L ; Gall, L. N.; Shkurov, V. A.; Pavlenko, V. A.; Krasnov, N. V.; Nikolaev, V. I. J Anal. USSR 1984, 39, 1268
26. Gomez, A.; Tang, K. Phys. Fluid. 1994, 65, 404
27. Dole, D.; Mack, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. D.; Alice, M. B. Chem. Phys. 1968, 49, 2240
28. Iribarne, J. V.; Thornson, B. A.; J. Chem. Phys. 1976, 64, 15
29. Rollgen, F. W.; Bramer-Wegner, E.; Butfering, L. J. Phys. 1987, 48, 253-256
30. Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1232
31. Siuzdak, G. Mass Spectrometry for Biotebhnology 1995, Academic Press, California
32. Clarke MJ. Ruthenium in biology: DNA interactions. 1997 American Chemical Society
33. Keppler BK, Berger MR, Heim ME. New tumor-inhibiting metal complexes.
34. Barton JK, Lolis E. Chiral discrimination in the covalent binding of bis(phenanthroline)dichlororuthenium(II) to b-DNA. J Am Chem. Soc, 1985, 107, 708-709
35. Grove N, Gupta N, Throp HH. Stereoaelective covalent binding of aquaruthenium(II) complexes to DNA. J Am Chem. Soc, 1992, 114, 3390-3393
36. Grove N, Welch TW, Fariley TA, Cory M, Throp HH. Covalent binding of aquaruthenium(II) complexes to DNA. Inorg Chem. 1994, 33, 3544-3548
37. Novakova O, Kasporkova J, Vrana O, van Vliet PM, Reedijk J, Brabec V. Correlation between cytotoxicity and DNA binding of polypridyl ruthenium complexes. Biochemistry 1995 Sep 26; 34(38): 12369-12378
38. Clarke JM, Zhu F, Frasca DR. Non-platium chemotherapeutic metallopharmaceuticals. Chem. Rev., 1999, 99,2511-1533
39. Van Vilet PM, Hassnoot JG, Reedijk J. Binding of 9-methylhypoxanthine and 9-ethylguanine to [cis-Ru(2,2’-bipyridine)2]+‧NMR and X-ray structure of cis-chlorobis(2,2’- bipyridine)( 9-ethylguanine-kN7)ruthenium(II) chloride. Inorg Chem. 1994, 33, 1934-1939
40. Zhao M, Clarke MJ. Effect of trans-pyridine ligands on the interactions of RuII and RuIII ammine complexes N7-coordination to purine nucleosides and DNA. J. Biol Inorg Chem. 1999, 4, 325-340
41. Fenn JB, Mann M, Meng CK, Wong SF, Whitehose CM. Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989, 246, 64-71
42. Smith RD, Loo JA, Edmonds CG, Barinaga CJ, Udseth HR. New developments in biochemical mass spectrometry: Electrospray ionization. Anal Chem. 1990, 62, 882-899
43. Van Berkel GJ, Glish GL, Mcluckey SA. Electrospray ionization combined with ion trap mass spectrometry. Anal Chem. 1990, 62, 1284-1295
44. Louris JN, Cooks RG, Syka JEP, Kelley PE, Stafford Jr. GC, Todd JFJ. Instrumentation, applications, and energy deposition in quadrupole ion trap tandem mass spectrometry. Anal Chem. 1987, 59, 1677-1685
45. MuLuckey SA, Van Berkel GJ, Glish GL. Tandem mass spectrometry of small, multiply charged oligonucleotides. J. Am. Soc. Mass Spectrom. 1992, 3, 60-70
46. MuLuckey SA, Habibi-Goudarzi S Decompositions of multiply charged oligonucleotide anions. J. Am.chem Soc. 1993, 115, 12085-12095
47 Boschenok J, Shei MM. Electrospray tandem mass spectrometry of nucledotides. Rapid Commun Mass Spectrom 1996; 10(1): 144-119
48. Ni J, Mathew MAA, McCloskey JA. Collision-induced dissociation of polyprotonated oligonucleotides produced by electrospray ionization. Rapid Commun Mass Spectrom 1997, 11, 535-540
49. Sherman SE, Gibson D, Wang AH, Lippard SJ. X-ray stucuture of the major adduct of the anticancer drug cisplatin with DNA: cis-[Pt(NH3)2(d(pGpG))]. Science 1985, Oct 25; 230(4724): 412-417
50. Esposito G, Cauci S, Fogolari F, Alessio E, Scocchi M, Quadrifoglio F, Viglino P. NMR structural characterization of the reaction product between d(GpG) and the octahedral antitumor complex trans-RuCl2(DMSO)4 Biochemistry 1992, Aug 11; 31(31): 7094-7103
51. Collins JG, Sleeman AD, Aldrich-Wright JR, Greguric I, Hambley TW. A 1H NMR study of the DNA binding of ruthenium(II) polypyridyl complexes. Biochemistry 1998, 37, 3313-3141
52. Proudfoot EM, Mackay JP, Karuso P. Probing site specificity of DNA binding metallointercalators by NMR spectroscopy and molecular modeling. Biochemistry 2001, Apr 17; 40(15): 4867-4878
53. Anagnostopoulou A, Moldrheim E, Katsaros N, Sletten E. Interaction of cis- and trans-RuCl2(DMSO)4 with the nucleotides GpA, d(GpA), ApG, d(ApG) and d(CCTGGTCC): high field NMR characterization of reaction products. J. Biol Inorg Chem. 1999,Apr 4(2): 199-208
54. Cheng CC, Lee WL, Su JG, Liu CL. Covalent interaction of Ru(terpy)(tmephen)Cl+ with DNA: A potential ruthenium-base anticancer drug. J. Chin Chem Soc. 2000, 47, 213-220
55. Ling LS, He ZK, Zeng YE. Spectral studies on the interaction of DNA and Ru(bipy)2 (dppx)2+. Spectrochimica Acta Part B 1999, 55, 1297-1302
56. Riley CM, Sternson LA, Repta AJ, Slyter SA. Monitoring the reactions of cisplatin with nucleotides and methionine by reversed-phase high-performance liquid chromatography using cationic and anionic pairing ions. Anal Biochem 1983, Apr 1; 130(1): 203-214
57. Troujman H, Chottard JC. Comparison between HPLC and capillary electrophoresis for the separation and identification of the platination products of oligonucledotides with cis-[Pt(NH3)2(H2O)2]2+ and [Pt(NH3)3(H2O)]2+ Anal Biochem 1997 Oct 1; 252(1): 177-185
58. Wu Q, Cheng X, Hofstadler SA, Smith RD. Specific metal-oligonucleotide binding studied by high resolution tandem mass spectrometry. J. Mass Spectrom. 1996, 31 669-675
59. Favre A, Gonnet F, Tabet JC. Locations of the Na+ cation in negative ions of DNA evidenced by using MS2 experiments in ion trap mass spectrometry. Intern. J. Mass Spectrom. 1999, 190/191, 303-312
60. Bartleet MG, McCloskey JA, Manalili SM, Griffey RH. The effect of backbone charge on the collision-induced dissociation of oligonucleotides. J. Mass Spectrom. 1996, 31, 1277-1283
61. Marzilli LA, Barry JP, Sells T, Law SJ, Vouros P, Harsch A. Oligonucleotide sequencing using guanine-specific methylation and electrospray ionization ion trap mass spectrometry. J Mass Spectrom 1999 Apr; 34(4): 276-280
62. Reedijk, J. Chem. Rev. 1999, 99, 2499-2510
63. Lippert, B. Bioinorganic Chemistry 1995, 179-199
64. Katsaros, N. B. Bioinorganic Chemistry 1995, 219-235
65. Clarke, M. J.;Stubbs, M. Interaction of Metallopharaceuticals with DNA; Sigel, A. and Sigel, H., Ed;Marcel Dekker, Inc.:New York, New York, U.S.A., 1996, 32, 727-775
66. Barca, A,; Pani, B.; Tamaro, M.; Russo, E. Mutation Research 1999, 423, 171-181
67.Moucheron, C.; Mesmaeker, A. K.-D.; Kelly, J. M. J. photochemistry and Photobiology B: Biology 1997, 40, 91-106
68. Vilchez, F. G.; Vilaplana, R.; Blasco, G.; Messori, L. J biological Chemistry 1998, 71, 45-51
69. Kratz, F.; Hartmann, M.;Keppler, B.; Messori, L. J biological Chemistry 1994, 269, 2581-2588
70. Sava, G.; Pacor, S.; Bregant, F.; Ceschia, V. Anticancer Research 1991, 11, 1103-1108
71. Kane, S. A.; Lippard, S. J. Biochemistry 1996, 35, 2180-2188
72. Clarke, M. J.; Zhu, F.; Frasca, D. R. Chem. Rev. 1999, 99, 2511-2533
73. Wong, E.; Giandomenico, C. M. Chem. Rev. 1999, 99, 2450-2466
74.Phillips, D. R. Advances in DNA Sequence Specific Agents 1996, 2, 101-137
75. Rajski, S. R.; Williams, R. M. Chem. Rev. 1998, 98, 2723-2795
76. E. Nordhoff, F. Kirpeaker, and P. Roepstorff Mass Spectrometry Review 1996, 15, 67-138
77. Sherman, S. E. and Lippard, S. J. Chem. Rev. 1987, 87, 1153-1181
78. Zijian Guo and Peter J. Sadler Angew. Chem. Int. Ed. 1999, 38, 1512-1531
79. Habibi-Goudarzi, S.; McLuckey, S. A. J. Am. Soc. Mass Spectrom. 1995, 6, 102
80. Trevor W. Hambly and Andrew R. Jones Coordination Chemistry Reviews 2001, 212, 35-39
81. Jan Reedijk Chem. Rev. 1999, 99, 2499-2510
82. Paul M. van Vliet, Jaap G. Haasnoot, and Jan Reedijk Inorg. Chem. 1994, 33, 1934-1939
83. Jirl Kozelka, Franck Legendre, Franziska Reeder, Jean-Claude Chottard Coordination Chemistry Reviews 1999, 190-192, 61-82
84.Valerie Gabelica and Edwin De Pauw J. Mass Spectrom. 2001, 36, 397-402
85. Paul D. Schnier, John S. Klassen, Eric F. Strittmatter, and Evan R. Williams J. Am. Chem. Soc. 1998, 120, 9605-9613
86. Zlatanova, J., Yaneva, J. & Leuba, S. H. Proteins that specifically recognize cisplatin-damaged DNA: a clue to anticancer activity of cisplatin. FASEB J. 1998, 12, 791-799
87. Gelasco, A. & Lippard, S. J. NMR solution structure of a DNA dodecamer duplex containing a cis-diammineplatinum(II) d(GpG) intrastrand cross-link, the major adduct of the anticancer drug cisplatin. Biochemistry 1998, 37, 9230-9239
88. Takahara, P. M., Frederick, C. A. & Lippard, S. J. Crystal structure of the anticancer drug cisplatin bound to duplex DNA. J. Am. Chem. Soc. 1996, 118, 12309-12321
89. Sherman, S. E., Gibson, D., Wang, A. H.-J. & Lippard, S. J. Crystal and molecular structure of cis-[Pt(NH3)2{d(pGpG)}], the principal adduct formed by cis-diamminedichloroplatinum(II) with DNA. J. Am. Chem. Soc. 1988, 110, 7368-7381
90. http://www.kfsyscc.org/patients/cure.html