合成5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以作為應用於原位合成之新穎性中性核苷酸之研究

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姓名

阮靖宇(Cing-Yu Ruan) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

合成5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以作為應用於原位合成之新穎性中性核苷酸之研究
(Synthesis of 5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite as a novel neutralized deoxynucleoside for in situ synthesis)

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摘要(中)

實現個人化醫療是現代醫療的新趨勢。配合基因資訊的分析，醫療人員能夠更精準地給予不同病患合適的療程。倘若倚靠基因晶片並配合大型基因資料庫，在臨床上便能快速且精確地得到病患特定表現的基因，可以在診斷時，可以更快地提供相關的基因資訊。目前的基因晶片仍舊有相當多問題，螢光標定的不穩定性、價格昂貴，基因晶片靈敏度及準確度不夠……等需要解決。透過矽奈米線場效電晶體(SiNWFET)其高靈敏度、免標定、及時偵測等特性作為新一代檢測DNA序列雜交的平台具有相當大的潛力。然而，場效電晶體在檢測DNA序列雜交的電訊號時，易受到環境鹽離子濃度影響造成檢測上的困難。根據本實驗室過去的研究，將DNA序列磷酸骨幹上的氧經由甲基化的改質，使帶負電的磷酸根轉變成甲基磷酸三酯鍵methyl phosphotriester (MPTE)而成為電中性，可以降低序列本身負電量，使得DNA序列得以於低鹽離子濃度下進行雜交，進而使場效電晶體檢測靈敏度上升。
現今的基因晶片主要是倚靠點陣法(spotting method)所製作出來，其晶片上探針密度相較於Affymetrix公司利用類似半導體製程的原位合成法(in situ synthesis)所生產之晶片低，若在臨床上需要更精準地得知病患的基因資訊，勢必需要更高密度的探針以檢測差異表現的基因。因此，本研究為了開發能夠在場效電晶體上進行原位合成nDNA(phosphate-methylated DNA)序列的原料，我們利用(R,S)-1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate (MeNPOC-Cl)作為原料，以兩步親核反應合成出5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite(光敏化中性核苷酸)，並藉由本校的600MHz核磁共振儀及質譜儀得到定性分析，然而其本身的不穩定性，導致我們目前尚無法得到乾淨的產物。此外，我們也利用紫外光光譜儀及質譜儀去分析目標產物照光後的特性及其產物分子量，並嘗試在溶液相中合成出二聚體(dimer)。
最終，5’-MeNPOC-2’-nucleoside p-methoxy phosphoramidite是可以利用本研究的合成途徑被合成出來，但仍需改善純化方法以得到乾淨產物。倘若未來能夠找到一個合適目標產物的純化方法，便能利用5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以原位合成的方式在矽奈米線場效電晶體上合成出相當高密度的寡核苷酸探針，以得到高通量的基因晶片，在臨床上為精準醫療提供更準確的基因資訊。

摘要(英)

Personal treatment has been a new medical trend recently. Based on the analysis of gene information, professional healthcare could give every individual a precise medical treatment. From the perspective of clinical diagnosis, if we could combine gene chip technology and a huge gene database, we would obtain the gene information immediately. However, there are still numerous problems in gene chip technology, such as the instability and the cost of fluorescent label, the sensitivity of gene chip. Silicon nanowire field effect transistor (SiNWFET) could be a huge potential for next generation detection platform due to its high sensitivity, label-free technique, and high response time…etc. However, the environmental ionic concentration would influence the sensitivity of SiNWFET when we detect the electronic signals of duplex formation. According to the previous studies in our lab, by neutralizing site-specific phosphate backbone with methyl phosphotriester (MPTE) inter-nucleoside linkage(s), we improved the electronic signals of SiNWFET when DNA/DNA hybridization under low ionic concentration.
The probe density on gene chip which fabricated with spotting method is lower than those fabricated with in situ synthesis that developed by Affymetrix. In order to have a more precise gene information on clinical diagnosis, there must be higher dense probes on microarray, which is called high throughput microarray. Therefore, in this study, we use (R,S)-1-(3,4-methylenedioxy-6-nitrophenyl)ethyl chloroformate (MeNPOC-Cl) as the raw material to develop novel deoxynucleosides for in situ synthesized oligonucleotides, which contain site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s), on SiNWFET. Using 2-step nucleophilic reactions, 5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite can be synthesized and can be qualitative analyzed with 600MHz NMR and ESI-QTOF-MS, but so far, we cannot get the pure products due to their instability. In addition, we characterized these deoxynucleosides by UV spectrum and MASS spectrum after solution phase illumination test, and try to synthesize dinucleotide in solution phase.
Eventually, 5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite can be synthesized. In the future, if we could improve the purification method for the desired products, we aim to fabricate a high-throughput FET-based DNA microarray with these novel deoxynucleosides by in situ synthesis method and provide a more precise gene information on clinical diagnosis.

關鍵字(中)

★ 光敏化基團
★ 中性核苷酸

關鍵字(英)

★ photolabile group
★ neutralized deoxynucleoside

論文目次

摘要 i
Abstract iii
誌謝 v
目錄 vii
圖目錄 x
表目錄 xv
第一章緒論 1
第二章文獻回顧 4
2.1基因檢測 4
2.2核酸及核酸類似物 5
2.2.1 去氧核醣核酸及其結構 5
2.2.2 肽核酸(Peptide Nucleic Acid, PNA) 8
2.2.3 鎖核酸(Locked Nucleic Acid, LNA) 9
2.2.4 中性去氧核醣核酸(oligonucleotides containing site-specific methyl phosphotriester(MPTE) inter-nucleoside linkage(s), nDNA) 10
2.3原位合成(in situ synthesis) 11
2.4 光敏化基團(Photolabile Protecting Group, PPG) 17
第三章實驗藥品、方法與儀器設備 20
3.1 實驗藥品 20
3.1.1 化學品 20
3.2.2 實驗耗材 23
3.2 儀器設備 24
3.3 合成方法 25
3.3.1 MeNPOC-thymidine與MeNPOC-guanosine(ibu)合成 25
3.3.2MeNPOC-thymidine p-methoxy phosphoramidite 與MeNPOC- guanosine(ibu) p-methoxy phosphoramidite 合成 36
3.4 照光反應 41
3.5 溶液相二聚體合成方法 43
第四章　結果與討論 44
4.1 MeNPOC-nucleoside 定性結果 44
4.1.1 MeNPOC-thymidine定性結果 44
4.1.2 MeNPOC- guanosine(ibu) 定性結果 48
4.2 MeNPOC-nucleoside p-methoxy phosphoramidite 定性結果 52
4.2.1 MeNPOC-thymidine p-methoxy phosphoramidite定性結果 52
4.2.2 MeNPOC-guanosine(ibu) p-methoxy phosphoramidite定性結果
55
4.3 MeNPOC-nucleoside p-methoxy phosphoramidite於萃取與純化時產物損壞討論 57
4.3.1 MeNPOC-thymidine p-methoxy phosphoramidite萃取比例的討論
58
4.3.2 MeNPOC-thymidine p-methoxy phosphoramidite不同純化方式結果 60
4.3.2.1 MeNPOC-thymidine p-methoxy phosphoramidite以前處理過的矽膠純化結果 62
4.3.2.2 MeNPOC-thymidine p-methoxy phosphoramidite以LH20中性樹脂純化結果 64
4.3.2.3 MeNPOC-thymidine p-methoxy phosphoramidite以再結晶純化結果 66
4.4 MeNPOC光敏化基團光化學動力學探討 67
4.5溶液相二聚體合成 76
第五章　結論與未來展望 78
第六章　參考文獻 80
第七章補充資料 85

參考文獻

[1] P. Berroy, M. Viriot, and M. Carre, "Photolabile group for 5′-OH protection of nucleosides: synthesis and photodeprotection rate," Sensors and Actuators B: Chemical, vol. 74, no. 1, pp. 186-189, 2001.
[2] C. G. Bochet, "Photolabile protecting groups and linkers," Journal of the Chemical Society, Perkin Transactions 1, 10.1039/B009522M no. 2, pp. 125-142, 2002.
[3] M. Beier and J. D. Hoheisel, "Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays," (in eng), Nucleic acids research, vol. 28, no. 4, pp. E11-E11, 2000.
[4] 李. 吳俊忠, 孫光蕙, 趙崇義,王美嘉,王聖帆,朱大成,江倪全,吳芳姿,李建宏,何鴻耀,林文昌,邱全芊,胡忠怡,林佳霓,林亮音,林淑容,林淑華,林景堉,施浤彰,孫建峰,陳佑誠,陳定平,陳怡伶,張長泉,黃家群,陳盈汝,張建國,陳桂添,陳泰龍,陳錫秉,許蕙玲,張懿欣,郭保麟,黃智生,黃溫雅,曾慶平,游雅言,楊正芬,楊雅倩,楊國梁,詹爾昌,鄭如茜,鄭恩加,鄧麗珍,蕭明裕,駱紀東,鍾明怡,羅時燕,蘇怡寧, 醫學分子檢驗(5版). 2017.
[5] W.-P. Hu, C.-C. Tsai, Y.-S. Yang, H. W.-H. Chan, and W.-Y. Chen, "Synergetic improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing neutralized DNA as probe," Scientific Reports, vol. 8, no. 1, p. 12598, 2018/08/22 2018.
[6] A. B. Wolf, R. J. Caselli, E. M. Reiman, and J. Valla, "APOE and neuroenergetics: an emerging paradigm in Alzheimer′s disease," Neurobiology of Aging, vol. 34, no. 4, pp. 1007-1017, 2013/04/01/ 2013.
[7] M. Stoneking, "From the evolutionary past," Nature, vol. 409, no. 6822, pp. 821-822, 2001/02/01 2001.
[8] L.-C. Li et al., "A Single Nucleotide Polymorphism in the <em>E-cadherin</em> Gene Promoter Alters Transcriptional Activities," Cancer Research, vol. 60, no. 4, p. 873, 2000.
[9] M. Cargill et al., "Characterization of single-nucleotide polymorphisms in coding regions of human genes," Nature Genetics, vol. 22, no. 3, pp. 231-238, 1999/07/01 1999.
[10] B. P. Hodkinson and E. A. Grice, "Next-Generation Sequencing: A Review of Technologies and Tools for Wound Microbiome Research," Advances in Wound Care, vol. 4, no. 1, pp. 50-58, 2015/01/01 2014.
[11] 李曜珊, "簡介次世代定序技術及美國的法規管理," 當代醫藥法規月刊, 2017.
[12] J. D. Watson and F. H. C. Crick, "Molecular Structure of Nucleic Acids: A Structure for Deoxyribose Nucleic Acid," Nature, vol. 171, no. 4356, pp. 737-738, 1953/04/01 1953.
[13] M. Egholm, O. Buchardt, P. E. Nielsen, and R. H. Berg, "Peptide nucleic acids (PNA). Oligonucleotide analogs with an achiral peptide backbone," Journal of the American Chemical Society, vol. 114, no. 5, pp. 1895-1897, 1992/02/01 1992.
[14] M. Egholm et al., "PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules," Nature, vol. 365, no. 6446, pp. 566-568, 1993/10/01 1993.
[15] S. Tomac et al., "Ionic Effects on the Stability and Conformation of Peptide Nucleic Acid Complexes," Journal of the American Chemical Society, vol. 118, no. 24, pp. 5544-5552, 1996/01/01 1996.
[16] R. E. L. Aly Farag El Sheikha , Jianping Xu Ed. Molecular Techniques in Food Biology: Safety, Biotechnology, Authenticity and Traceability. 2018.
[17] K. Oliveira, G. W. Procop, D. Wilson, J. Coull, and H. Stender, "Rapid Identification of <em>Staphylococcus aureus</em> Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes," Journal of Clinical Microbiology, vol. 40, no. 1, p. 247, 2002.
[18] J. Wang et al., "Peptide Nucleic Acid Probes for Sequence-Specific DNA Biosensors," Journal of the American Chemical Society, vol. 118, no. 33, pp. 7667-7670, 1996/01/01 1996.
[19] S. Obika et al., "Synthesis of 2′-O,4′-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering," Tetrahedron Letters, vol. 38, no. 50, pp. 8735-8738, 1997/12/15/ 1997.
[20] G. Obernosterer, J. Martinez, and M. Alenius, "Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections," Nature Protocols, vol. 2, p. 1508, 06/14/online 2007.
[21] S. Fang, H. J. Lee, A. W. Wark, and R. M. Corn, "Attomole Microarray Detection of MicroRNAs by Nanoparticle-Amplified SPR Imaging Measurements of Surface Polyadenylation Reactions," Journal of the American Chemical Society, vol. 128, no. 43, pp. 14044-14046, 2006/11/01 2006.
[22] L. H. Koole et al., "Synthesis of phosphate-methylated DNA fragments using 9-fluorenylmethoxycarbonyl as transient base protecting group," The Journal of Organic Chemistry, vol. 54, no. 7, pp. 1657-1664, 1989/03/01 1989.
[23] W. H. A. Kuijpers, J. Huskens, L. H. Koole, and C. A. A. van Boeckel, "Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent," Nucleic Acids Research, vol. 18, no. 17, pp. 5197-5205, 1990.
[24] M. H. P. G. L.H. Koole, van, R.G. Reiniers, H.M. Buck, "Enhanced stability of a Watson and Crick DNA duplex structure by methylation of the phosphate groups in one strand," 1987.
[25] M. H. P. van Genderen, L. H. Koole, and H. M. Buck, "Hybridization of phosphate-methylated DNA and natural oligonucleotides. Implications for protein-induced DNA duplex destabilization," Recueil des Travaux Chimiques des Pays-Bas, vol. 108, no. 1, pp. 28-35, 1989.
[26] 陳奕儒, "探討中性DNA與一般DNA雜交反應熱力學與結合機制之研究(Studies of thermodynamic and mechanism for neutralized DNA (nDNA)/DNA and DNA/DNA duplex formation)," 2016.
[27] L. Mu, Y. Chang, S. D. Sawtelle, M. Wipf, X. Duan, and M. A. Reed, "Silicon Nanowire Field-Effect Transistors—A Versatile Class of Potentiometric Nanobiosensors," IEEE Access, vol. 3, pp. 287-302, 2015.
[28] R. Bumgarner, "Overview of DNA microarrays: types, applications, and their future," (in eng), Current protocols in molecular biology, vol. Chapter 22, pp. Unit-22.1., 2013.
[29] K. L. Michael, L. C. Taylor, S. L. Schultz, and D. R. Walt, "Randomly Ordered Addressable High-Density Optical Sensor Arrays," Analytical Chemistry, vol. 70, no. 7, pp. 1242-1248, 1998/04/01 1998.
[30] D. R. Walt, "Bead-based Fiber-Optic Arrays," Science, vol. 287, no. 5452, p. 451, 2000.
[31] J. A. Ferguson, F. J. Steemers, and D. R. Walt, "High-Density Fiber-Optic DNA Random Microsphere Array," Analytical Chemistry, vol. 72, no. 22, pp. 5618-5624, 2000/11/01 2000.
[32] F. J. Steemers, J. A. Ferguson, and D. R. Walt, "Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays," Nature Biotechnology, vol. 18, no. 1, pp. 91-94, 2000/01/01 2000.
[33] S. Fodor, J. Read, M. Pirrung, L. Stryer, A. Lu, and D. Solas, "Light-directed, spatially addressable parallel chemical synthesis," Science, vol. 251, no. 4995, pp. 767-773, 1991.
[34] A. C. Pease, D. Solas, E. J. Sullivan, M. T. Cronin, C. P. Holmes, and S. P. Fodor, "Light-generated oligonucleotide arrays for rapid DNA sequence analysis," Proceedings of the National Academy of Sciences of the United States of America, vol. 91, no. 11, pp. 5022-5026, 1994.
[35] G. H. McGall, A. D. Barone, M. Diggelmann, S. P. A. Fodor, E. Gentalen, and N. Ngo, "The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates," Journal of the American Chemical Society, vol. 119, no. 22, pp. 5081-5090, 1997/06/01 1997.
[36] T. K. Karakach, R. M. Flight, S. E. Douglas, and P. D. Wentzell, "An introduction to DNA microarrays for gene expression analysis," Chemometrics and Intelligent Laboratory Systems, vol. 104, no. 1, pp. 28-52, 2010/11/15/ 2010.
[37] M. Dufva, "Fabrication of high quality microarrays," Biomolecular Engineering, vol. 22, no. 5, pp. 173-184, 2005/12/01/ 2005.
[38] J. B. Legutki, Z.-G. Zhao, M. Greving, N. Woodbury, S. A. Johnston, and P. Stafford, "Scalable high-density peptide arrays for comprehensive health monitoring," Nature Communications, Article vol. 5, p. 4785, 09/03/online 2014.
[39] S. Singh-Gasson et al., "Maskless fabrication of light-directed oligonucleotide microarrays using a digital micromirror array," Nature Biotechnology, vol. 17, no. 10, pp. 974-978, 1999/10/01 1999.
[40] P. Wang, "Photolabile Protecting Groups: Structure and Reactivity," Asian Journal of Organic Chemistry, vol. 2, no. 6, pp. 452-464, 2013.
[41] N. Kretschy, A.-K. Holik, V. Somoza, K.-P. Stengele, and M. M. Somoza, "Next-Generation o-Nitrobenzyl Photolabile Groups for Light-Directed Chemistry and Microarray Synthesis," Angewandte Chemie International Edition, vol. 54, no. 29, pp. 8555-8559, 2015.
[42] M. Sack et al., "Express photolithographic DNA microarray synthesis with optimized chemistry and high-efficiency photolabile groups," Journal of Nanobiotechnology, vol. 14, p. 14, 03/02
[43] D. Wöll et al., "More Efficient Photolithographic Synthesis of DNA-Chips by Photosensitization," Nucleosides, Nucleotides and Nucleic Acids, vol. 22, no. 5-8, pp. 1395-1398, 2003/10/01 2003.
[44] S. S. Wijmenga, M. Kruithof, and C. W. Hilbers, "Analysis of 1H chemical shifts in DNA: Assessment of the reliability of 1H chemical shift calculations for use in structure refinement," Journal of Biomolecular NMR, journal article vol. 10, no. 4, pp. 337-350, December 01 1997.
[45] S. Zhang and J. C. Chaput, "Synthesis of Glycerol Nucleic Acid (GNA) Phosphoramidite Monomers and Oligonucleotide Polymers," Current Protocols in Nucleic Acid Chemistry, vol. 42, no. 1, pp. 4.40.1-4.40.18, 2010.
[46] S. Zhang and J. C. Chaput, "Synthesis of Threose Nucleic Acid (TNA) Phosphoramidite Monomers and Oligonucleotide Polymers," Current Protocols in Nucleic Acid Chemistry, vol. 50, no. 1, pp. 4.51.1-4.51.26, 2012.
[47] 林耀能, "Phosphate-Methylated DNA as Neutralized DNA (nDNA)： Synthesis, Properties and Potential Applications " 2015.
[48] J. S. Hargreaves, R. Kaiser, and P. K. Wolber, "The Degradation of dG Phosphoramidites in Solution," Nucleosides, Nucleotides and Nucleic Acids, vol. 34, no. 10, pp. 691-707, 2015/10/03 2015.
[49] A. H Krotz et al., Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile. 2004, pp. 767-75.
[50] E. W. T. M. Rulka, "A combinatorial library, a method for preparation of that combinatorial library, a method for sequence identification, a method for sequencing the elements of combinatorial libraries of oligonucleotides and/or oligonucleotide analogues, the use of a linker to generate combinatorial libraries and a sequence identification set ", 2011.
[51] A. Roget, H. Bazin, and R. Teoule, "Synthesis and use of labelled nucleoside phosphoramidite building blocks bearing a reporter group: biotinyl, dinitrophenyl, pyrenyl and dansyl," (in eng), Nucleic acids research, vol. 17, no. 19, pp. 7643-7651, 1989.
[52] S. M. Gryaznov and R. L. Letsinger, "Selective O-phosphitilation with nucleoside phosphoramidite reagents," Nucleic Acids Research, vol. 20, no. 8, pp. 1879-1882, 1992.
[53] I. Aujard et al., "o-Nitrobenzyl Photolabile Protecting Groups with Red-Shifted Absorption: Syntheses and Uncaging Cross-Sections for One- and Two-Photon Excitation," Chemistry – A European Journal, vol. 12, no. 26, pp. 6865-6879, 2006.

指導教授

陳文逸(Wen-Yih Chen)

審核日期

2019-7-25

推文