- 學位論文
Bis(1-methyl-3-vinylpyrazinium-4-phenyl) amine diiodide與寡核苷酸作用之共振拉曼光譜研究
Investigation of the Interactions Between Bis(1-Methyl-3-Vinylpyrazinium-4-Phenyl) Amine Diiodide and Oligonucleotides by Resonance Raman Spectroscopy
摘要
Bis(1-methyl-3-vinylpyrazinium-4-phenyl) amine diiodide (BMVPA4)為一新開發與DNA作用之藥物分子，由於BMVPA4螢光強度微弱，所以適合以共振拉曼進行研究。BMVPA4被發現會選擇性針對連續的AT鹼基序列作用，並可能以minor groove binding形式與雙股螺旋DNA作用。因BMVPA4共振拉曼光譜振動譜峰的研究不完整，且此分子對不同DNA構形會有選擇性作用，我們希望藉由光譜訊號改變並搭配理論計算進而了解BMVPA4與DNA四重及雙股結構作用形式。 我們發現BMVPA4與人類端粒的四重結構d[TAGGG(TTAGGG)3](Hum23)及線性雙股螺旋d[(GC)2AATT(GC)2](LD12)，兩種不同DNA構形混合後其吸收光譜產生不同程度的變化：BMVPA4與Hum23作用後480 nm的吸收譜峰紅移27 nm；而BMVAP4與LD12作用後則紅位移67 nm。表示LD12影響BMVPA4的電子能階較大。此外，我們將理論計算一般拉曼振動正交模式(normal modes)對應於實驗共振拉曼光譜得知BMVPA4各個譜峰振動模式。分析BMVPA4/LD12共振拉曼光譜，發現BMVPA4上的吡嗪(pyrazine)基團有關的振動譜峰會有強度或頻率上的變化(如：941 cm-1、1027 cm-1、1184 cm-1、1329 cm-1、1545 cm-1等譜峰)。由於吡嗪基團帶正電，且BMVPA4分子角度及長度符合LD12的minor groove寬度，又minor groove寬度(3-4 Å)較窄，帶負電的磷酸骨架能以靜電作用力將BMVPA4夾在其中。另一方面，由於BMVPA4/Hum23共振拉曼光譜變化較無BMVPA4/LD12來的明顯，且Hum23的groove最窄處約5-6 Å，所以推論BMVPA4可能橫跨在Hum23的磷酸骨架上。 接著我們探討BMVPA4是否會與水生成氫鍵。藉由一系列的非質子性(aprotic)溶劑實驗，由共振拉曼光譜發現BMVPA4可能形成氫鍵的振動譜峰頻率變化不大，由此推測BMVPA4並不會與水形成氫鍵。 最後我們以不同波長的雷射激發BMVPA4不同電子能階，並比較共振拉曼光譜之變化。發現激發BMVPA4的第二個電子激態能階(378 nm)，與第一個電子激發能階(483 nm)相比，其含吡嗪基團的振動譜峰相對強度下降。這反應吡嗪的電子結構在第二個電子激發能階相對於電子基態的變化較第一電子激發態小。吸收光譜也反應這性質：當BMVPA4與LD12作用後，第一個電子激發能階位移程度大於第二個激發電子能階。 綜合上述討論，我們發現BMVPA4與LD12或Hum23作用皆是以靜電作用力方式結合，且未涉及氫鍵的生成。BMVPA4第一電子激態能階在吡嗪電子結構的變化較第二電子激態能階大，所以在與DNA混合時，第一電子能階能量曲面位移程度較大。
並列摘要
An AT-rich region of DNA often involved in DNA transcription and DNA-drug interactions. Thus a molecular probe that can recognize a particular DNA sequence is important. The previous research indicated bis(1-methyl-3-vinylpyrazinium-4-phenyl) amine diiodide (BMVPA4) selectively interacting with AT-rich region of DNA through minor groove binding. Since BMVPA4 is lack of fluorescence, we measured resonance Raman spectra of BMVPA4 complexed with a G-quadruplex d[TAG3(T2AG3)3] (Hum23) and a double helix d[(GC)2A2T2(GC)2] (LD12). In comparison with calculated Raman spectra by the density functional method, we attempted to identify the interacting moieties of BMVPA4 with these DNA sequences. The absorption peak of BMVPA4 at 480 nm shows a red shift of 27 nm when BMVPA4 mixed with Hum23 while the peak is red shifted 67 nm upon interacting with LD12. The difference resonance Raman spectrum of BMVPA4/LD12 relative to the BMVPA4 excited by 488 nm laser light shows more substantial deviations than the difference resonance Raman spectrum of BMVPA4/Hum23 relative to BMVPA4. We utilized the theoretical normal Raman modes to assign the experimental resonance Raman peaks. Several vibration frequencies of the pyrazine group are red shifted (e.g. 941、1027、1481、1545 cm-1 peaks) upon interacting with LD12. Some peaks intensity decreased. They are assigned as the vibrational modes of pyrazine group (e.g. 1027、1184 cm-1 peaks). These results reflect that the pyrazine groups play an important role in binding to LD12. The angle and length of the BMVPA4 structure allow BMVPA4 snugly fitted into the minor groove of the double helix of LD12. LD12 and BMVPA4 bind together through the electrostatic interaction. When BMVPA4 was mixed Hum23, the spectra varied slightly. We speculated that BMVPA4 combined with periphery phosphate backbone of Hum23. We also use several aprotic solvents to discussion hydrogen bond of BMVPA4. The resonance Raman spectra of BMVPA4 dissolved in different solvents showed no large shift of Raman peaks. So, we think BMVPA4 does not form the hydrogen bond with water. Finally, we use a different laser (405 nm) to excite the second electronic transition band (378 nm) of BMVPA4. One resonance Raman peak of about pyrazine group decreased in intensity (compared with 488 nm excited). The result indicated that the second excited electronic state has a similar potential surface relative to the electronic ground state along that pyrazine vibrational coordinate. In constrast, the first excited electronic transition state has a substantial change relative to the electronic ground state along this pyrazine vibrational mode.
參考文獻
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