利用飛秒時間解析可見/紅外吸收光譜法研究細菌視紫質和綠色螢光蛋白發光團衍生物之光激發動態學

Abstract

本論文主要利用瞬態可見/紅外吸收光譜法以及螢光上轉移技術，研究細菌視紫質分子之視黃醛以及綠色螢光蛋白發光團在不同溶劑中之光激發動態學。 吾人利用飛秒時間解析可見光光譜儀，以波長570 nm雷射光激發紫膜懸浮溶液以及分別加入不同表面活性劑後之紫膜懸浮溶液，量測細菌視紫質分子於偵測波長460 nm (中間體I)之吸收度隨時間的變化情形。由結果可知，當加入離子性表面活性劑(CTAB和SDS)後，中間體I之生命期變長約20 %；但加入中性表面活性劑(C6E2)後之生命期僅增加約3 %，在量測誤差範圍內。而由細菌視紫質分子之電子吸收光譜、螢光光譜和可見光圓二色光譜得知，在加入表面活性劑後，細菌視紫質分子仍為三聚體結構，但部分在紫膜中之脂質已被表面活性劑移除。由於部分脂質被表面活性劑移除後，吾人推測細菌視紫質分子之結構已改變；而此構形的變化可能造成中間體I之生命期變長。 吾人利用螢光上轉移技術和實驗室自行搭建之時間解析紅外光吸收光譜儀，以400 nm雷射光激發對位胺基取代之綠色螢光蛋白發光團衍生物p-ABDI以及p-CFABDI，量測在質子性(CH3OH和CD3OD)及非質子性(CD3CN)溶劑中之螢光生命期和瞬態紅外吸收光譜。此外，結合楊吉水教授量測其在不同溶劑中之Z→E光異構化量子產率以及螢光量子產率之資訊以了解其光激發動態學。由量測結果得知，p-ABDI在非質子性溶劑中之Z→E光異構化量子產率(0.5)以及基態回復時間常數(18.3 ps)較在質子性溶劑中大(0.17，7.3 ps)，但其螢光生命期在不同溶劑中大致相同(< 1 ps)；而p-CFABDI在不同溶劑中量測之結果與p-ABDI相近。根據上述量測之結果以及單一雙鍵扭轉(OBF)之光異構化反應機制，吾人推測p-ABDI和p-CFABDI在質子性以及非質子性溶劑中主要以C=C雙鍵扭轉之方式進行非輻射衰減過程。其中在質子性溶劑中，分子雙鍵扭轉位能面受到溶劑-溶質間氫鍵效應(SSHB)的影響，在小於90度扭轉角即可發生內轉換回到基態。因此，SSHB效應造成Z→E光異構化量子產率的減少以及加快分子回到基態之時間，但對於螢光生命期並無明顯的影響。分子以內轉換的形式形成振動激發的電子基態後，其振動激發弛緩速率與溶劑之質子性質有關。 利用相同之瞬態光譜法量測m-ABDI在不同溶劑中經400 nm雷射光激發至電子激發態之螢光生命期和瞬態紅外吸收光譜，並且配合其螢光量子產率和Z→E光異構化量子產率來研究m-ABDI之光激發動態學。於氘取代乙腈溶劑中，其總量子產率約為1.0。吾人在1613、1557、1531和1513 cm−1觀測到m-ABDI之激發態吸收譜帶，其衰減時間常數(> 5 ns)與其在600 nm之螢光衰減時間常數為7.9 ns一致。於甲醇溶劑中，m-ABDI之總量子產率僅為0.16；其瞬態螢光譜帶之積分強度的衰減曲線，可利用雙指數衰減函數適解得到~16 ps以及~62 ps之螢光衰減時間常數。此外，吾人觀測到在1608、1560、1531和1512 cm−1有四個正吸收，其衰減時間常數約為15.5 ps；而隨著上述四個譜帶的消失，在1592、1554、1532和1513 cm−1位置觀測到新譜帶的生成，其生成時間常數為15.5 ps，而衰減時間常數為~58 ps。再者，於氘取代甲醇溶劑中，吾人亦觀測到在1615、1557、1530和1515 cm−1之正吸收於雷射光激發後，強度即達最大值，其後以衰減時間常數為16.0 ps衰減至零；隨著上述四個譜帶的消失，在1596、1553、1528和1512 cm−1亦觀測到新生成之譜帶，其生成時間常數為16.0 ps，而衰減時間常數為~247 ps。此結果表示m-ABDI在質子性溶劑中於電子激發態生成新的中間體。吾人以DFT和TDDFT理論計算m-ABDI與溶劑形成之氫鍵錯合物和質子化之陽離子分子所得之電子基態和激發態振動波數為輔助，進行譜帶之指派。根據上述之結果，吾人推測m-ABDI在氘取代乙腈溶劑中是以C=C雙鍵扭轉進行Z→E光異構化反應回到電子基態；而在甲醇和氘取代甲醇溶劑中，因溶劑-溶質間氫鍵作用力的影響，可能誘發m-ABDI於電子激發態生成質子轉移中間體，與C=C雙鍵扭轉之反應競爭，加快電子由激發態回到基態之時間，造成其Z→E光異構化量子產率的減少。

In this thesis, the ultrafast photochemical reactions, such as cis-trans photoisiomerization or proton/electron transfer process, of bacteriorhodopsin and GFP-like chromophores in varied solvents were investigated with pump-probe transient absorption technique in visible and infrared spectral regions and fluorescence up-conversion technique. The dynamics of the reactive excited-state of bacteriorhodopsin treated with various surfactants have been investigated through the measurement of visible pump-probe absorption spectra. Upon excitation of bR at 570 nm in the presence of ionic surfactants CTAB and SDS, the lifetime of the reactive excited-state of the all-trans protonated Schiff base, probed at 460 nm, was observed to increase up to 20 %, whereas an insignificant change was observed upon addition of neutral surfactant C6E2. Measurements of steady-state absorption spectra, fluorescence, and circular dichroism indicated that the bR suspensions retain their trimeric configuration with partial delipidation. This removal of lipids causes a structural alteration and the varied excited-state dynamics. The excited state dynamics of GFP-like chromophores p-ABDI and p-CFABDI in protic and aprotic solvents have been investigated through the measurements of fluorescence lifetimes and visible-pump/infrared-probe absorption spectra. In combination with the reported quantum yields of the Z→E isomerization and fluorescence, three data were used to investigate the nonradiative decay mechanism of two molecules in selected solvents. When CD3CN was replaced with CH3OH, the observed isomerization yield and time constants of ground-state recovery of p-ABDI decreased from 0.50 to 0.17 and 18.3 to 7.3 ps, respectively, but similar fluorescence lifetimes (< 1 ps) were observed in all solvents. Similar results were observed for p-CFABDI in varied solvents. According to those observations and one-bond-flip mechanism, we propose that the nonradiative decay of p-ABDI and p-CFABDI is governed by the t-torsion in both protic and aprotic solvents, but in protic solvent, such as CH3OH, the solvent-solute hydrogen binding (SSHB) interactions perturbs the potential energy surface (PES) for the t-torsion in a way that the internal conversion (IC) occurs at a distribution of t-torsion angle smaller than 900. Therefore, the SSHB diminishes the quantum yields for the Z→E isomerization and fastens the recovery of ground state, but has a negligible effect on the fluorescence quenching kinetics. The IC results in a hot ground electronic state that exhibits solvent-dependent cooling dynamics. The excited-state dynamics of meta-amino analogue of GFP chromophore m-ABDI in selected solvents were also investigated with time-resolved fluorescence and transient infrared absorption. The quantum yields of the Z→E isomerization and fluorescence have been measured for m-ABDI in selected solvents. For solutions in CD3CN, the total isomerization yield is 1.0 and the fluorescence decay lifetime is ~7.9 ns. The IR absorption lines near 1513, 1531, 1557, and 1613 cm-1 of m-ABDI in its electronically excited state were observed with a decay time > 5 ns. For solutions in CH3OH, the yields decrease to 0.16 and the fluorescence decay is double exponential with time constants ~16 and 62 ps. The IR absorption lines near 1512, 1531, 1560 and 1608 cm-1 appeared immediately upon excitation, followed by a decay with a time constant of 15.5 ps. New features near 1513, 1532, 1554 and 1592 cm-1 were observed to have a rise time of 15.5 ps and a decay constant of ~58 ps, indicating formation of an intermediate. Furthermore, for solutions in CD3OD, new features near 1512, 1528, 1553 and 1596 cm−1 were also observed to have a rise time of 16 ps and a decay constant of ~247 ps. The assignments for the IR spectra of the ground- and excited-states were assisted with DFT and TDDFT calculations, respectively. We conclude that the t-torsion of the exocyclic C=C bond is responsible for the nonradiative decay of electronically excited m-ABDI in CD3CN. In contrast, in CH3OH and CD3OD, formation of a reaction intermediate competes successfully with the t torsion, consistent with the diminished yield observed for Z→E photoisomerization; this intermediate is likely associated with excited-state proton transfer (ESPT) from the solvent to m-ABDI.