激發態質子轉移之基礎研究與生物影像的應用

在我的論文中分成兩部分，第一部分為圍繞在激發態質子轉移的基礎研究與應用，第二部分為發光材料於細胞影像上的應用。第一部分: 激發態質子轉移的基礎研究與應用質子轉移可大致上分為兩類，一種是分子內本身就含有質子提供者與接受者所形成的分子內氫鍵。另外一種則是會跟溶劑(e.g water 、methanol)反應進行分子間的質子轉移。第一部分的三個章節就圍繞在這兩種質子轉移的研究上。第一章分子內激發態雙質子轉移之基礎研究分子內單質子轉移的研究已經非常的廣泛，在我的研究中主要想探討有沒有兩個或多個質子轉移的可能性。分子內雙質子轉移是屬於同步(concert)或是非同步(stepwise)反應一直是我們有興趣且廣泛被討論的。在此研究中我們首次利用1,8-Dihydroxy-2-naphthaldehyde (DHNA)此分子證實了非同步(stepwise)進行的雙質子轉移的可能性。當光激發分子後，第一個OH質子會快速進行質子轉移而得到520 nm的放光(TA*)，而第二個OH質子再接著進行第二次質子轉移得到650 nm 的放光(TB*)。在光譜動力學的研究下我們可以得知TA*跟TB*的質子轉移是可逆的。詳細的理論計算也支持我們的實驗結果。延續DHNA的研究，我們多引入一個醛基形成了一個對稱的結構(DHDA)，探討了氫鍵的方向性對於雙質子轉移的影響。這種結構使氫鍵的發生不再是單一方向。我們從光譜上可判斷出對稱結構(DHDA-23_OO)與不對稱結構(DHDA-23_OI)在ground state形成平衡。當受光激發後，DHDA-23_OI跟DHNA的情形一樣可以進行stepwise 的雙質子轉移而分別放出~500 nm及660 nm的tautomer放光，而對稱結構的DHDA-23_OO則不進行質子轉移而產生很強的Normal放光(450 nm)。在單晶結構下其中一個醛基的氧原子會跟另一分子的醛基的氫形成的氫鍵影響而使得結構不會形成對稱的DHDA-23_OO，故光譜中可很明顯的看出stepwise雙質子轉移的特徵放光。第二章 N-H 類型之分子內激發態質子轉移之基礎研究及細胞影像上的應用近年來我們實驗室發展了一系列NH 類型的激發態分子內質子轉移(ESIPT)的研究，我們知道NH 質子的酸度(pKa~31)比一般OH質子的酸度(pKa~18)要弱上許多。我們嘗試利用2-(2′-aminophenyl)-benzothiazole (ABT)當作發光團，將胺基上的其中一個質子修飾成不同拉電子性質的取代基，藉此改變另一個質子的酸度使其在激發態時可進行質子轉移。我們知道ESIPT的分子容易受外在溶劑的影響，尤其是在水中，質子容易受水的氫鍵影響而不形成tautomer。我們將其修飾上不同鏈長的Ethylene Glycol，使其在高於臨界微胞濃度下可以形成micelles而降低了水干擾而進行ESIPT並將其應用在細胞影像上。另外我們研究了一系列新穎的NH 類型2-(imidazo[1,2-a]pyridin-2-yl)aniline (PIP)。PIP 系列的樣品在照光後會形成Localized Zwitterionic Tautomer，當我們將胺基上的其中一個質子取代成不同拉電子性質的取代基，在拉電子性質越強可發現樣品吸收與tautomer的放光會逐漸藍移。PIP結構與ABT類似，但研究其質子轉移速率發現其與一般形成semi-conjugate tautomer的樣品(例如: ABT系列)不同。當其修飾上越高拉電性質的取代基時質子轉移的速率則越慢。第三章分子間質子轉移應用於偵測蛋白質與胜肽結合時界面水環境的變化我們實驗室發展了色胺酸(Trp)的類似物(2,7-diazaTrp or 7-azaTrp)其可和水進行催化而進行分子間質子轉移。這些類似物的生物相容性佳，可以簡單的將我們有興趣的蛋白質中的色胺酸取代成我們的樣品，藉此偵測蛋白質微環境的水變化。而我的研究中，我將胜肽(calmodulin binding peptide)中的色胺酸取代成2,7-diazaTrp或是7-azaTrp藉此觀察其和攜鈣蛋白(calmodulin)結合後的界面水環境的變化，結合這兩個探針的結果，發現當胜肽與攜鈣蛋白結合後是無水但高極性的環境。第二部分: 發光材料於細胞影像上的應用第四章利用新穎的發光材料研究細胞的微環境黏度的變化延續在生物影像的應用，我們知道疾病與細胞中黏度的變化息息相關。我們發展了一系列樣品(N,N′-disubstituted-dihydrodibenzo[a,c]phenazines)其在基態時呈現彎曲的結構而經光激發後構型會改變形成平面的結構而放出紅光。此新穎的材料不受外在極性的變化而有很大的放光波長位移，但在改變環境黏度時，構型的改變會受黏度變化影響使得光譜會呈現ratiometric的放光變化。此優點在應用於偵測黏度的變化時不會受環境極性變化的干擾且可藉由顏色改變，例如從藍到紅，來判別不同位置的黏度是其他偵測黏度的光學探針無法做到的。

關鍵字

激發態雙質子轉移；胺類質子轉移；類色胺酸；細胞影像；黏度

並列摘要

Part I: Chapter 1 Portion I: Optically Triggered Stepwise Double Proton Transfer in an Intramolecular Proton Relay: A Case Study of 1,8-Dihydroxy-2-naphthaldehyde (DHNA). 1,8-Dihydroxy-2-naphthaldehyde (DHNA) having doubly intramolecular hydrogen bonds was strategically designed and synthesized in an aim to probe a long-standing fundamental issue regarding synchronous versus asynchronous double proton transfer in the excited state. In cyclohexane, DHNA shows the lowest lying S0 S1 (*) absorption at ~400 nm. Upon excitation, two large Stokes shifted emission bands maximized at 520 nm and 650 nm are resolved, which are ascribed to the tautomer emission resulting from the first and second proton transfer products, denoted by TA* and TB* respectively. The first proton transfer (DHNA*TA*) is ultrafast (< system response of 150 fs), while the second proton transfer is reversible, for which the rates of forward (TA*  TB*) and backward (TA*  TB*) proton transfer were determined to be (1.7 ps)1 and (3.6 ps)1, respectively. The fast equilibrium leads to identical population lifetimes of ~54 ps for both TA* and TB* tautomers. A comprehensive 2-D plot of reaction potential energy surface further proves that the sequential, two-step proton motion is along the minimum energetic pathway, firmly supporting the experimental results. Chapter 1 Portion II: A Study of Competitive Multiple Hydrogen Bonding Effect and Its Associated Excited-State Proton Transfer Tautomerism. 1,8-Dihydroxynaphthalene-2,7-dicarbaldehyde (DHDA) has been strategically designed and synthesized in a aim to study the competitive multiple hydrogen bonding (H-bonding) effect and the associated excited-state intramolecular proton transfer reaction (ESIPT). In nonpolar solvents such as cyclohexane, equilibrium exists between two H-bonding isomers DHDA–23_OO and DHDA–23_OI, which all possess double intramolecular H-bonds. In polar, aprotic solvents such as CH2Cl2, DHDA–23_OO becomes the predominant species. Due to various degrees of H-bond induced changes of electronic configuration each isomer reveals distinct absorption feature and excited-state behavior, in which DHDA–23_OI in cyclohexane undergoes double ESIPT in a stepwise manner, giving the first and second proton-transfer tautomer emissions maximized at ~500 nm and 660 nm, respectively. As for DHDA–23_OO both single and double ESIPT are prohibited, resulting in an intense normal 450 nm emission band. In a single crystal DHDA–23_OI is the dominant species, which undergoes excited state double proton transfer, giving intense 530 nm and 650 nm emission bands. The mechanism associated with competitive multiple H-bonding energetics and ESIPT was underpinned by detailed spectroscopy/dynamics and computational approaches. Chapter 2 Portion I: Ethylene Glycol Modified 2-(2′-Aminophenyl)Benzothiazoles at The Amino Site: The Excited-State N-H Proton Transfer Reactions in Aqueous Solution, Micelles and Potential Application in Live-Cell Imaging. Triethylene glycol monomethyl ether and poly(ethylene glycol) monomethyl ether modified 2-(2’-aminophenyl)benzothiazoles, varied by different chain length of poly(ethylene glycol) (ABT-PnEG, n=3,7,12), were synthesized to probe their photophysical and bio-imaging properties. In polar, aprotic solvents such as CH2Cl2 ultrafast excited-state intramolecular proton transfer (ESIPT) takes place, resulting in a large Stokes shifted tautomer emission in the green-yellow (550 nm) region. In neutral water, ABT-P12EG forms micelles with diameters of 153 nm under a critical micelle concentration (CMC) of ~80 µM, in which the tautomer emission is greatly enhanced free from water perturbation. Live-cell imaging experiments were also performed, the results indicate that all ABT-PnEGs are able to enter HeLa cells and in part penetrate to the nuclei, adding an additional advantage for the cell imaging. Chapter 2 Portion II: A New Class of N–H Excited-State Intramolecular Proton Transfer (ESIPT) Molecules Bearing Localized Zwitterionic Tautomer. A series of new amino (NH)-type intramolecular hydrogen-bonding (H-bonding) compounds have been strategically designed and synthesized. These molecules comprise a 2-(imidazo[1,2-a]pyridin-2-yl)aniline moiety, in which one of the amino hydrogens was replaced with substituents of different electronic property. This, together with the versatile capability for modifying the parent moiety, makes feasible the comprehensive spectroscopy and dynamics studies of excited-state intramolecular proton transfer (ESIPT) as a function of N-H acidity. Different from other (NH)-type ESIPT systems where ESIPT rate and exergonicity increase as increasing the N-H acidiy and hence the H-bonding strength, the results reveal irregular relationship among ESIPT dynamics, thermodynamics and H-bond strength. This discrepancy may be rationalized by the localized zwitterionic nature of 2-(imidazo[1,2-a]pyridin-2-yl)aniline in the proton-transfer tautomer form, which is different from the -delocalized tautomer form in other (NH)-type ESIPT systems. Chapter 3 Probing the Polarity and Water Environment at The Protein-Peptide Binding Interface Using Tryptophan Analogues. 7-Azatryptophan and 2,7-diazatryptophan are sensitive to polarity changes and water content, respectively, and should be ideal for studying protein-protein and protein-peptide interactions. In this study, we replaced the tryptophan in peptide Baa (LKWKKLLKLLKKLLKLG-NH2) with 7-azatryptophan or 2,7-diazatryptophan, forming (7-aza)Trp-Baa and (2,7-aza)Trp-Baa, to study the calmodulin (CaM)-peptide interaction. Dramatic differences in the (7-aza)Trp-Baa and (2,7-aza)Trp-Baa fluorescence properties between free peptide in water and calmodulin-bound peptide were observed, showing a less polar and water scant environment at the binding interface of the peptide upon calmodulin binding. Part II: Luminescent Materials in Cell Image Application. Chapter 4 Using Novel Butterfly-Like Molecule to Probe the Intracellular Microenvironment. In biological specimen, changes in viscosity have been linked to disease and malfunction at the cellular level. Microscale measurements of the viscosity change remain a challenge. Until now, viscosity maps of single cells have been hard to obtain. Taking the advantage of ratiometric spectral imaging, we develop a new butterfly-like molecule that revealing significant nonplanar distortions (i.e., a saddle shape) and remarkably large Stokes-shifted emission independent of the solvent polarity. These unique photophysical behaviors are rationalized by electronic configuration coupled conformation changes en route to the geometry planarization. Taking this advantage in mind, we can exclude the polarity change to probe different microenvironment.