一氧化氮與半胱胺酸/高半胱胺酸螢光探針之設計、合成及其細胞造影之研究

Abstract

分子影像技術可提供疾病在細胞層級與分子層級變化之研究能力，而分子影像技術之一的螢光造影技術由於具有儀器價格低廉、靈敏度高及操作簡單等優勢，因此被廣泛使用。在螢光造影中，為了要增加對目標分子造影的特異性，螢光探針的開發是至關重要的。本論文闡述了螢光探針的組成、訊號傳遞原理及探針設計策略的基本概念，並配合實例說明。本論文是由一氧化氮螢光探針及半胱胺酸/高半胱胺酸螢光探針兩部分所組成。在論文的第一部份發展了RH及FA-OME兩個一氧化氮螢光探針，期望對一氧化氮進行造影。RH的設計策略是基於一氧化氮誘導其結構上spirolactum進行開環，而造成螢光訊號的出現。當RH與一氧化氮反應後，可獲得大約1000倍的螢光上升倍數，明顯高於目前商業化的探針DAF-2。另一方面，FA-OMe的設計策略是基於一氧化氮誘導芳香族一級單胺化合物進行脫胺反應，進而抑制光誘導電子轉移效應，促使螢光訊號上升。在本研究中，利用密度泛函理論計算並支撐FA-OMe結構上的光誘導電子轉移效應。此外，FA-OMe成功地改善了DAF-2容易受抗壞血酸及脫氫抗壞血酸影響之缺點，提高對一氧化氮的專一性。由濃度效應研究可看出此二探針的螢光強度對一氧化氮濃度有極高的線性關係，RH及FA-OMe對一氧化氮的偵測極限分別為20及44 nM。此二探針在生理環境的pH值下具有高度的穩定性，並且能成功地進行細胞內一氧化氮的螢光造影。在論文的第二部份則是發展了半胱胺酸/高半胱胺酸的螢光探針NBD-SCN，當探針上的−SCN被半胱胺酸/高半胱胺酸所取代，能提高探針分子內電荷轉移特性而使螢光強度上升。NBD-SCN與半胱胺酸及高半胱胺酸作用分別產生470倍及745倍的螢光上升，而偵測極限則分別是2.99及1.43 nM。在時間效應研究中可發現NBD-SCN對半胱胺酸反應極快，只須20秒即可令螢光強度達到飽和狀態，此速率優於文獻上大部分的硫醇探針；對高半胱胺酸則需要10分鐘。最後，本研究也成功地利用NBD-SCN對Raw 264.7巨噬細胞中的硫醇進行螢光造影。

Molecular imaging provides the ability to study cellular and molecular processes that have the potential to impact many facets of biomedical research. Fluorescence imaging, one technique of molecular imaging, is generally superior in terms of sensitivity, low-price, and ease of use. To promote the specificity of the target molecule, the developments of fluorescent probes is essential. In this thesis, the basic concepts including components, signal transduction principles, and design strategies of fluorescent probes were illustrated. This thesis is composed of two parts, nitric oxide fluorescent probes and cysteine/homocysteine fluorescent probe. In the first part of this thesis, two fluorescent probes, RH and FA-OMe, were developed for the visualization of nitric oxide (NO). The design strategy of RH is based on the NO-induced spirolactam ring opening. The ring opening reaction gives rise to strong fluorescence emission. RH possesses a ~1000-fold fluorescence turn-on from a dark background, which is higher than that of the commercialized probe, DAF-2. In the other hand, FA-OMe is designed by the concept of NO-induced reductive deamination of aromatic primary monoamine. After reacting with NO, the photoinduced electron transfer (PeT) effect is suppressed due to the disappearance of the electron donating amino group, and the fluorescence is restored. The PeT effect was demonstrated by density functional theory (DFT) calculations of the components forming FA-OMe and dA-FA-OMe. FA-OMe successfully improves the shortcoming of the condensation of o-phenylenediamine moiety by dehydroascorbic acid and ascorbic acid. Compared to the DAF-2, FA-OMe is more specific to NO. The concentration-dependent studies of these two probes showed an excellent linearity between fluorescence intensities and NO concentrations. The detection limit of RH and FA-OMe for NO is 20 and 44 nM, respectively. Furthermore, both probes are highly stable at physiological pH, and can be applied to monitor the endogenous nitric oxide. In the second part of this thesis, a push-pull fluorogenic reagent, NBD-SCN, was developed and applied for specific detection of cysteine (Cys) and homocysteine (Hcy). Replacing thiocyanato group with Cys/Hcy increased the intramolecular charge transfer (ICT) characteristic of the probe and resulted in emission of fluorescence. The fluorescent response of the probe toward Cys/Hcy was significantly higher than toward glutathione and other amino acids. The probe showed 470- and 745-fold fluorescence enhancement at 550 nm and detection limit of 2.99 and 1.43 nM for Cys and Hcy, respectively. Time-dependent fluorescence assays showed that the fluorescence intensity reached a plateau within 20 seconds after addition of Cys and within 10 minutes after addition of Hcy. Furthermore, the fluorescence images of biothiol in Raw 264.7 cells were obtained successfully by the employment of NBD-SCN.