發展氧化還原應答型比例螢光探針並應用於偵測生物硫醇

Abstract

含有硫醇基的小分子如半胱胺酸 (Cysteine)、高半胱胺酸 (Homocysteine)、穀胱甘肽 (Glutathione) 在生理上的濃度若有異常現象發生時，通常與特定疾病有密切的關聯性。因此為了能提早診斷疾病，即需發展一個能有效偵測生物硫醇的探針。本論文發展兩個基於氧化還原應答型比例螢光變化的探針AO-3-DSF、TF-AO-3-DSF。探針選用的螢光團為具雙放射特性的3-羥基黃酮，當在3號位置以碳酸酯引入一個能專一性被硫切斷的基團乙基-雙硫鍵，使探針螢光放射以電荷轉移之藍色螢光為主。待與生物硫醇作用後，先進行硫交換反應隨即引發分子內合環，分別釋出產物AO-3-OHF、TF-AO-3-OHF，此時的螢光放射則會改以質子轉移之綠色螢光為主。值得一提的是在3-羥基黃酮的2’號位置引入一個三氟甲基，不僅可以增加探針的水溶性，亦能調節TF-AO-3-DSF在水中能以ESIPT的螢光放射為主，使探針在生理環境中與生物硫醇反應前後的螢光，能具有顯著的改變。探針AO-3-DSF與TF-AO-3-DSF在穀胱甘肽濃度於正常至異常範圍內 (2 uM至10 mM)，均展現不同程度的比例螢光變化，前後比例螢光變化最高分別可達97倍與69倍，最低偵測極限分別可達0.013 uM與0.61 uM，因此能有效區分穀胱甘肽是否已達病理水平。此外，探針AO-3-DSF與TF-AO-3-DSF對於生物硫醇均具有優良的反應性，且不會受其他胺基酸與生理常見的物質影響。最後共軛焦雷射細胞顯影實驗顯示探針TF-AO-3-DSF能成功進入海拉細胞，並偵測細胞內的生物硫醇。

Biothiols including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) play pivotal roles in pathological cells. The abnormal level of intracellular biothiols is commonly associated with diseases. Therefore, developing an efficient fluorescent probe to detect biothiols is beneficial for the early diagnosis of diseases. Herein, we report two redox-responsive ESIPT-based ratiometric fluorescent probes denoted by AO-3-DSF and TF-AO-3-DSF, respectively, in which ESIPT/ESICT fluorophore 3-hydroxy flavone (3-HF) is chosen as a dual-colored reporter. Thio-specific cleavable ethyl-disulfide group is introduced to the hydroxyl group of flavone probe at the 3-position to block ESIPT pathway at the initial state exclusively giving blue ESICT fluorescence. Upon exposure to biothiols, AO-3-DSF and TF-AO-3-DSF undergo the thiol exchange reaction followed by an intramolecular cyclization to generate demasked AO-3-OHF and TF-AO-3-OHF, respectively, resulting in a green ESIPT-dominative fluorescence. It is noteworthy that the introduction of trifluoromethyl group on 2’-position of 3-HF fluorophore not only effectively enhances the water solubility, but also rationally modulate TF-AO-3-DSF to maintain proton transfer band in aqueous media. Therefore, when TF-AO-3-DSF reaction with biothiols under biological environment, it can demonstrate a significant change of fluorescence. Both of AO-3-OHF and TF-AO-3-DSF show blue-to-green ratiometric detection of GSH with a 97- and 69-fold enhancement, respectively, and thus successfully distinguished between normal and pathological levels (2 uM to 10 mM) of GSH with a detection limit of 0.013 uM and 0.61 uM, respectively. Furthermore, AO-3-OHF and TF-AO-3-DSF both displayed remarkable reactivity toward biothiols over other amino acids. Confocal imaging show that TF-AO-3-DSF can get into HeLa cell and detect intracellular biothiols.