次硝酸及半胱胺酸螢光化學感測分子之合成與應用

Abstract

本論文內容是設計合成出兩個新穎的螢光感測分子BDPB與ANP，分別可用來偵測次硝酸 (HNO) 和生物硫醇分子 (biothiol)。 化合物 BDPB 是在發光團 HPBI 上修飾 2-(diphenyl- phosphino)-benzoate 基團，以此作為 HNO 辨識端，當環境中存在 HNO 時會觸發裂解反應，2-(diphenylphosphino)benzoate 基團會脫去還原成 HPBI，而 HPBI 本身具有 ESIPT 效應，會放出強烈的藍色螢光 (QY = 0.459)。而相較於其他的生物性相關物質，BDPB 對 HNO 具有良好的選擇性，且偵測極限可達到 18.5 nM (S/N = 3)。此外，在HeLa cells 的螢光顯影實驗中，成功觀察到 BDPB 對 HNO 的偵測能力，並由細胞毒性檢測得知 BDPB 對細胞不太具有毒性。 化合物 ANP 是以 6-acetyl-N-methyl-2-naphthylamine 為基底，在其酮基的位置與 2-pyridinecarbaldehyde 縮合作為偵測硫醇的辨識端。ANP 在胺基酸中對 Cys 和 Hcy 具有選擇性，其中，又以 Cys 的反應性為佳。進一步研究 ANP 與 Cys 反應後的產物，發現 Cys 會與 ANP 上 unsaturated ketone 進行 Michael addition，導致共軛系統改變，產生強烈的綠色螢光。此外，化合物本身具有能夠經由雙光子進行激發之特性，因此，在子宮頸癌細胞的螢光顯影實驗中，分別經由單光子以及雙光子去進行激發，皆成功觀察到化合物 ANP 在細胞內對 Cys 的偵測能力。

In this thesis, two novel chemosensors were designed and synthesized that could be used to detect nitroxyl and biothiols, respectively. A new fluorescent probe BDPB based on the excited state intramolecular proton transfer (ESIPT) mechanism for specific detection of nitroxyl (HNO) was designed and synthesized. BDPB displays a weak fluorescence ( = 0.014). Upon addition of HNO, the cleavage of the carboxylate group of the 2-(diphenylphosphino)benzoate moiety to make the probe BDPB back to the HPBI which emits strong fluorescence (QY = 0.459). BDPB is highly selective to HNO compared to other biologically relevant species. It also shows high sensitivity to HNO with low detection limit, 18.5 nM (S/N = 3). In addition, BDPB is successfully applied to fluorescent imaging in HeLa cells. According to cytotoxicity test, we found that BDPB is low toxicity for cells. A new fluorescent probe ANP for detection of biothiols is designed and synthesized using 6-acetyl-N-methyl-2-naphthylamine with an attached pyridine group. ANP is selective to Cys and Hcy among amino acids, but it exhibites higher sensitive to Cys than Hcy. Further studies show that Cys undergoes Michael addition through the thiol group to unsaturated ketone. The product emits strong fluorescence enhancement at emission peak 508 nm. Moreover, the probe ANP exhibits two-photon excited fluorescence. And it is successfully used to detect Cys in HeLa cells by one-photon and two-photon fluorescence microscopy.