Development Of Fluoroscent Probes With Double Recognition Factors And Their Applications In Biological Systems

Generally, a fluorescent probe contains only one recognition unit, and the change of the fluorescence signal was realized by single molecular recognition reaction. However, the above mentioned probes fail to discriminate those molecules with similar structure. In order to improve the selectivity of probes, this work took full advantage of the structural and chemical characteristics of analytes, and fluorescent probes with double molecular recognition factors have been developed, which could modulate the fluorescent signal of sensing system by the double molecular recognition reaction, and thus realized the specific detection for biological molecular.H2S and some biological thoil (Cys, Hcy and GSH) play crucial roles in many physiological and pathological processes, and abnormal levels of them are associated with a series of diseases. In order to unravel the complicated biomedical mechanisms and improve the cognition on related diseases, fluorescent probes with high sensitivity and selectivity are highly desirable.This dissertation synthesized fluorescent probe Ⅱ-1and Ⅲ-1with double recognition factors based the structural features of H2S and biological thoil. In addition, this work synthesized another environment-sensitive fluorescent probe Ⅳ-1. Upon conjugation to the bovine serum albumin(BSA) via a transthioesterification reaction, the fluorescence intensity of the sensing system increased dramatically. This paper is divided into four chapters as follows:In chapter1, a general introduction to the research progess on the fluorescent probe with double recognition factors was summarized. Based on referring to the relative literatures, the objective of this dissertation was proposed.In chapter2, a ratiometric fluorescent probe for H2S has been developed based on modulation of the excited-state intramolecular proton transfer (ESIPT) process of2-(2’-hydroxy-3’-methoxyphenyl)benzothiazole (HMBT). The probe was prepared by coupling HMBT with3,3’-dithiodipropionic acid, and it only displays enol-like fluorescence emission at374nm. However, upon introducing H2S in neutral solution, the protecting groups of the probe was removed via the tandem nucleophilic substitution/cyclization reaction, thereby retrieving the ESIPT process of HMBT, which resulted in a decrease of the emission band at374nm along with a concomitant increase of a newfluorescence peak at478nm. The fluorescent intensity ratio at478and374nm (I478/I374) increases linearly with H2S concentration in the range0.5—10μM. The proposed probe shows excellent selectivity toward H2S over other common anions and biothiols.In chapter3, we synthetic a thioester (Ⅲ-1) that responds to GSH and Cys/Hcy with distinct fluorescence emissions in neutral media. Probe Ⅲ-1reacts with Cys/Hcy to form the corresponding deconjugated spirolactam via a tandem native chemical ligation (NCL) reaction. This intramolecular spirocyclization leads to the "quinone-phenol" transduction of rhodol dyes, and an ESIPT process between the phenolic hydroxyl proton and the aromatic nitrogen in the benzothiazole unit occurs upon photoexcitation, thus affording2-(2’-hydroxyphenyl) benzothiazole (HBT) emission (454nm). In the case of the tripeptide GSH, only transthioesterification takes place removing the intramolecular photo-induced electron transfer (PET) process caused by the electron deficient4-nitrobenzene moiety giving rise to a large fluorescence enhancement at the rhodol emission band (587nm). The simultaneous detection of GSH and Cys/Hcy is attributed to the significantly different rates of intramolecular S,N-acyl shift of their corresponding thioester adducts derived from Ⅲ-1. The utility of probe Ⅲ-1has been demonstrated in various biological systems including serum and cells.In chapter4, an environment-sensitive fluorescent probe IV-1for BSA has been developed based on the unique spectroscopic behavior of compound IV-3. The probe was prepared by coupling fluorophore IV-3with2-mercaptobenzothiazole, and it exhibits very weak fluorescence in the aqueous buffer. However, upon introducing BSA in this solution, a transthioesterification reaction between probe IV-1and BSA would undergo rapidly. Therefore, the environment-sensitive fluorophore would closer to the hydrophobic pocket of BSA, and the proximity of the hydrophobic environment could cause the fluorophore to emit stronger fluorescence at470nm. The fluorescent intensity at470nm increases linearly with BSA concentration in the range2.5—70μg mL-1. The proposed probe shows excellent selectivity toward BSA over other common anions and biothiols.