Fluorescent probes for thiol and hydrogen sulfide detection in living cells

Abstract

Small molecule thiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play crucial roles in maintaining cellular redox environment and mitigating damages from free radicals and toxins. Recent research has showed that hydrogen sulfide (H2S) can be produced from sulfur-containing biomolecules such as Hcy and Cys. H2S has been linked to a series of diseases, including Alzheimer's disease, Down's syndrome, diabetes and liver cirrhosis. Consequently, development of highly sensitive and selective fluorescent probes for detecting thiols and H2S is intensely sought after because of their critical roles in physiological and pathological processes. After introducing the background on fluorescence, fluorescent probes for thiol and H2S detection were summarized in chapter 1. In chapter 2, a new fluorescent probe was developed for selective detection of thiols based on 1,4-Michael addition reaction of α,β-unsaturated acyl sulfonamide. The probe has good water solubility and reacts with thiols under aqueous conditions without using organic solvents and additional catalysts. More importantly, the probe could selectively detect thiols rather than other strong nucleophiles such as amines. We also proved that the probe could be applied to detect thiols in living cells. The probe was subsequently applied to detect intracellular thiols. In chapter 3, we proposed a novel strategy, namely, a fluorescent probe installed with dual-reactive and dual-quenching groups, for rational design of highly selective and sensitive fluorescent thiol probe. The principle of the strategy is as follow: 1) based on the dual-quenching effects, the fluorescence of the probe can be maximally inhibited, and the turn-on response upon thiol activation is much larger which leads to high sensitivity of the probe towards thiols; 2) based on the dual-reactive groups, the selectivity of the probe towards thiols is greatly improved, because the thiol detection can only be obviously interfered by molecules that can react with both two different reactive units. As a proof-of-concept study, we prepared such a new fluorescent thiol probe installed with dual-reactive and dual-quenching groups. The experimental results indicated that sensitivity of the probe towards thiols was significantly improved by dual-quenching effects, whereas selectivity of the probe was also greatly enhanced by installation of dual-reactive groups. Chapter 4 provides a novel and remarkably probe 4F-2CN, (tetrafluoroterephthalonitrile), which was discovered to be an efficient fluorescent probe for detecting thiol under physiological condition. Interestingly, fluorescent probe for detecting thiol under physiological condition. Interestingly, reacts with Hcy/GSH and generates blue fluorescence. Bioimaging experiments proved that Cys and Hcy/GSH can be selectively detected using two distinct mission channels. Addition of CTAB (cetyl trimethylammonium bromide) was observed to alter the fluorescent color of the reaction product of 4F-2CN and Hcy (from blue to green), but no alteration of fluorescent color occurred for Cys and GSH. Therefore the three commonly occurring thiols (Cys/Hcy/GSH) can be discriminated through the combined use of 4F-2CN and CTAB. Furthermore, with the use of CTAB, Cys, Hcy and GSH can be selectively detected using two distinct emission channels. In addition, the reaction product of 4F-2CN and Cys exhibits two-photon property, offering potentially useful tools for tissue imaging studies. Chapter 5 reports a novel fluorescent probe for detecting H2S based on iminocoumarin benzothiazole scaffold. The probe utilizes a H2S-induced cascade reaction consisting three consecutive steps: reduction, elimination and cyclization. The probe is highly sensitive and displayed around 80-fold increment in fluorescence signal after reacting with H2S under physiological condition. The probe also showed excellent selectivity towards H2S over other biologically relevant species including ROS, RSS and RNS. It displayed 32-fold of selectivity for H2S compared to that of other reactive sulphur species. Furthermore, the probe has been applied for imaging H2S in living cells. Cell imaging experiments demonstrated that the probe is cell-permeable and can be used to monitor the alteration of H2S concentration in living cells. Chapter 6 has developed a fluorescent probe based on rhodamin 110 to detect H2S in aqueous buffer at physiological pH. Because of the dual azide quenching effect, the probe exhibited high sensitivity for H2S detection. The fluorescence intensity increases by approximately 120-fold when H2S is added. The detection limit can be as low as 1.12×10-7 M. The probe is highly selective to H2S over other biologically relevant species. It displays 32-fold selectivity over reactive sulfur species. The probe also has good cell-permeability and can be used for live-cell imaging of H2S.

Date of Award	2 Oct 2015
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hongyan SUN (Supervisor)