Development Of Fluorescence Probes For Biologically Reactive Small Molecules And Application In Biological Systems

Biologically reactive small molecules play crucial roles in various physiological processes. For example, as the protonated form of well-known gaseous signal molecule nitric oxide, Nitroxyl ?HNO? can react with thiols in aldehyde dehydrogenase, leading to the inhibition of the activity of the enzyme. In addition, HNO can induce vasorelaxation through upregulation of calcitoningene-related peptide and regulate cardiovascular function to treate heart failure. Hydrogen sulfide ?H₂S? has been identified as an important endogenous gasotransmitter in addition to nitric oxide and carbon monoxide. H₂S plays key roles in many physiological processes, including relaxation of vascular smooth muscles, mediation of neurotransmission, inhibitionof insulin signaling, and regulation of inflammation. As oxidized forms of hydrogen sulfide, hydrogen polysulfides ?H₂S_n, n>1? possess stronger nucleophilicity and reducibility than that of H₂S. So H₂S_n may be more effective in activating tumor suppressors, ion channels, and transcription factors. On the other hand, abnormal levels of these biologically reactive small molecules are usually related to various diseases. For example, nitrosative stress induced by reactive nitrogen species is closely related to cardiovascular disease. Reactive sulfur species are important antioxidant in living body, but abnormal levels of H₂S are associated with a variety of human diseases including Parkinson's, Alzheimer's, and Huntington's diseases. Therefore, developing high sensitive and selective methods for detection of these species reactive small molecules have great significance in monitoring their production and elucidating their biological roles. In this work, we design several kinds of fluorescent probes for sensitive and selective detection of HNO, H₂S, as well as H₂S_n based on their special chemical properties. The main research contents are as following: Chapter 1. The chemical properties and biological function of HNO, H₂S and H₂S_n are summarized. Recent advances in detection method focused on fluorescence analysis are also presented. Chapter 2. For the first time, we develop a mitochondria-targetable near-infrared fluorescent probe based on a merocyanine skeleton. 2-?diphenylphosphino?benzoyl group is introduced as the recognition unit for nitroxyl and a lipophilic benzo[e]indole cation used as the mitochondria-targeted site. The probe shows weak fluorescence in the absence of HNO. However, a significant turn-on response at 727 nm ?up to 38-fold? can be observed after addition of nitroxyl. There is good linearity between the fluorescence intensity and the concentrations of HNO in the range of 0-10 ?M with a detection limit of 60 nM. The probe is high sensitive and selective towards nitroxyl and can specifically locate in the mitochondria. It is proved that the probe is suitable for visualizing nitroxyl in the mitochondria of living cells.Chapter 3. Taking the advantage of nitroso group can be reduced into amino group by H₂S under mild conditions, we design a new fluorescent probe for H₂S detection based on a flavylium dye with nitroso group as recognition unit. The as-prepared probe shows weak fluorescence owing to the intramolecular charge transfer ?ICT? process being blocked. However, the nitroso group is reduced to amine group and the ICT process restore after addition of H₂S, which induce the fluorescence emission increased. The probe has rapid response to H₂S and fluorescence intensity can reach to the maximum within 2 minutes. Furthermore, the probe shows high sensitivity to H₂S with a detection limit of 40 nM. Probe has good membrane penetrability and is capable of detecting exogenous and endogenous H₂S in living cells.Chapter 4. H₂S_n possess stronger nucleophilicity and reducibility compared with that of H₂S, cysteine ?Cys? and glutathione ?GSH?. Based on this fact, we design a turn-on fluorescent probe for detection of H₂S_n. Flavylium dye and nitro group is used as skeleton and recognition unit respectively in the probe. The probe shows weak fluorescence because of ICT process being blocked by introducing the strong electron-withdrawing group. After addition of H₂S_n, nitro group is reduced to amino group and the ICT process restore, which induce a dramatic fluorescence enhancement. The probe displays favorable stability and high selectivity, as well as is suitable for detection of H₂S_n in living cells.Chapter 5. We design a dye-assembled upconversion luminescent nanoprobe for detection of H₂S. The upconversion nanoparticles are used as fluorescence donor and organic dye molecule designed as fluorescence acceptor. The organic dye molecule with specific recognition site for H₂S is loaded to upconversion nanoparticles through electrostatic interaction. The organic dye molecule can absorb the emission of upconversion nanoparticles located at 580-660 nm, and thus the fluorescence of the upconversion nanoparticals is quenched. After addition of H₂S, H₂S can be added to organic molecule through nucleophilic addition and alters the conjugated system of organic molecule. The absorption of organic molecule is lowered. Thus the upconversion luminescence of the nanoprobe can be restore. There is good linearity between the fluorescence intensity and the concentrations of H₂S in the range of 100-700 ?M. The probe is high selective towards H₂S over other biologically relevant species.