Design, Synthesis And Biological Applications Of Novel Fluorescent Probes For Reactive Species

Reactive species play an important role in maintaining the homeostasis of intracellular redox.The abnormal concentration and distribution of reactive species is commonly related to various diseases.Therefore,in-situ monitoring of reactive species is of great significance for diagnosis and treatment of diseases,as well as the related biomedical study.Fluorescence imaging has attracted much attention because it is a noninvasive,highly sensitive and highly sensitive in-situ analysis technology with high spatial and temporal resolution.In recent years,various fluorescent probes have been developed and widely used in bio-labeling and bio-analysis,which is an important tool in the study of life science and diseases diagnosis.Developing fluorescent probes for reactive species with high sensitivity,good selectivity and fast response still is a hot topic in the field of bio-analysis.In this thesis,we have designed and synthesized three fluorescent probes to detect the hydrogen sulfide?H₂S_n?,hydrogen peroxide?H₂O₂?and biothiols,which provided important tools for further exploring their functions in the biological system and related medical study.The detailed contents are described as follows.?1?Hydrogen sulfide?H₂S_n?is one of important reactive species and has gain much attentions in recent years.In some biological processes,H₂S_n plays more important roles than H₂S.In Chapter 2,a phenothiazine coumarin-based red fluorescent probe PZC-S_n was developed for the detection of H₂S_n.The phenothiazine coumarin was introduced as the fluorescence unit,and 2-fluoro-5-nitrobenzoate moiety was used as the recognition group.H₂S_n could react with 2-fluoro-5-nitrobenzoate moiety via nucleophilic substitution,which then triggered the substitution-cyclization cascade reaction to release the fluorescent moiety.PZC-S_n had a large Stokes shift?140 nm?,and displayed a high sensitivity and selectivity,which has a detection limit of 1 nmol/L.This probe also showed a low cytotoxicity,and the red emission also facilitated the fluorescence imaging of exogenous and endogenous H₂S_n in living RAW 264.7cells and zebrafish.?2?Hydrogen peroxide?H₂O₂?is a typical reactive oxygen species,which plays important functions in many physiology and pathology processes.In Chapter 3,based on the specific reaction of hydrogen peroxide to boric acid ester group,we developed a novel red fluorescent probe PZC-HP with large Stokes shift for H₂O₂ detection by employing boric acid ester to phenothiazine coumarin dyes.The reaction between PZC-HP and H₂O₂ triggered the cleavage of boric acid ester group to release the quinone derivative.And as a result,the fluorescent phenothiazine coumarin derivative was formed through the intramolecular cyclization.PZC-HP showed a high sensitivity with linear detection range of 1-50?mol/L and a detection limit of 60nmol/L.This probe also exhibited a high selectivity,low cytotoxicity and good cell permeability.The fluorescence imaging of exogenous and endogenous H₂O₂ in living RAW 264.7 cells was also achieved by using this probe.?3?Cysteine ?Cys?,homocysteine ?Hcy? and glutathione ?GSH? are the most abundant small molecular biothiols in cells.Because the chemical structures and properties of Cys,Hcy and GSH are very similar,developing fluorescent probes for simultaneous detection and discrimination of these biothiols is important.In Chapter 4,based on intramolecular charge transfer mechanism,we employed the derivative of 2-dicyanomethylen-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran ?TCF-OH? as the fluorescence moiety and 4-Nitro-1,2,3-benzoxadiazole ?NBD? as the recognition group,and then constructed a novel fluorescent probe TCF-O-NBD for biothiols with dual emissions.The-SH groups in Cys,Hcy and GSH could react with the NBD moiety in TCF-O-NBD via nucleophilic substitution to release the fluorescence dye TCF-OH.At the same time,Cys/Hcy could further react with NBD through intramolecular cyclization and a green fluorescent product was generated.But,due to the lack of proximal amino group,GSH could not trigger the intramolecular cyclization with NBD,thus forming a non-fluorescence product.According to the different fluorescence mode between two channels,the simultaneous detection and discrimination of Cys/Hcy and GSH could be achieved.This probe had a high selectivity and sensitivity,the detection limits of Cys,Hcy and GSH were 18 nmol/L,25 nmol/L and 22 nmol/L,respectively.Furthermore,TCF-O-NBD exhibited a low cytotoxicity and was applied in the fluorescence imaging of Cys/Hcy and GSH in living cells.