Cyanine Skeleton-Based Fluorescent Probes For Imaging Analysis Of Biological Reactive Species Under Hypoxia Stress

Increasingly grim environmental pollutions are closely related with the occurrence and development of diseases.There are also migration,transformation and interaction in various pollutants,which are easy to form secondary environmental stress,such as environmental hypoxia,threatening the ecological environment and life health.However,it’s obscure how environmental stress disturbs the normal physiological process,and then how endogenous reactive species mend the cases remains unclear.Fluorescence imaging technology has been widely used in chemical biology,environmental science,medical imaging and other fields with the advantages of high sensitivity,good selectivity and in situ analysis,which can achieve non-destructive,real-time and high spatial and temporal resolution monitoring of analytes.In this work,with the help of fluorescence imaging technology,the novel cyanine fluorescent probes were used as analysis and detection tools to in situ monitor and image the fluctuations of endogenous biology thiols（glutathione（GSH）,cysteine（Cys））,hydrogen polysulfides（H₂S_n ）,SO₂ and its derivatives in reactive sulfur species（RSS）,as well as the fluctuation of reactive oxygen species（ROS）under environmental hypoxia stress.The meaning of work is to assess the physiological and pathological functions of these bioreactive molecules in hypoxia-related injury and disease,revealing the relationship between environment and health from the molecular levels.The research contents of this thesis are as follows:1.Detecting and evaluating the protective effects of endogenous glutathione on cerebral ischemia/reperfusion stress via near-infrared fluorescence probeThe ischemia/reperfusion（I/R）process frequently arises in stroke and other common diseases.Cerebral I/R-induced stroke could lead to hypoxia,oxidative stress and produce a lot of ROS.As the most abundant biology thiol and antioxidant,the content of intracellular GSH is up to millimole concentration,which the fluctuations of endogenous level can reflect the redox state of cell.The antioxidant properties of GSH play important roles in cerebral I/R process in cells and tissues as well as in vivo.To explore the changes of endogenous GSH under cerebral I/R stress,we have designed and synthesized two distinctive“off-on”near-infrared（NIR）fluorescent probes BCy-SeSe and BCy-SS based on a new fluorophore BCy-Keto for real-time imaging and evaluation of mitochondrial GSH in cells and in vivo during cerebral I/R.The probes included two components:the NIR fluorophore BCy-Keto and GSH-specific recognition group diselenide or disulfide.The probes were cleaved the diselenide or disulfide bond via selenium-sulfur or sulfur-sulfur exchange and then underwent intramolecular cyclization to release the fluorophore.The double charges of the probes exhibited powerful mitochondria targeting capability,and the probes exhibited high selectivity and sensitivity properties toward GSH,which exhibit“turn-on”fluorescence response.The in situ imaging results in cells I/R models and mice middle cerebral artery occlusion indicated that cellular I/R injury and cerebral infarction in mice were closely related to the down-regulation of GSH levels,which confirmed that GSH is effective on the cerebrum protection from I/R.The probe provides a visual tool for studying the physiological and pathological mechanisms and the prevention and intervention of I/R damage under cerebral I/R stress.2.Visualizing and evaluating endogenous cysteine（Cys）in cells,tissues and in vivo under hypoxia/reperfusion（H/R）stressCys,another biology thiol,is not only an endogenous metabolite that can indicate the health status of organism,but also an important bioreactive molecule in the living system,which is correlated with GSH in complex biological system.Based on our previous study on bioeffects of GSH against to damage induced from cerebral ischemia/reperfusion stress,a fluorescent probe BCy-AC was designed to monitor Cys and evaluate the biological function of Cys under environmental H/R stress in this wok.The probe was fabricated with recognition unit of acrylate group and a NIR fluorophore BCy-Keto.The unique Enol-Keto tautomerization of fluorophore BCy-Keto improves the sensitivity and potential application performance of the probe.The probe showed good mitochondria target ability,NIR fluorescence spectra,tissue penetration,and in situ visualization characteristics.The visualization results of Cys in hippocampal neuronal HT-22 cells,zebrafish and mice hippocampi tissues showed that BCy-AC can be used as an effective monitor tool for Cys levels under environmental hypoxia stress.BCy-AC was applied to investigate the connection between Cys concentration and H/R-induced damage,which confirmed that Cys could resist H/R stress induced mitochondria-related damage.Therefore,the study of Cys fluctuation and its bioeffect evaluation under hypoxia stress provides a basis for finding effective ways to resist environment stress.3.A near-infrared fluorescent probe for observing hydrogen polysulfides formation and fluctuation in cells and in vivo under hypoxia stressHydrogen polysulfide（H₂S_n ,n>1）is a member of RSS,which has the function of cell protection and signal transduction.It can be metabolically produced by polysulfide and hypersulfide of biothiols（including GSH and Cys）and protein amino acid residues in organisms.H₂S_n can also generate from the third type of gas signal molecule H₂S via ROS interaction and enzymatic catalysis.Hypoxia stress can disturb the redox balance and homeostasis of cells,and various reactive molecules balance and regulate each other in complex biosystems.H₂S_n connects RSS and ROS in biological systems where the formation and fluctuation of H₂S_n under hypoxia stress is of great significance for the damage study of hypoxia in cells and in vivo.In this work,we designed a NIR probe BCy-FN for in situ observing mitochondrial H₂S_n formation mediated by HSNO in cells and zebrafish under hypoxia stress.The probe was composed of NIR cyanine fluorophore BCy-Keto and 2-fluoro-5-nitrobenzoate recognition group,which featuring mitochondria localization.The probe has low cost and high selectivity,sensitivity and improved response rate towards H₂S_n .In both H/R models of ZF4 cells and zebrafish,BCy-FN was successfully applied to in situ imaging H₂S_n changes including HSNO-mediated H₂S_n generation.The results revealed that HSNO-mediated H₂S_n could resist hypoxia stress to some extent.Penetration imaging H₂S_n in mice peritoneal cavity via BCy-FN suggests that our probe has excellent NIR properties for in situ visualization with noninvasiveness.The real-time imaging of H₂S_n in living zebrafish assist to evaluate the state of oxidative stress during hypoxia process,which will help to better understand the biological effects of HSNO-mediated H₂S_n formation under hypoxia stress.BCy-FN provides a potential tool for exploring HSNO-mediated H₂S_n generation and their roles in biological systems in the future.4.Discovering new specific recognition moiety for H₂S_n fluorescent probe designReactive sulfur species（RSS）are involved in regulation of redox balance and homeostasis in organisms as a class of key antioxidants and biological signaling molecules.The current researches of H₂S_n mainly focused on developing fluorophores or improving the performances of the probes,and less attention was paid to explore new specific recognition moiety for H₂S_n fluorescent probe design.In this work,six simple compounds containing 2,4-dinitrobenzene as recognition moiety were initially prepared based on nitrophenols,4’-Hydroxyacetophenone,P-Bromophenol,P-methoxy phenol,and P-dimethylamino phenol and other phenols with different substituents.A small molecule library was constructed to recognize H₂S_n ,and verified the specific response of 2,4-dinitrobenzene groups to H₂S_n .The results indicated that the higher electron-withdrawing effect of the substituent in phenol,the better response of the probe for H₂S_n .Based on coumarin fluorophore,we designed and synthesized fluorescent probe 7 to verify the results.This work provided more potential candidate for the research of H₂S_n and development of H₂S_n fluorescent probes.5.Insight into sulfur dioxide and its derivatives metabolism in living system with visualized evidences via ultra-sensitive fluorescent probeSulfur dioxide（SO₂）and its derivatives are not only environmental pollutants but also widely used food additives,as well as the metabolites of sulfur-containing substances in living systems,which can form water-soluble sulfate and be discharged with urine to maintain the biological sulfur balance under the action of sulfite oxidase.Endogenous SO₂ can be produced by GSH under enzymatic catalysis or generated from H₂S.A variety of sulfur-containing substances can be converted into SO₂ and exist in the form of HSO₃^-and SO₃^2-.However,due to the lack of effective detection tools,the molecular mechanisms underlying the generation and metabolism of SO₂ and its derivatives in many physiological and pathological processes remain unclear.In this work,we designed an ultra-sensitive fluorescent probe MS-Bindol for recognition and detection SO₂ and its derivatives as well as elucidate the physiological and pathological mechanisms of the close association and mutual transformation of RSS in biological systems.The probe took advantage of large stokes shift,mitochondrial localization,good water solubility,fast response,and low detection limit properties（0.2 n M）,which respond sensitively to SO₂ in water solution and air.In situ visualization results of SO₂ derivatives in cells and zebrafish indicated that SO₂ derivatives could be synthetized from thiosulfate and GSH and it was found that when sulfite oxidase was inhibited by ferricyanide or arsenite,SO₂ derivatives were not easily metabolized and then accumulated in cells and zebrafish.Moreover,the probe MS-Bindol also exhibits excellent practicability in food as well as environmental water samples.The probe will be an effective sensor tool for monitoring SO₂derivatives in cells,in vivo,food,and environmental water samples.6.In situ imaging analysis of H₂O₂ under hypoxia stress via fluorescence probeNot only do RSS levels fluctuate,ROS can also be significantly affected in damage and complex physiological and pathological processes under hypoxia stress.Endogenous ROS levels are closely related to the redox balance,the activity and concentration of biothiols such as GSH,Cys,as well as other antioxidants.As an important ROS,hydrogen peroxide（H₂O₂）involved in various physiological processes of cells,and precisely regulated to activate intracellular signal transduction pathways,promote cell proliferation and differentiation under normal circumstances.However,as a source of oxidative stress,abnormal expression of H₂O₂ would attack intracellular biomolecules and related to DNA damage,senility,cancer,cardiovascular and neurodegenerative diseases.In this work,the ratiometric fluorescent probe MSO-Bindol was designed by coupling of 4-（methylsulfoxide）phenyl moiety and benzindole to form a carbon-carbon double bond,which was used to observe H₂O₂ variations in cells and zebrafish exposed to various stimulations or environmental stress especially hypoxia stress.The probe was used to study the fluctuation of H₂O₂ in biological systems under oxidative stress and hypoxia stress,revealing the relationship between LPS,PMA and Hg²⁺induced oxidative stress and H₂O₂ up-regulation in living cells and zebrafish.Cell and zebrafish hypoxia stress models with different degrees of hypoxia were constructed to explore the change of H₂O₂ induced by different hypoxia time.The results suggested that hypoxia stress-induced H₂O₂ up-regulation may be a manifestation of ROS outburst and the hypoxia induced injury may be directly related to H₂O₂ levels.The occurrence H₂O₂ outburst may cause oxidative stress in a certain extent.This work provides a new way and idea for studying damage mechanism of hypoxia stress and H₂O₂ monitor.