Synthesis Of Reactive Inorganic Sulfur Species Molecular Fluorescent Probes And Study Of Their Cell Imaging

During a series of physiological processes,RSSs can be further metabolized to H₂S,S_n^2-,SO₃^2-,HSO₃^-,S₂O₄^2-,and S₂O₅^2-(Reactive Inorganic Sulfur Species,RISSs)with the specific enzyme.RSSs and their metabolites(RISSs)play a crucial role in the cushion against oxidative stress and the other physiological events.The molecular mechanisms how they affect cellular signaling and other physiological events remain largely unclear.Although H₂S,S_n^2-,SO₃^2-,HSO₃^-have been detected by several methods and their possible functions have been studied in detail.But S₂O₄^2-and S₂O₅^2-are barely studied.S₂O₄^2-is irritating to human eyes and respiratory mucosa;meanwhile people with allergies or asthma are very sensitive to S₂O₅^2-.Because of the importance of RISSs in biological systems and the lack of research on S₂O₄^2-and S₂O₅^2-,it is necessary to develop better detection technology to track the level of RISSs in cells.However,due to the similar reductivity between RISSs,it is very difficult to design and synthesize RISS probes that specifically identify a single RISS.Therefore,it is necessary to design and synthesize RISSs probes for the detection of total RISSs.The characteristics of visualization,high sensitivity,fast response,and good selectivity of fluorescence biological imaging technology overcome the shortcomings of traditional detection.Therefore,the development of fluorescent probes for the detection of cellular RISSs can be used to elucidate the role of RISSs in cell signaling.Chapter 1:In this chapter,six colorimetric and ratiometric fluorescent probes CH-RISSs based on coumarin hemicyanine scaffolds were designed and synthesized,and whose structures were characterized with NMR and Mass.Their absorption and fluorescence spectrum were performed and the results showed that CH-RISS-1/2/4/5could quickly respond to H₂S,S_n^2-,SO₃^2-,HSO₃^-,S₂O₄^2-,and S₂O₅^2-(RISSs)within 30 s in PBS or PBS/ACN(7/3),and showed good stability between p H2-10.As the ratiometric fluorescent probes,with the addition of RISSs,the fluorescence of CH-RISS-1/2/4/5disappeared at 650-660 nm and increased at 480-505 nm,the emission wavelength blue-shift(??=150 nm)was showed in the fluorescence spectrum.CH-RISS-1/2/4/5showed strong responses upon RISSs,the ratio of Em_{505 nm}/Em_{651 nm}of CH-RISS-4 upon SO₃^2-increased 705 folds.The fluorescence intensity of CH-RISS-1/2/4/5 has a good linear relationship with RISSs,which meant they could quantitatively detect RISSs.Meanwhile,the sensitivity of CH-RISS-1/2/4/5 was very high,the detection limit of CH-RISS-1 for Na₂S₂O₅was as low as 6.7 n M.In addition,CH-RISS-1/2/4/5 had good selectivity for RISSs.Analog CH-RISS-3/6 should have similar spectral characteristics.In live cell imaging,the fluorescent probes CH-RISS-3/6 with C12 alkyl chains could be used to detect RISSs in L929 cells.Chapter 2:Herein,six RISSs two-photon fluorescent probes PH-RISSs based on1-Pyrenaldehyde hemicyanine scaffolds were designed and synthesized.Two-photon microscopes have the advantages of increased penetration depth,local excitation,and extended observation time.PH-RISS-1/4 showed good stability in glycerol.Their fast(1min)and strong responses upon RISSs were clarified in the absorption spectra.PH-RISS-1/4 showed excellent selectivity.PH-RISS-1/4 were excellent sensors for detecting RISSs in cell imaging with low cytotoxicity.PH-RISS-1/4 will be used for further potential biology study related to H₂S and biothiol metabolism.Two photo imaging of cellular RISSs will be investigated further due to the limited time.In this thesis,CH-RISSs and PH-RISSs fluorescent probes have been developed ab initio.The structures of the fluorescent probes were characterized by NMR and LC-MS.The optical properties of RISSs fluorescent probes were detected using absorption and fluorescence spectroscopy.In the live-cell imaging,the fluorescence responses and chemical selectivity of fluorescent probes to RISSs were investigated.CH-RISSs showed strong stability,high sensitivity,good selectivity,robust and fast responses(30 s)to RISSs,and CH-RISSs could quantitatively monitor RISSs in cuvette.CH-RISS-3/6were successfully used to detect RISSs in living cells.PH-RISSs,as two-photon fluorescent probes,had near-infrared emission wavelengths,which could easily penetrate cells with the low cytotoxicity.Access to the fluorescent probes for RISSs sets the stage for applying the developing technologies to probe reactive sulfur biology in living systems.