Development And Application Of Fluorescent Probes For Reactive Sulfur Species

Reactive sulfur species ?RSS? are a family of sulfur-containing molecules found in biological systems, which have various biological functions. These molecules include biological thiols ?-SH?, hydrogen sulfide ?H₂S?, persulfides ?H₂S2?, polysulfides ?H₂S_n?, sulfur dioxide ?SO2?, and S-modified cysteine adducts such as sulfenic acids ?HOSOH? and S-nitrosothiols ?RSNOs?. Development fluorescent probes for RSS are important because these molecules show a variety of physiological functions, including neuroregulation, inflammation and blood vessel tension. It is significant to develop highly selective and sensitive technique to investigate the role of RSS in biological systems.Hydrogen sulfide ?H₂S? has attracted increasing attention in biochemical research because the gaseous signaling molecule plays a critical role in many physiological and pathological processes. In contrast, hydrogen polysulfides ?H₂S_n, n>1?, which was recognized as oxidized forms of endogenous H₂S, have received less attention. However, some recent studies imply that some biological activities that were originally ascribed to H₂S may actually executed by H₂S_n. In order to better differentiate H₂S_n from H₂S and understand the functions of H₂S_n, it is important to provides a possible mechanism of H₂S_n and develop new methods for their detection.Taking the advantage of two free-SH groups in H₂S_n, two nucleophilic reactions can be occured in H₂S_n with compounds containing bis-electrophilic groups. Based on the idea, three kinds of fluorescent probes using different mechanism for H₂S_n were developed in this thesis. The thesis consists of four chapters as following:The biological functions and chemical properties of RSS were introduced in the first chapter. In addition, a general introduction to the research progress on the fluorescent probe for RSS was summarized. On this basis, the progress of this dissertation was proposed.In chapter 2, we screened a series of coumarinyl cinnamate esters with different electron-withdrawing groups on the phenyl ring of cinnamoyl moiety ?probe A1-A6? and found that 3-?trifluoromethyl? cinnamate ester showed a desirable turn-on fluorescence response to H₂S_n with high specificity. Based on this result, a turn-on fluorescent probe for H₂S_n was constructed by incorporating a 3-?trifluoromethyl? cinnamoyl group to N,N-diethylrhodol ?DER?. Probe A containing bis-electrophilic groups (carbonyl carbon and ?3-sulfido carbonyls? exhibits no fluorescence in the aqueous buffer. Upon treating with H₂S_n, two nucleophilic reactions between H₂S_n and the probe were occurred, and the fluorophore DER was released to emit stronger fluorescence at 548 nm. The fluorescence intensities were linearly related to the H₂S_n concentrations in the range of 0-40 ?M. The limit detection was calculated to be 0.40 ?M. Moreover, the probe proves to be able to assessing H₂S_n levels in serum as well as in live cells.In chapter 3, we have developed a long wavelength fluorescent probe B for H₂S_n by using aziridine as the recognition unit and rhodamine B as the fluorophore. Due to the large ?-conjugated system of the probe, it has a strong fluorescence emission at 590 nm. Upon introducing H₂S_n in neutral solution, the aziridine ring was attacked to afford an asymmetric ring-opening reaction and to form an intermediate containing an amide group. The 9-carbon in rhodamine can then be arracked by the nitrogen-atom in amide group to form the corresponding spirolactam. Thus, the large conjugated system of probe B was interrupted. The fluorescence intensity is decreased via a ring-closing reaction. The fluorescence intensity changes at 590 nm showed a good linear relationship with H₂S_n concentration 0-50 ?M. The limit detection was calculated to be 0.54 ?M.In chapter 4, we have developed a probe C for H₂S_n by using pyrene butyric acid as the fluorophore. The pyrene butyric acid is general fluorophore with high fluorescence quantum yield and long fluorescence lifetime. Upon treating with H₂S_n, the aziridine was attacked to occur asymmetric ring-opening reaction and to form intermediate containing-S-SH group. The strong fluorescence emission at 378 nm is decreased because of photoinduced electron transfer ?PET? between-S-SH and pyrene butyric acid. The fluorescence intensities were linearly related to the H₂S_n, concentrations in the range of 0-50?M. The limit detection was calculated to be 0.48?M.