Detection Of Important Physiological Active Substances In The Cell Design Synthesis And Applications Of Fluorescent Probe

Hydrogen sulfide （H₂S） has been considered to be a toxic chemical. H₂S is mainlyproduced by geological or microbial activity. H₂S is a colorless gas with rotten-egg smelland is strongly irritative to eyes and respiratory tract. Excessive inhalation of H₂S willlead to loss of consciousness, respiratory failure, and heart damage. Recent studies havefound that H₂S in the mammalian body widely participate in the regulation ofphysiological and pathological processes. H₂S is mainly produced by the enzymaticreaction of cysteine, methionine and homocysteine that catalyzed bycystathionine-β-synthase （CBS）, cystathionine-γ-lyase （CSE）, andcysteine transferenzymes. The second source of H₂S is a non-enzyme route that produced during theoxidation of the sugar by the elemental sulfur. Endogenous H₂S plays a wide range ofbiological effects in the biological systems, including nervous system, respiratory system,cardiovascular system, and digestive system, etc.The relative stability of pH in the living cells plays a crucial role in the maintainance ofsurvival and health of living organism. Intracellular pH anomalies often induces celldysfunction, cell growth and division, and may lead to diseases such as cancer andAlzheimer. Therefore, the detection of intracellular pH is important and is vital to thephysiological and pathological information that reflecting diseases.Fluorescence probe-based fluorescence analysis has been widely used to thedetermination of active species in the living organism with high sensitivity and selectivity.Moreover, it is easy to operate and is non-destructive to the living organism. Whencombined with laser scanning confocal microscopy, fluorescence probe can facilitate tothe realization of "real-time” visual detection of bioactive species in the living organism.Based on the design and synthesis of small organic fluorescent probes, this thesiscarried out the research work of the following two aspects:（A） We describe the design and synthesis of a cyanine-based near-infrared ratiometric fluorescent probe, HS-Cy, for H₂S detection, which features rapid response, highsensitivity, and mitochondria targeting. After a rapid quenching at780nm by initialnucleophilic addition on aldehyde group, HS-Cy experienced a polymethine π-electronconjugation modulation triggered by a second nucleophilic addition on ester, releasing thecyanine fluorophore which underwent tautomerism from enol form to ketone form.Therefore, gradual emergence of a new peak at625nm was observed, constructing aratiometric signal for H₂S with a detection limit of5.0-10nM, which is the most sensitiveamong the reported H₂S-sensing fluorescent probes. HS-Cy was proved to selectivelylocate into mitochondria with faster trapping kinetics towards H₂S. Based on this, theendogenously generated H₂S in human A549cells was ratiometrically detected andimaged by HS-Cy.（B） Based on the difluoroborane derivative structure,we designed and synthesized a pHfluorescent probe. The probe reversibly responded to pH in chemical and biologicalsystems, which is modulated by photoinduced electron transfer （PET）. The pH titrationexperiments showed that probe was pink under acidic conditions （pH <7.0） and was paleyellow at pH>7.0. The probe was found to locate to Golgi apparatus. The probe showedgood photostability and good cell membrane permeability, as well as low cytotoxicity.The probe was successfully applied to the imaging of pH variation in human HepG2cellswith good sensitivity and selectivity.