| Objective: A range of physiological activities in cells are closely associated with the intracellular pH.Subtle changes in intracelluar pH may cause cell dysfuction and diseases.It has been shown that pH fluctuation in acidic organelles including lysosome and endosome has been implicated into many important diseases such as cancer and Alzheimer's.Thus,accurate detection of pH in the acidic organelles,especially real-time and high-resolution imaging of pH,can provide important information on the physiological and pathological functions of these organelles.The methods for detection of the intracellular pH values include microelectrodes,nuclear magnetic resonance spectroscopy and fluorescence techniques.Compared with other methods,fluorescence techniques can provide high sensitivity,high time resolution and high spatial resolution.In addition,it can monitor couples of cells or plenty of cells,but is not limited by cell concentrations and sizes.In addition,the fluorescent techniques also have the advantage of easy operation which is suitable for routine laboratory use.Fluorene and its derivatives have the following merits: high fluorescence quantum yield,excellent optical and chemical stability,and high two-photon absorption(TPA)cross-section.As such,it has been proved to be excellent two-photon fluorescence(TPF)material.In order to detect the pH values in the weakly acidic organelles such as lysosome and endosome,we synthesized a fluorene-based fluorescent pH probe and studied its photophysical properties.We believe that this acidic fluorescent probes could provide a new possibility for the detection of weakly acidic environments.Methods: Based on a fluorescent core 2,7-dibromofluorene,a donor-p-acceptor system was developed via conjugation with the electron donor diethylamine and with the electron acceptor benzothiazole,respectively.To improve the solubility,the hydrophilic diethylene glycol methyl ether ethyl group was introduced to the methylene position of fluorene.The fluorescent probe AF-1 was obtained via four steps.In brief,dialkylation of the 9-position of 2,7-dibromofluorene was achieved under basic conditions to give the compound 2,which then reacted with n-BuLi and DMF to afford the compound 3.Then,nucleophilic substitution of 3 with diethylamine gave rise to the compound 4.Finally,the compound 4 was condensed with 2-aminothiophenol to give the target compound AF-1.All the intermediates and target compound AF-1 were well characterized by NMR.UV-Vis and fluorescence spectroscopies were used to examine its response to pH.Accordingly,the p Ka of AF-1 was calculated.Results: In this work,we successfully synthesized the desirable compound AF-1.The molecular structures of the target compound AF-1 and its intermediates were consistent with their NMR data.The UV-Vis and fluorescence experiments were carried out to study the response of the compound AF-1 towards designated pH values(3.5-7.50).The results showed that the UV-vis absorbance and fluorescence intensity of AF-1 were pH-dependent,and its pKa was determined to 5.88 by nonlinear regression to these data.Conclusion: The fluorene-based fluorescent pH probe AF-1 was designed and successfully synthesized.The results showed that UV-vis absorbance and fluorescence intensities of AF-1 were pH-dependent at the pH range of 3.5 to 7.5,and its p Ka was determined to be 5.88.Thus,it can be concluded that AF-1 is a weak acidic fluorescent pH indicator,showing great promise in the detection of pH at weakly acidic environments such as endosome and lysosome. |
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