Development And Recognition Property Of Fluorescein Based Fluorescent Probe For Nitric Oxide

Objective: To develop fluorescein-based fluorescence probe for detecting Nitric Oxide,and study its recognition property.Methods: 1,2-Dimethyl-4-nitrobenzene was used as the starting material to prepare 5-Aminofluorescein?L₁?by reactions of oxidation,dehydration,addition,reduction and so on.Then L₁ and 4-?2-Aminoethyl?morpholine were used to obtain 5-Aminofluorescein morpholine derivatives?L₂?.The compositions and structures of L₁ and L₂ were characterized by modern analysis technologies such as ¹H-NMR,¹³C-NMR,HRMS,FT-IR,single crystal diffraction and so on.The recognition property and mechanism of L₁ and L₂ towards NO were investigated by UV-Vis absorption spectroscopy and fluorescence spectrophotometry in Tris-HCl buffer solution.Results: The L₁ and the L₂ were confirmed to be the expected compounds by ¹H-NMR,¹³C-NMR,HRMS,FT-IR and single crystal diffraction.NO can be recognized by L₁ and L₂ selectively.L₁ identifies NO by the way of deamination lead to fluorescence enhanc ing and the fluorescence intensity showed a good linear relationship with the nitric oxide donor named NOC-18 in the range of 0-8.0×10^-5 mol/L.Hypochlorite as one of the active oxygen that was chosen in the study showed slight interference to the recognition of L₁ towards NO.Moreover,ascorbic acid and dehydrogenated ascorbic acid showed great interference.The mechanism that L₁ identifies NO was confirmed to be deamination by ¹H-NMR and HRMS.C ytotoxicity assay showed that L₁ has low toxicity to Raw 264.7 cells and be able to achieve fluorescence imaging of NO in Raw 264.7 cells.L₂ identifies NO by the way of deamination and lead to fluorescence quenching.The L₂ shows blue fluorescence under the UV light,and the fluorescence would be quenched when it reacted with NO.But the quenching effect is affected by NO₃^-,which is one of the active oxygen species and nitrogen species in the scope of the study.C ytotoxicity assay showed that L₂ has low toxicity to Raw 264.7 cells.Conclusion: The two obtained 5-aminofluorescein derivatives,L₁ and L₂,both of the two probes can detect NO by the reaction of deamination.L₁ is a type of fluorescent probe that enhanced fluorescence by NO.But L₂ is just the opposite,it is a type of fluorescent probe that quenched fluorescence by NO.