Synthesis Of Fluorescent Probes Of Naphthalene Derivatives And Theirs Biological Applications

Organic small molecule fluorescent probes have the advantages of structure modifiability,high sensitivity,rapid response,high applicability and non-invasive visualization analysis.In recent years,it has been well received by researchers in all fields in disease detection,surgical navigation and other areas.In this paper,by reviewing the relevant literatures and combining the existing small molecule fluorescent probes and the difficult and hot issues on the detection of bioactive substances,6-(2-benzothiazolyl)-2-naphthol dyes were selected as an ideal platform for the construction of fluorescent probes.Three novel organic small molecule fluorescent probes were designed and synthesized based on the specific chemical properties of the target analytes for the analysis,detection,and imaging studies of hypochlorous acid(HClO),nitroxyl(HNO),and selenocysteine(Sec),respectively.The structures of the three probes were characterized and confirmed by FT-IR,1H NMR,12C NMR,and HR-MS methods,respectively.The main studies are as follows.(1)A novel naphthalene derivative fluorescent probe(BNA-HClO)specifically recognizing HClO was designed and synthesized using 6-(2-benzothiazolyl)-2-naphthol as the fluorescent reporter group and N,N-dimethylthiocarbamate as the recognition group.In vitro optical experiments showed that the probe BNA-HCIO responds quickly(<30 s)to HClO in the PBS buffer solution system and the detection limit of probe BNA-HClO was 37.5 nM,which was far below the threshold limit set by the National Standardization Administration of China(8.6μM).The mechanism of HClO recognition by the probe was also verified by HPLC and HR-MS methods.In addition,the probe BNA-HClO could be directly used for the detection of HClO in tap water,medical wastewater and fetal bovine serum with recoveries in the range of 96.1%一 102.7%.The probe BNA-HClO was successfully made into portable test strips for the direct visualization of HCIO in real water samples for in situ semi-quantitative detection.In terms of bioimaging applications,the cytotoxicity of the probe BNA-HClO was evaluated by the CCK-8 method.When the probe BNA-HClO concentration was 0～12 μM,the cell survival rate of the probe BNA-HClO treatment was 102.50%～94.24%,indicating that the probe BNA-HClO has good cell compatibility;further through the fluorescence confocal imaging technology,it is proved that the probe BNA-HClO can directly track the exogenous and endogenous properties in HeLa cells in situ HClO.(2)A"turn-on" organic small molecule fluorescent probe(BNB-HNO)was designed and synthesized for the specific recognition of HNO using 6-(2-benzothiazolyl)-2-naphthol as a fluorescent reporter group connected to a strong electron-absorbing group,diphenylphosphine o-benzoic acid.The spectroscopic properties(response time,pH stability,selectivity,interference resistance,qualitative and quantitative analysis ability)of the probe BNB-HNO were studied in a PBS buffered solution system.The results show that the probe BNB-HNO is sensitive to HNO with a low detection limit of 54.6 nM;the fluorescence response of the probe BNB-HNO to HNO is stable in the presence of common interfering substances in various organisms and has excellent anti-interference performance;The mechanism of action of the probe in recognizing HNO was verified by HPLC.In addition,the probe BNB-HNO could be directly used for the quantitative detection of HNO in serum samples;the results of in vivo HNO imaging studies also showed that the probe BNB-HNO could be successfully used for fluorescence confocal imaging of exogenous HNO in live zebrafish.(3)A novel two-photon fluorescent probe BNT-Sec was designed and synthesized by selecting 6-(2-benzothiazolyl)-2-naphthol as the fluorescent reporter group,2,4-dinitrophenylsulfonyl as the bursting group and Sec-specific recognition group.In vitro optical experiments showed that the fluorescence signal-to-back ratio of the probe BNT-Sec in PBS buffer solution showed a good linear relationship with the Sec(0～10 μM),and the detection limit was as low as 10.6 nM,which was lower than most of the reported fluorescent probes for Sec detection.Secondly,the recognition mechanism of the probe BNT-Sec for Sec was verified by HPLC and TLC methods.In addition,the cytotoxicity of probe BNT-Sec was evaluated by MTT method,and the survival rate of probe BNT-Sec-treated cells ranged from 99.56%to 92.1%at concentrations of 0～12 μM of probe BNT-Sec,indicating that probe BNT-Sec has good cytocompatibility.Further,by combining two-photon fluorescence confocal imaging technique as well as in vivo imaging,it was demonstrated that the probe BNT-Sec could be used to directly target exogenous and endogenous Sec in Hela cells and exogenous Sec in adult zebrafish in situ.Three novel fluorescent probes(BNA-HClO,BNB-HNO,BNT-Sec)were designed and synthesized using the unique chemical properties of the target analytes.By further using the probes for dynamic real-time tracking of target analytes in cells and living organisms,we provide a theoretical basis and technical means to investigate the pathopharmacological mechanism of action of target analytes in life sciences as well as the early diagnosis of related diseases and the implementation of precision medicine.