The Synthesis And Study Of Aminocyanine Fluorescent Probes For Hypoxia

Hypoxia,an important feature of malignant tumor,plays a significant role in tumor growth and regulation.Study on tumor hypoxia can provide a powerful tool for tumor diagnosis.For hypoxia imaging,fluorescent probes have been widely applied in the detection of tumor hypoxia owing to their advantages of high sensitivity.Since nitroreductase(NTR)is generally overexpressed in hypoxic tumors,it can be an important target for hypoxia probes.To date,although many hypoxia probes have been reported to detect NTR in hypoxic cells,near-infrared(NIR)fluorescent probes for hypoxia are still few,especially the fluorescent probes with absorbance and fluorescence wavelength both in the near-infrared region.In addition,aminocyanine-based fluorescent probes are rarely reported for hypoxia,thus novel NIR fluorescent probes for hypoxia based on aminocyanine dyes are needed to promote the development of tumor imaging.In this paper,a wide range of fluorescent probes for hypoxia is reviewed,and then based on the current situation,a series of novel NIR fluorescent probes are designed to detect NTR and tumor hypoxia.First,two novel NIR fluorescent probes CyNP1 and CyNP2 were synthesized to detect NTR based on p-nitrobenzyl-substituted aminocyanine,and the mechanism of the response to NTR was verified through mass spectrum and HPLC test.CyNP1 is fluorescence-quenched in the buffer system,however it demonstrates a significant fluorescence recovery in the presence of NTR and NADH in a very short time,and the fluorescence enhancement can be 40-fold;in comparison,CyNP2 shows only 2.5-fold fluorescence enhancement after its response to NTR,and fluorescence intensity becomes weak after the enhancement.Mass spectrum and HPLC test show that,in the presence of NTR and NADH,CyNP1 would become aminobenzene and then transform to its parent dye CyNP through rearrangement and elimination reaction,while CyNP2 can be only reduced to nitrosobenzene,which is an intermediate through catalytic reduction process.Therefore,CyNP1 is a more excellent probe for NTR detection.Moreover,CyNP1 shows no fluorescent change in the presence of other reductive species,which means it has a specific response to NTR.In addition,when it comes to cell imaging experiment,obvious fluorescence can be detected when CyNP1 is cultured in hypoxic Hela cells,while no fluorescence show up when the probe is cultured in normoxic cells.Besides,when dicoumarin-an NTR inhibitor is added to hypoxia cells before the probe,nearly no fluorescence can be detected.All those results show that CyNP1 can be an excellent fluorescent probe for nitroredctase and tumor hypoxia imaging.To explore other types of hypoxia probes with longer wavelength,two novel probes CyN1 and CyNP3 were then synthesized based on p-nitrobenzoyl-substituted aminocyanine dyes.And the catalytic mechanism of NTR to nitrobenzene was investigated through mass spectrum for the first time.Results show that the maximum absorption and emission wavelength of the two probes are in the near infrared region above 700 nm,and the maximum emission of CyN1 is even more than 800 nm,which is the longest fluorescent wavelength among the current fluorescence enhanced hypoxic probes.Two probes both show an obvious fluorescence recovery after their response to NTR,and fluorescence enhancement can both be 5-fold.In addition,mass spectrum reveals that catalytic reduction process of NTR to nitrobenzene involves three main species,including nitrosobenzene,hydroxyaminobenzene and aminobenzene.However,fluorescence enhancement of two probes mainly comes from two intermediates nitrosobenzene and hydroxyaminobenzene.The fluorescent response of CyN1 and CyNP3 to NTR indicates a potential application on tumor hypoxia imaging.