Photoactivated Generation Of Pentamethine Cyanine Dyes And Its Application In In Situ Near Infrared Biological Imaging

Pentamethine Cyanine dyes(Cy5)are an important group of cationic cyanine dyes.The maximum absorption and fluorescence emission wavelength of Cy5 have entered the near infrared region.Therefore,Cy5 are widely used in tumor therapy,molecular detection,fluorescence imaging or other biomedical fields.However,the classical synthesis method of Cy5 needs several reaction steps,complex post-processing and condensation agent,so it is urgent to develop a simple and novel strategy for the synthesis of Cy5.Hence,a new synthesis method of Cy5 induced by visible light is designed in this paper.This method no longer needs condensation agent and photosensitizer and has good air-tolerance,the final product Cy5 dye could be obtained in one pot.On the other hand,after incubating the monomers with He La cells,Cy5 fluorophores can be generated in-situ in the cells under mild conditions,which could realize spatiotemporally controlled near-infrared fluorescence imaging and provide a new idea for lysosomes spatial resolution imaging.In the first part,we designed a new synthesis method of Cy5 induced by visible light.We synthesized a kind of indole heterocyclic quaternary ammonium salt molecule(TMI).The amino group of TMI could react with active ester molecules,and TMI can be regarded as condensation agent under the irradiation of visible light.The final Cy5 product was obtained by dissolving TMI,triethylamine and active ester in chloroform at the equivalent ratio of 2:4:1 and stirring at 65 ? for 12 h.The optical properties of Cy5 were studied.The highest yield was 8% and four Cy5 dyes were successfully synthesized by this method.The generation of photogenerated carbon radical(TMI·)was verified by electron spin resonance assay.The visible-light-activated coupling product(TMI-DMPO)was obtained by preparation-level HPLC separation.Combined with the other experimental results,the reaction mechanism mediated by visible-lightinduced free radical was proposed.The reaction process was similar to trimerization and the TMI was used as condensation agent under visible light irradiation to generate Cy5 product.However,the visible-light-induced synthesis of Cy5 could not be achieved when replacing the amino group with methyl group.Second,Cy5 fluorophores was generated in-situ in the cells under mild conditions for near-infrared fluorescence imaging.TMI was incubated with He La cells,and the red fluorescence signal was detected after irradiation.The in-situ generation of AmCy5 was identified by fluorescence spectrum and mass spectrometry.The colocalization experiment showed that the red fluorescence was generated from lysosomes but not other organelles.In addition,visible-light-induced Cy5 synthesis could also be achieved in QSG cells,and the biocompatibility is good.Endocytosis inhibition and isothermal titration calorimetric assay showed that TMI was binding with serum components and thus endocytosis happened.Cy5 was formed in-situ at mild temperature in the unique microenvironment of lysosome.When amino group was replaced by methyl or trimethylamino or bromine group,and co-incubated with He La cells at higher concentrations,Cy5 could not be synthesized in He La cells,which was consistent with in vitro assays.This strategy of in-situ generation of Cy5 fluorophores can be used for spatiotemporally controlled near-infrared fluorescence imaging and lysosome spatially resolved imaging.