Synthesis And Application Of PY-NBD Dyes Based On Pyronine

Fluorescent dyes play a key role in the field of biological imaging.With the continuous development of imaging technology,especially the emergence of super-resolution fluorescence imaging technology,the demand for high-performance fluorescent dyes has also increased.Super-resolution imaging technology not only requires the dye to have higher luminous intensity,but also has higher stability.Among the common fluorescent dyes,the 9-position aryl substituted pyronine dyes have become a popular dye in the field of optical imaging due to their high fluorescence quantum yield and stability.However,such dyes are generally prepared by aryl substitution of the 9th position of pyronine.This method has many synthetic steps,harsh reaction conditions and difficult synthesis,which greatly limits the further application of such fluorescent dyes.In response to this problem,in this paper,we explored a new synthetic method of 9-aryl substituted pyronine dyes.The research contents of this paper are as follows:1.Using the nucleophilic substitution reaction of singlet carbene,a 9-position aryl substituted pyronine dye was prepared by a one-step method,and the target compound PY-NBD was successfully obtained.The reaction conditions were systematically optimized and explored,and the optimal synthesis conditions were determined(reaction time 12 h;temperature 65 0C;solvent THF;reactant ratio Pyronin Y:t-BuOK:NBD-Cl=1:2:2).Compared with the synthesis method reported in the literature,this method is simple in operation(from the previous 5 steps to 1 step);greatly reduced the reaction time(from 5 days to 12 hours);Avoiding the use of the highly toxic reagent potassium cyanide and the flammable reagent butyl lithium greatly reduces the risk factor of the reaction and is beneficial to mass production.2.The luminescence performance of PY-NBD was further investigated by ultraviolet and fluorescence spectroscopy.The maximum absorption of PY-NBD is located at 545 nm and 575 nm,respectively.When excited at 575 nm,its fluorescence emssion at 610 nm is clearly quenched;when excited at 545 nm,the compound emits intense yellow-green fluorescence with a maximum emission wavelength at 575 nm.This hypothesis was also confirmed by calculations at the B3LYP/6-31G(d)level,including the PY-NBD excitation energy E(eV),array intensity f and corresponding value,as well as the electronic configuration.When PY-NBD reacts with reducing compounds such as GSH,sodium sulfide and sodium bisulfite,the nitro group in PY-NBD is reduced to an amine group,which causes the intramolecular TICT effect to be blocked.3.In cell experiments,PY-NBD dye exhibits good cell permeability,and 1 ?M can stain cells well.The cytotoxicity test by MTT method showed that the compound had almost no cytotoxicity at 1 ?M,which further indicated that the compound had the possibility of live imaging.Cell experiments with mitochondrial colocalization showed that,compared with the commercially available mitochondrial marker Rhodamine 123 and Deep Red,the Pearson coefficient of PY-NBD is 0.95,indicating that PY-NBD can be used as a mitochondrial marker.