Based On Benzoxazine And Rhodamine Near Infrared Fluorescence Probes Design Synthesis And Biological Applications

Fluorescence imaging technology has been widely used in many fields because of its small interference,easy operation and high sensitivity.In the past six years,this technology has won the Nobel Prize in Chemistry twice,which is sufficient to illustrate the importance of the technology.And near infrared fluorescence probe is one of the most core technology.The ideal near-infrared fluorescent probe should have the advantages of long wavelength absorption and emission,high fluorescence quantum yield,high light stability,and good biocompatibility,etc.Hydrogen ions are the basis of the life system and participate in most cell activities such as cell growth,proliferation,and apoptosis.The acidity of cancer cells is stronger than that of normal ones,and lysosomes are the most acidic organelles in cells.Therefore,the pH of the lysosomes of cancer cells may be smaller than that of normal ones,but few researchers study the difference between them.Nitrite is also a signal molecule in the human body,with the importance of antibacterial antivirus,dilation of blood vessels,maintenance of the role of cardiovascular system balance.At the same time nitrite is also a carcinogen.Therefore,the development of new pH or nitrite near infrared fluorescent probes is very important.In this paper,three kinds of fluorescent molecular probes were designed and synthesized with benzoxazine and rhodamine as maternal,and their optical properties and biological properties were in a thorough study.The details are as follows:1.Based on a novel fluorophore-based photo-induced effect?FBR-PET?mechanism,three benzoxazine-based near-infrared pH probes 5a-c were designed and synthesized.The results showed that the three probes 5a-c showed the maximum fluorescence emission was near 634 nm,and the pK_a were 5.63,5.41 and 5.65 respectively,and the fluorescence intensities were 2.6 times,9.6 times and 18.2 times respectively.Fluorescence confocal images were performed with HeLa,KB cancer cells and V79 normal cells.The fluorescence emission of probe 5c exhibited near-OFF at pH=4.0 and a pH response that exhibited ON at pH=5.0-6.0.Cell experiments show that probe 5a has a strong emission in the lysosomes of cancer cells?HeLa and KB?and normal cells?V79?,is a near-infrared lysosomal targeting probe.Whereas probes 5b and 5c are shown to emit in the lysosomes of normal cells V79 and show only a very weak background emission in cancer cells?HeLa and KB?,indicating that probe 5b-c has potential applications in selective differentiationof normal cells from cancer cells.2.Based on the photoinduced electron transfer mechanism?PET?,a near-infrared nitrite probe based on benzoxazine was designed and synthesized,and its optical response to sodium nitrite was studied in detail.When the nitrite is added,the UV absorption of probe 8 changes in the rate of change,and the change of the sample solution from blue to purple is observed by the naked eye,and the near OFF-ON fluorescence is observed.The fluorescence intensity is at the maximum emission of 636 nm,and there is a linear relationship between the fluorescence intensity of probe 8 and the concentration of NaNO₂when the NaNO₂ concentration is in the range of 0.0-2.6 equivalents.The detection limit of probe 8 for nitrite was 45 nM.HeLa and V79 cell imaging experiments show that probe 8has the selective use of exogenous nitrite.3.Two rhodamine-based near-infrared pH probes?probes 10a-b?were designed and synthesized by a simple one-step reaction.pH titration experiments show that the probe10a-b exhibits a near OFF-ON fluorescence response to the hydrogen ions as the acidity enhances,breaking the mechanism of rhodamine's simple open-loop ringing.The pK_a of the probe 10a is 8.28 and the pK_a of the probe 10b is 12.1 because the hydrogen bond is contained in the probe 10b,so that the deprotonation of the hydroxyl group is more difficult leading to pKa increased.Ion selectivity and competitiveness indicate that both common ions and amino acids do not interfere with hydrogen ions.Finally,HeLa and V79cell experiments show that probe 10a can be used to detect cytoplasmic pH.