Synthesis And Biological Application Of Two Types Of Organic Fluorescent Probes Targeting Subcellular Structures

The visualizations of subcellular structures in live cells,such as organelles and endogenous proteins,are beneficial for studies of the dynamics and the relationship between structure and function,which is of great significance for the study of bioscience.With the fast development of fluorescence microscopy for live-cell imaging,labeling methods for live cells are also constantly developing.At present,the main labeling methods for live cells include fluorescent proteins,chemical tags,organic small molecule fluorescent probes,quantum dots and nano-particles.On account of the the larger size of the fluorescent protein,the target protein could be affected functionally and overexpressed,which is not beneficial for visualization of subcellular structure of live cells.Quantum dots and nano-particles cannot be the best choice for live-cell labeling,because of the cytotoxicity for cells.Chemical tags,such as SNAP-tag and CLIP-tag,can covalently attach to different organic fluorescent dyes by the substrates BG and BC derivatives.Organic small molecule fluorescent probes can be bound covalently to different organic fluorescent dyes by the different recognition groups,such as 3P and docetaxel.According to the above-mentioned,a series of organic fluorescent probes can be designed and synthesized for live-cell labeling.However,most commercial organic fluorescent dyes cannot directly enter the living cell due to the impermeability,limiting their applications in live-cell labeling.Based on the above problems,a permeable organic fluorescent dye,two impermeable organic fluorescent dyes and four recognition groups were synthesized.Finally,organic fluorescent probes for microtubules,microfilaments,endoplasmic reticulum and nucleus were synthesized by the dyes covalently attached to recognition groups.In addition,organic fluorescent probes for mitochondria and lysosomes were designed and synthesized by using commercial organic fluorescent dyes,and these probes were used to study the labeling of living cells.The main contents are shown as follows:?1?A permeable organic fluorescent dye SiR was synthesized by using some simple initiators.In addition,two impermeable organic fluorescent dyes TMR and 5?6?-carboxyl Rho110 were synthesized by using some simple initiators for different imaging channels.?2?The recognition group BG of SNAP-tag and its derivatives CBG,SNAP-BHQ2and the recognition group BC of CLIP-tag were synthesized by using some simple initiators.Finally,the recognition group Docetaxel-C₆-NH₂ of targeted microtubules was designed and synthesized,which can reduce the difficulty of microtubule probe designed and synthesized,and can be connected with commercial organic fluorescent dyes to form a series of microtubule probes.?3?Organic fluorescent probe Tubulin-SiR was synthesized by the organic fluorescent dye SiR and the recognition group Docetaxel-C₆-NH₂,which can label microtubules specificity for the first time in live clinical human platelet cells.organic fluorescent probes SNAP-SiR and CLIP-SiR which were also synthesized by the organic fluorescent dye SiR and the recognition group BG and BC achieve the labeling specifically for endoplasmic reticulum,nucleus and microfilament.Furthermore,a new type organic fluorescent probe SNAP-Q-Rho110 was designed and synthesized,which can improve the fluorescence background for live-cell imaging.In addition,Mitochondrial probes 3P-FITC,3P-Cy3B and lysosome probe Morph-FITC were designed and synthesized by commercial organic fluorescent dyes FITC and Cy3B,which were used for live-cell labeling.The co-localization study between Morph-FITC and commercial lysosomal probe LysoTracker Red demonstrated the excellent specificity of Morph-FITC for lysosomes.