Synthesis Of Organic Fluorescent Probes For Subcellular Structures And Their Applications In Live-cell Imaging

Due to the limit of optical diffraction,traditional optical microscopes cannot obtain spatial resolution below 200 nm.Super-resolution fluorescence microscopy techniques successfully break the optical diffraction limit and improve the spatial resolution.Among super-resolution fluorescence microscopy techniques,structured illumination microscopy（SIM）is distinct in high imaging speed and low illumination intensities;thus,it is a standout tool for observing subcellular structures and their dynamics in live cells.However,the applications of SIM for observing subcellular structures remain challenging,as they need fluorescent probes not only can specifically label various subcellular structures but also possess high fluorescence intensity and excellent photostability.Now,there are two kinds of fluorescent groups mainly used in live-cell fluorescence imaging:fluorescent proteins and organic fluorescent dyes.Organic fluorescent dyes generally offer several advantages over fluorescent proteins,including high fluorescence intensity,broader spectral coverage and better photostability,thus they have potential advantages in super-resolution imaging technology.Numerous small-molecule organic fluorescent dyes with excellent properties have been commercialized,but most of them lack cell permeability.Therefore,the applications of organic fluorescent dyes for live-cell imaging are severely restricted.In order to solve this problem,twenty one cell-impermeable organic fluorescent probes with excellent optical properties were constructed by covalently linking organic fluorescent dyes with recognition units at first.This new probe design has the advantages of cost saving and flexibility in choosing dyes and recognition units.Secondly,five new peptide vehicles（PV-1、PV-2、PV-3、PV-4 and PV-5）were designed and synthesized.Then the best peptide vehicle PV-1 was selected,by simply co-incubating with PV-1,many cell-impermeable organic fluorescent probes were delivered into live cells and specifically labeled various subcellular structures.Using cell-impermeable fluorescent probes and PV-1,microtubule and ER dynamics were obtained in live cells by SIM.The main contents are shown as follows:（1）Commercially available recognition units Docetaxel and Hoecsht 33258 were modified to synthesize Docetaxel-C6-NH₂ and Hoecsht-C3-NH₂.Cell-permeable fluorescent dye Si R was synthesized.Tubulin-Si R and thirteen new cell-impermeable fluorescent probes for microtubules,lysosomes or nucleus were constructed by covalently linking recognition units Docetaxel-C6-NH₂,Morph-C2-NH₂or Hoecsht-C3-NH₂ with organic fluorescent dyes.（2）The recognition ligands BG-NH₂,BC-NH₂ and CA-NH₂ for Snap-tag,Clip-tag and Halo-tag were synthesized with simple compounds.Eight fluorescent probes for Snap-tag,Clip-tag and Halo-tag were constructed by covalently linking recognition units BG-NH₂,BC-NH₂or CA-NH₂with organic fluorescent dyes.（3）The design and synthesis process of peptide vehicles were introduced.Rh6GD with exposed carboxyl group was synthesized from commercially available fluorescent dye Rh6G.Using solid-phase synthesis and liquid-phase synthesis we synthesized control peptide vehicle df TAT and five new peptide vehicles PV-1,PV-2,PV-3,PV-4,PV-5.（4）The best peptide vehicle PV-1 was selected.By simply co-incubating with PV-1,synthetic cell-impermeable organic fluorescent probes were delivered into live cells and specifically labeled various subcellular structures.Then the dynamics of microtubule and endoplasmic in live cells were observed by SIM.Here we report a new simple but effective fluorescence labeling strategy that using non-covalent peptide vehicles cell-impermeable organic fluorescent probes were delivered into live cells and specifically labeled various subcellular structures.The successful application of PV-1 delivery cell-impermeable organic fluorescent probes in the specific fluorescent labeling of subcellular structures in living cells and the SIM imaging of living cells,proved this new labeling strategy is universal.These results expand the applications of fluorophores in the field of living cell research,provide an effective tool for super-resolution fluorescence imaging of living cells and a new idea for the design and applications of peptide vehicles.