Design And Synthesis Of Novel Fluorescent Probes For Distinguishing Lipid Rafts And Non-raft Microdomains And Detecting Intracellular Microenvironments

Intracellular micro structures and microenvironments play crucial roles in various and complex physiological processes.Lipid raft and non-raft domains are two kinds of functional microdomains in plasma membranes.Amongst,lipid rafts are involved in signaling,virus entry,endocytosis,exocytosis,formation of protein clusters and many other important processes.Meanwhile,some kinds of immune-related proteins tend to insert into non-raft domains,and thus non-raft domains play essential roles in immunization.The efflux of cholesterol was also found to be related to non-raft domains,and abnormal levels of non-raft domains would lead to arteriosclerosis and some other diseases.Furthermore,both lipid rafts and non-raft domains are involved in Alzheimer's disease.The pH value is an important parameter of intracellular conditions,and the conformation change of proteins and the catalytic efficiency of enzymes can be directly influenced by pH value.Consequently,the pH values in cytoplasma,mitochondria,and lysosomes are usually maintained in a narrow range.Abnormal changes in pH values would result in disaster and even the cell apoptosis,which are also a sign of many diseases,including cancer.Viscosity can influence the rates of energy and object transduction and biochemical reactions.Especially,viscosity can control the reaction rates in which protein-protein contact processes,enzymes,and transmembrane transports are involved.Abnormal changes of viscosity are closely related to arteriosclerosis,Alzheimer's disease,diabetes,and many other diseases.Therefore,in situ and real-time observation of cellular microstructures and microenvironments are of great significance for biology,pathology,and diagnose.Fluorescence imaging method has unique advantages in the observation and monitoring of cellular micro structures and microenvironments.In comparison with other methods,including microelectrode detection,atom field microscope,and scanning electronic microscope,fluorescence imaging method enables the in situ,real-time,and dynamic visualization of microstructures and microenvironments with negligible injury to biosamples,which is essential for biological investigations.Consequently,the probes for intracellular microenvironments and micro structures have gained intense attention during the past decades.For example,polarity sensitive dyes have been modified as probes for dual-color imaging lipid rafts and non-raft domains in plasma membrane.Viscosity sensitive dyes have been modified as probes exclusively illuminating lipid rafts.Many groups capable of accepting and donating protons have been used for the design of pH probes,based on intramolecular charge transfer(ICT)or photo-induced energy transfer(PET)mechanisms.Viscosity sensitive molecular rotors have been used to detect the intracellular viscosity changes.Although plenty of works have been delivered to exploit and improve the probes,there are still limitations in their application in living cells.For instance,the emission spectra difference of polarity probes in lipid rafts and non-raft domains is modest,which makes it hard to clearly distinguish the two microdomains in plasma membrane of living cells.The emission spectra difference of ratiometric pH probes in basic and acid conditions is modest,which increases the difficulty in collecting emission signals in two channels during ratiometric imaging experiments.Accordingly,to break through these limitations,it is essential to improve the properties of current probes,and especially to develop novel fluorescent probes based on new mechanism.To overcome the problem that emission spectra difference of polarity probes in lipid rafts and non-raft domains is modest,in this work,aggregate/monomer type of probes,2,7-9E-BHVC12 and 3,6-9E-BHVC12,were developed with a special kind of fluorescent dyes whose emission spectra are different in monomer and aggregation states,based on the intrinsic difference in packing state of lipid rafts and non-raft domains.Lipid rafts were closely and packed in order,while non-raft domains were loosely packed out of order.Consequently,many fluorogens can insert into non-raft domains,while be refused by lipid rafts.We have constructed and synthesized the two fluorescent probes by linking two long alkyl chains to such fluorogens.According to the experimental results from giant unilamellar vesicles,the two probes can partition into loosely packed non-raft domains and give yellow fluorescence,while form aggregates on the surface of lipid rafts and give red fluorescence.In particular,the emission spectra difference in lipid rafts and non-raft domains of 2,7-9E-BHVC12 is up to 110 nm,much larger than that of polarity probes(50 nm).Consequently,2,7-9E-BHVC12 can clearly distinguish the two microdomains,and the lipid rafts and non-raft domains of plasma membrane in living cells have been successfully and clearly imaged with dual colors.The imaging results indicate that the distribution of the two microdomains in normal cells was different from that of cancer cells.Therefore,2,7-9E-BHVC12 is a qualified probe and powerful tool for the investigation of lipid rafts and non-raft domains.Moreover,the strategy to design dual-color probes for the two microdomains based on aggregate/monomer mechanism is feasible and reasonable,which can guide the design of corresponding probes.On the other hand,although numerous pH probes have been delivered,the emission spectra difference in basic and acid conditions of most ratiometric ones is confined to 100 nm.Moreover,two-photon ratiometric pH probes were rarely reported.The monotonous design strategies are responsible for this.To overcome this bottleneck,we have designed and synthesized six probes based on a pH sensitive ring opening/closing reaction,using carbazole,triphenylamine,and pyrene as the basic blocks.These probes were in ring opened form in acid conditions and gave long-wavelength emission,and would experience ring closing reaction in basic conditions,which could interrupt the conjugated system and largely blue-shift its absorption and emission wavelength.According to the experimental results,all the six probes can respond to the pH values in a ratiometric manner.Taking the excitation source of confocal and two-photon microscope,we have chosen three out of them for the ratiometric imaging of intracellular pH values under one-and two-photon microscopes.These probe exhibit different pKa values,and stain mitochondria or lysosomes in cells,dependent on their chemical structure.Consequently,the lysosomal pH values have been imaged in a ratiometric manner under one-and two-photon microscopes.Moreover,the emission spectra differences in basic and acid conditions are above 150 nm,even 210 nm.The targets and pKa values of these probes can be changed by tuning their chemical structures.Therefore,this kind of ring opening/closing reaction can be used to design one-and two-photon ratiometric probes with large emission spectra difference in basic and acid conditions.Molecular rotors are sensitive to viscosity.They exhibit very weak fluorescence in low-viscosity conditions,and the fluorescence can be largely enhanced in high-viscosity conditions.Therefore,these fluorescent dyes are greatly useful in imaging the high-viscosity targets with high signal to noise ratio(SNR)and monitoring the intracellular viscosity changes.In this work,using amino group as strong electron donor,and organic cation as strong electron acceptor,we have designed and synthesized three molecular rotors,R-1,R-2,and V-1,with D-?-A structures.The three probes can respond to viscosity.They exhibit very weak fluorescence in methanol,a low-viscosity solvent.The fluorescence can be greatly enhanced with the increasing amount of glycerol,a high-viscosity solvent.Amongst,probe R-1 and R-2 exhibit certain affinity to RNA,and can be used to image nucleolus with high SNR under one-and two-photon fluorescent microscope.Moreover,the nucleolus inside muscle tissues has been successfully imaged with R-2 under two-photon microscope.Considering that R-1 and R-2 are sensitive to viscosity,they have potential to image viscosity changes of nucleolus in living cells and tissues.Probe V-1 can target mitochondria in living cells.Moreover,it is very sensitive to viscosity,and its fluorescence can be increased to 60-fold in glycerol compared with methanol.Therefore,the viscosity change in mitochondria induced by nystatin treatment has been successfully imaged with probe V-1 under confocal microscope.These results indicate that V-1 can be used to monitor the viscosity change in mitochondria.Fluorescent dyes that are insensitive to pH values,viscosity,and polarity can serve as a platform for the design of fluorescent probes.Therefore,based on the experiences to construct environment-sensitive probes,we have designed a one-and two-photon excited green-emission fluorescent dye.By linking a weak electron donor and a strong electron acceptor to a simple conjugated system,we have constructed a small-structure two-photon fluorescent dye,which exhibits strong fluorescence in low-viscosity solvents.It possesses certain two-photon absorption cross sections(350GM),small chemical structure,high permeability to plasma membranes,and thus is applicable in bioimaging experiments.To testify it,a lysosomal probe and a mitochondrial probe have been designed and synthesized based on the fluorescent dye.Using the two probes,the lysosomes and mitochondria have been successfully imaged under one-and two-photon microscope.The two probes can pass through the plasma and organelle membranes in 20 min,which demonstrates the good permeability.Furthermore,the dye exhibit high photo-stability and low cytotoxicity,and therefore can serve as a qualified platform for probes design.And the strategy to construct fluorescent dyes with two-photon excited long-wavelength emission and suppressed TICT process using weak electronic donor and strong acceptor is valid,and can guide the design of small-structure dyes.In a nutshell,we have designed and synthesized aggregate/monor type of probes for clearly dual-color imaging lipid rafts and non-raft domains in plasma membranes.Using a pH-dependent ring opening/closing reaction,we have synthesized pH probes with large shift in emission spectra for imaging lysosomal and mitochondrial pH under one-and two-photon microscope.We have constructed molecule rotors sensitive to environmental viscosity for imaging nucleolus in cells and tissues with high SNR,and detecting the viscosity changes in mitochondria.We have also designed a small-structure two-photon fluorescent dye,serving as a platform for the design of membrane permeable probes.I think these probes can serve as powerful tools for the investigation on cellular microstructures and microenvironments,and our design strategies and the experimental results can give references for the following design of relative probes.