Syntheses Of Two-photon Red-emitting Organelle/RNA Probes And Their Application In High-fidelity Fluorescence Imaging In Living Cells

For bioassay,high-fidelity fluorescent images can help researchers obtain the accurate information on targets and observe their changes.Improving signal-to-noise ratio(SNR)of fluorescent probes is an efficient method to improve the fidelity of images.For cell membrane or membrane bounded organelles,a molecular rotor is a good choice to obtain ultrahigh SNR probes.In nonviscous environments,the intramolecular rotation of the rotor relaxes the excitation energy resulting in significant quenching of fluorescence,whereas in viscous medium,the rotation is inhibited,thereby reducing the probability of nonradiating pathways and thus restoring fluorescence.Membrane just can provide a highly viscous microenvironment.We imagine that if a rotor can selectively target membrane,it should exhibit little background emission in their unbinding states and strong emission once gathered by the membrane.Thus,fluorescent probes based on rotor mechanism will possess ultrahigh SNR.Otherwise,developing the red-emitting two-photon fluorescent probes is another method to obtain ultrahigh SNR probes.First,red-emission can minimize the background noises and increase SNR due to avoiding intracellular autofluorescence in blue/green regions when imaging.Also,red fluorescence in the optical window of biological tissue with low Rayleigh scattering can penetrate the thick specimen.Second,two-photon fluorescence microscopy(TPM)possesses high detection sensitivity,deep penetration,no image distortion,low photodamage,and reduced photobleaching in comparison with confocal microscopy.Although endogenous fluorophores could produce fluorescence under two-photon excitation,their intensities are always weak due to very small two-photon absorption cross sections(?).If the ? value of aprobe is much larger than that of the endogenous fluorophores,the probe can provide a high SNR when imaging under TPM on the premise of keeping the advantages above.However,as single-photon excitation and two-photon excitation obey different selection rules,the ? values of conventional single-photon probes are too small to be applied in TPM.To meet current situation,this thesis mainly designs and synthesizes red-emitting two-photon fluorescent probes on a rotor mechanism for high-fidelity imaging organelles or RNA.First,we designed and synthesized red-emitting two-photon mitochondrial probes.In living cells,the morphology and numbers of mitochondria are changing,which is closely related to various damages and diseases.High-fidelity fluorescent images can help us observe their changes effectively.Currently,reports about red-emitting two-photon mitochondrial probes are still lack and fidelities of fluorescent images obtained by these probes arelowunder TPM.In chapter 2,we designed and synthesized four two-photon cationic compounds BCVPI,BCVTI,BCVII,and HJVPI.The rotor character of HJVPI which was synthesized by modifying the classic rotor 9-(dicyanovinyl)julolidine(DCVJ)was better than that of other three compounds.Also,HJVPI showed relatively red-shifted emission.As a rotor,its single-photon excited fluorescence(SPEF)and two-photon excited fluorescence(TPEF)emissions were very weak in low-viscosity medium,but both of them were very strong in high-viscosity glycerol(Gly).HJVPI with ultrahigh SNR was capable of clearly imaging mitochondria in immortalized and normal cells.The double assay of HJVPI and S-11348 showed that HJVPI could stain mitochondria in living cells.Moreover,the new probe possessed good membrane permeability,low toxicity,high photo stability,large Stokes shift,and excellent counterstain compatibility with Hoechst 33342.All these properties have ranked it as one of the best red-emitting mitochondrial TPEF probes.Second,we designed and synthesized wash-free red-emitting two-photon plasma membrane probes.As cell barrier,plasma membrane plays an important role in biological systems.It not only can protect the integrity of the cell through selectively allowing certain substances to pass through,but also is closely linked to signal transduction,cell differentiation,cell fusion,and other biological activity.For living cells,the morphologies of cells will change in response to external stimuli.The outline of the plasma membrane is just the embodiment of the cell morphology.Mild stimulation may cause cell morphology changes,even physiological changes,such as washing.Washing could alter the cellular environment,and affect the physiological and structural integrity of living cells.Also,the unbound probe molecules inside the cells are difficult to be completely removed by washing.In addition,although modern medicine is advanced,some diseases are still stay in research stage.So the frozen tissues which could be retained for a long time are needed for research.If one probe can label the plasma membrane,it will help us to easily identify the cell.Currently,reports about red-emitting two-photon plasma membrane probes are still lack.In chapter 3,we designed and synthesized four two-photon cationic compounds BCVOP?,BCVO?,ASOP?-1,and HQVOP?.ASOP?-1 and HQVOP?,which were based on the classic rotor,could label the plasma membranes in the living cells,fixed cells,and tissues without washing.Third,we designed and synthesized a wash-free red-emitting two-photon RNA probe.As the genetic information carrier of cells,RNA is involved in the synthesis of protein and regulating gene expression.Compared with mitochondrial probes and plasma membrane probes,RNA probes based on small organic molecules are rare,let alone red-emitting two-photon probes.Some of the facts that restricted its development include the diminished affinity to RNA than to double-stranded DNA,the nonspecificity in binding with proteins due to the hydrophobicity of probe molecules.Moreover,there is limited knowledge of the interaction mechanisms between RNA and fluorescent probes,including outside,groove and intercalative binding,compared with the wealth of papers on DNA biosensors.All increased the difficulties to develop an RNA biosensor,which also promotes the demand for a library of compounds.Currently,among 18 RNA probes reported,only 15 probes could be used in living cells,but some of these probes should need 12 h to enter living cells.Especially,they need to be washed when label cellular RNA.As we all known,washing could alter the cellular environment,and affect the physiological and structural integrity of living cells.In chapter 4,we screened 8 molecules ultimately obtained only one RNA probe MOBTI.As a rotor,MOBTI could light up RNA in SiHa,HeLa and BMSC cells within 20 min incubation time without washing in LSM and TPM.It is much more photostable than commercial RNA probe SYTO RNA-Select.Last,we designed and synthesized wash-free red-emitting two-photon probes for sensing mitochondrial membrane potential(MMP).MMP is a key parameter representing mitochondrial functions,and its decrease can imply the disruption of mitochondrial electron transport chain,which results in cellular dysfunction or even death.So,the feedback from different situations of MMP,such as normal,decreasing,and vanishing,is practically useful in biomedical research and diagnosis of the related diseases.To date,there are mainly two kinds of probes:(1)fluorescence intensity probes,such as rhodamine 123,TMRE,and TMRM.But it is difficult to distinguish MMP decreasing from vanishing just by the fluorescence image.(2)Fluorescent dichromatic probes,such as JC-1.However,the staining concentration needs to be adjusted repeatedly before every experiment:it is difficult to form J aggregates when using too low staining concentrations,and high concentrations may produce block precipitates due to its very poor water solubility,which brings great inconvenience.As we all known,washing could alter the cellular environment,and affect the physiological and structural integrity of living cells.So washing will affect MMP.In chapter 5,we designed and synthesized two two-photon cationic compoundsHMOBT? and ASHTI.As rotors,HMOBT? and ASHT? could not be washed when staining cells.HMOBT? and ASHT? would target RNA or NA when MMP changed.The changes of MMP could be easily observed by the fluorescence position.In conclusion,a series of red-emitting two-photon probes for high-fidelity imaging organelles or RNA have been designed and synthesized.When staining cells,these wash-free probes could not only reduce the injury to cells,but also give the accurate information on targets,especially for mitochondria/plasma membrane whose morphology and numbers are changing.This thesis will lay the foundation to get the commercialization of mitochondrial probes,membrane probes and RNA probes.