Design,Synthesis And Imaging Applications Of Asymmetric Rhodamine-based Fluorescent Dyes

Benefited from the advant ages of adjustable properties and easy metabolism,etc,rhodamine dyes have been widely us ed in protein labeling,biomolecular detection,fluorescence-guided surgical,etc.However,as imaging techniques evolving and biomedical research being increasingly complex,classical rhodamine dyes are not equal to modern imaging applications.In response to this problem,chemists have developed various met hods to improve the optical properties of rhodamine dyes,such as redshifti ng wavelengths,improving brightness and photostability,increasing Stokes shifts,enhancing water solubility,and adding modification sites,etc.Most improvements,however,can only enhance one property of dyes,or sacrifice one property of dyes for another one.Although the fluorescent dye s with excell ent property in a single respect have a good perform ance in some applications,the unbalaced natures also limit their scope of application.Therefore,it is important to develop rodamine dyes or rodamine-like dyes with excellent overall proper ties and that meet the needs of different imaging applications.To meet the requirements of the advanced imaging technologies and complex biomedical applications,we developed a series of novel high-performance rhodamines through small structural modificat ions in the asymmetric rhodamine dyes（DQFs）.And applying these dyes as fluorescent reportors,we constructed a variety of fluorescent probes for proteins labeling and biomolecular det ection.These probes were then used for cell and in vivo imaging.The s pecific researches include the following aspects:1)For the reported modification methods,it is difficult to simultaneously improve brightness and photostability,and Stokes shift,which limits the application area of these rhodamines.To address this pr oblem,on the basic of the asymmetric rhodamine dyes with large Stokes shift developed by our group earlier,we precisely regulate the electron cloud density of the tetrahydropyrazine group by introducing substituents with different push-pull electron capabilities,and explore the effect of the push-pull electron capabilities of the substituents on the fluorescence brightnes s of asymmetric rhodamine dyes.The experimental results show that the trifluoroethyl-substituted asymmetric rhodamine dye YL578 has low solvent sensitivity and can effectively inhibit the formation of t wisted intramolecular charge transfer（TICT）.As a result,YL578 displays the higher brightness and photostability,and larger Stokes shi ft than the classical rhodamine dye（Rh B）.Moreover,this strategy can be ext ended to dyes such as pyronin,Rhodol,coumarin and BODIPY,and enhances the multiple photophysical properties of these dyes.The strategy generat es a variety of fluorescent dyes with excellent properties for advanced imaging tech niques and complex biomedical applications.2)On the basic of YL578,we further constructed a fluorescent probe（YL578-Halo）for labeling Halo proteins.The probe exhi bits significantly improved fluorescence brightness,phot ostability,Stokes shift and hi gher signal-to-back ratio compared to the Rh B-based probe Rh B-Halo.In stimulated emission depletion microscopy（STED）imaging,applying the same depletion laser（775 nm）,the probe has better photos tability than commercial probes such as JF608-Halo,and m aintains more than 50%fluorescence brightness over consecutive 9 frames of STED photography with imaging resolution of 57±5nm.Benefited from t he outstanding photostability,YL578-Halo was used for3D-STED super-resolution imaging of mitochondrial import receptor Tomm20-Halo Tag.Moreover,thanks to large Stokes shift,the probe can be used multicolour nanoscopy through combining with thaditional short-Stokes-shift probes under a single depletion laser（775 nm）,which is important for studying the function and interaction of proteins.3)Although the fluorescent dyes devel oped in the above work have superior photophysical properties and have promising applications in super-resolution imaging,activated probes constructed based on t hese dyes have strong back ground fluorescence,low response sensitivity and small fluorescence increas e multiples.To address the issue,in t his chapter,we proposed a strategy combining molecular design and structural screening,transformed the carboxylic acid in the Rhodol dyes d eveloped in Chapter 2 into ring-closing amide,and synthesized a s eries of novel dyes.Spectroscopic analysis shown that the introduction of N,N-dimethylsulfamide allows the dye to remain open-loop state but the hydroxymethylated dyes to remain close-loop state,significantly improving response multiples and detecti on sensitivity of probes.Moreover,the strategy can be extended to classic rhodol dyes,and Chang Sha（CS）dyes with near-infrared fluorescence,providing a variety of ideal fluorophores for th e design of fluorescent probes det ecting trace and low-activity analytes in cells and living organi sms.4)The trifluoroethyl group,while conferring superior photophysical properties to YL578,also occupies a m odification sites,which limits the widespread applicati ons of the dye.In t his chapter,using physical organic chemistry as a guide,we replaced-CF₃（0.54）in YL578 with-CH₂NH₃⁺（0.53）,which has a similar Hammett constant,and successfully developed another class of fluorophores NH2-Yue Lu dyes（NYLs）with high brightness,high photostability and large Stokes shift.The experimental results indicated that amino protonation plays a dominant role in driving the brightness of these dyes.Imging experiments showed that NYLs can be taken up more by canc er cells（5.4-16.4-fold）,which endows them the capability of distinguishing cancer and normal cells.Subsequently,taking advant age of the reduced brightness of the dye caused by aminoacylation,we constructed an activated probe（NYL2-NQO1）for further in creasing the discimination multiples between cancer and normal cells（56-fol d）.Notably,the probe can retain in cancer cells after activation,enabling long-term imaging of cancer cells and tumor.The research provides a novel des ign way for the construct ion of fluorescent dyes/probes that specifically and l ong-term image cancer cells/tissues.