Synthesis And Fluorescence Imaging Properties Of Organelle-Targeted Probes

Some species such as metal ions,reactive oxygen species,reactive sulfur species reactive carbonyl species and enzymes play crucial roles for participating the metabolism of intracellular materials,regulating the corresponding biological functions,and maintaining the homeostasis and normal physiological functions of the living cells because of their high reactivity and activity.Therefore,the detection and monitoring of the species is of great biological and medical significance for researching their biological functions and effects,and revealing the physiological function and pathology related to the human health and diseases.It is also important to monitor changes in environmental indicators.In recent years,thanks to the development of the fluorescence imaging techniques,the fluorescent probes techniques have been emerged as a powerful means to detect and track species.Some conventional dyes have short emission wavelengths and narrow stokes shifts,which can generally results in photo-damage and self-quenching due to back scattering from biological samples.Herein,to solve some key problems of the reported probes,the highly selective fluorescent probes based on the original near-infrared fluorescent dyes synthesized by using different strategies to modify the cyanine dyes and xanthene skeleton.Meanwhile,the organelle-targeting labeling of dyes/probes is achieved without introducing additional targeting groups.The main contents from following aspects:With the above concerns in mind,we developed a new lysosome targeting NIR xanthene-benzothiozolium dye DQ1,by the substitution of the dimethylmethylene group with the S atom to regulate the absorption and emission wavelengths of the dye.The dye DQ1 has good photostability,chemical stability,large stokes shifts and other fluorescence characteristics.Its absorption and emission wavelengths are located in the light transmitting region of biological tissue,and the maximum emission wavelength is about 730 nm.A series of lysosomal targeting probes（DQ1-Pd,DQ1-Cys,DQ1-H₂S）were respectively prepared.And their spectral properties and cell imaging properties were investigated.The novel fluorescent probes based on ICT mechanism were developed for imaging in living cells,exhibiting long emission wavelength,high selectivity,rapidly response time,low detection limit,membrane permeability,low cytotoxicity.The color changes dramatically with specific recognition,from purple to blue.The probe DQ1-Pd was used to detect the level of nM,and the strip was successfully used to detect water samples and drugs.More importantly,it did recognize palladium in lysosomes.The probe DQ1-Cys can respond to cysteine rapidly（10 min）,and the detection limit（5.9 nM）is far lower than the cysteine content in cells,which can locate endogenous cysteine in lysosomes.The probe DQ1-H₂S has high sensitivity（detection limit of 0.12μM）and rapid recognition（15 min）.It can target endogenous hydrogen sulfide in lysosomes and can be used for fluorescence imaging in living cell lysosomes.The thioxanthene-benzothiozolium dye DQ2 has been synthesized by substituting the O atom in xanthene with S atom.The maximum absorption wavelength and emission wavelength of the dye DQ2 in different solvents are in the near infrared region,and the maximum emission wavelength is about 830 nm.The dye DQ2 has good photostability,chemical stability,cell membrane permeability,low cytotoxicity,and obvious lysosomal targeting.The derived targeting probes（DQ2-Pd and DQ2-Cys）showed good selectivity and sensitivity,fast response time and wide pH range.They can recognize exogenous and endogenous substances in lysosomes,and can be used in fluorescence imaging in biology and chemistry fields.The maximum absorption wavelength of the probe DQ2-Pd is 585 nm,and the maximum emission wavelength is795 nm.It was developed for imaging palladium,exhibiting low detection limit（21.3nM）,rapid response time（200 s）.It can not only detect trace palladium in actual water samples and drugs,but also target palladium in lysosomes of He La cells.The probe DQ2-Cys could select cysteine specifically and quickly（15 min）with good sensitivity and low detection limit（0.31μM）.It can locate cysteine of lysosome in He La cells,with good membrane permeability and cellular compatibility,and realize cell fluorescence imaging.Reversible probes FY-pH and EY-pH were synthesized by common cyanine dyes and barbituric acid,they responded to pH based on protonation and deprotonation of nitrogen atoms.Their maximum emission are located near 690 nm.They displayed fluorescenceenhancement,anti-interference ability and stability.Their pKa values are3.32 and 2.96,which can be used for pH detection under extremely acidic conditions.In the absence of introducing additional positioning groups,the probe FY-PH which had no lysosomal localization ability showed mitochondrial targeting,and the probe EY-PH with benzothiazole structure is lysosome-targetable.The dyes（YY1,YY2,YY3）have the characteristics that the quinone structures have weak absorption and long fluorescence,and the enol structures have strong absorption and short fluorescence.Moreover,the absorption and emission wavelengths of the dyes（YY1,YY3）can be fine-tuned by changing substituents to provide a direction for accurate detection of a certain wavelength.The thioxanthene-quinoline dyes（YY1,YY3）containing sulfur atoms is lysosome-targeted.The xanthene-quinoline dye（YY2）is not lysosome-targeted.The probe YY2-Pd for the detection of Pd²⁺has been successfully synthesized,and preliminarily realized its application in biology.