Synthesis And Cell Imagings Of Xanthene-based Reactive Fluorecence Probes

Characterized by its high sensitivity,real-time spatial imaging and detection of targets in living cells or tissues with minimal damaging effects,the probe-based fluorescence imaging technology has become a facile and powerful tool for the detection of biologically relevant species with the ability to visualize morphological details and monitor various physiological processes in living systems.However,the imaging quality of this technique is strictly dependent on the photophysical properties of fluorescent dyes used for sample staining.To overcome the defects of rhodamine fluorescent dyes as optical signal reporters in the fluorescence imaging and lower organelle-targeting abilities of the reported fluorescent probes,in this thesis,we have carried out the following works:We present a facile synthetic approach for building novel rectilinearly ?-extended rhodamine chromophore TJ730.The maximum absorption wavelength is more than 600 nm and the maximum fluorescence emission is in near infrared region??730 nm?,and with a peak-to-peak Stokes shift larger than 100 nm.The TJ730 is cell permeable,low toxicity and biocompatibility,which can be used directly as a targetable NIR fluorescent tracer for lysosome staining with low background signal.To demonstrate the feasibility of the dye for designing of probes,fluorescent probes for ClO-with high sensitivity and selectivity have been designed and developed,and the fluorescence property and cell imaging of these probes have been investigated in detail.In addition,TJ730 possesses a rhodamine-like spirolactam structure,which provides a versatile platform for designing various probes by introducing an analyte-responsive factor on the spirolactone structure.To demonstrate the utility of this dye for probe design,NIR probes LC1,LC2,LC3 and LC4 are designed and synthesized for sensing Cu²⁺ and Hg²⁺ by using the spirolactam scaffold.Synthesis and properties of lysosome-targetable fluorescence probes.For the detection of ClO-,we synthesize a novel lysosome-targetable HOCl-selective probe PT-1 and its control compound PT-2 based on the photoinduced electron transfer?PET?mechanism.They show high selectivity and high sensitivity toward ClO-?the detection limits were 0.32 and 0.88 nM?.The smart sensing properties of the probe PT-1 enabled it to be used in living cells for monitoring of lysosomal-HOCl in live cells.In addition,a novel ratiometric lysosome-targetable fluorescence probe PT-3 by using the NIR dye TJ730 as signal reporter has also been designed and synthesized for ClO-detection and imaging in living cells.The probe demonstrates an excellent selectivity toward ClOwith fast and sensitive response?detection limit 3.2 nM?.Moreover,PT-3 exhibits accurate lysosome-targeting ability and real-time imaging of ClO-in lysosomes.At the same time,we design a bifluorophore-based universal platform with FRET nature and simultaneously incorporating a lysosome-targeting morpholine moiety within the platform.By taking advantage of the versatile spirolactone structure within the platform,a novel lysosome-targetable FRET-based probe for sensing Cu²⁺ ions in the lysosome of living cells has been synthesized.The probe exhibited high selectivity and sensitivity towards Cu²⁺.Meanwhile,using the dye TJ730 as a signal reporter,a novel lysosome-targetable NIR probe for highly selective and sensitive detection of Cu²⁺ has been synthesized.The probe is capable of imaging lysosomal Cu²⁺ in living cells,which further demonstrated the value of our NIR fluorescent dyes.A novel reaction-based fluorescent probe L,an ortho-diaminorhodamine derivative,for discrimination and detection of formaldehyde,methylglyoxal from other aldehydes.The detection principle of L is based on the reaction kinetics between the probe and analytes,as well as the difference of the fluorescence characteristics of reaction products.The probe shows distinct responsive patterns for formaldehyde?turn-on?and methylglyoxal?turn-off?by single wavelength excitation,which can be used for imaging of formaldehyde and methylglyoxal in living cells.