Construction Of Fluorescent Probes Based On Pyran-coumarin And Rhodamine For The Detection Of Disease-related Biomolecules

Fluorescent probes have been widely used in various fields due to their unique advantages.Small molecular fluorescent probes play an important role in the detection of biomarkers in biological systems due to their easy synthesis and modification and easy entry into cells.In this thesis,we constructed six new types of fluorescent probes using pyran-coumarin and rhodamine fluorophores to detect various biomarkers and successfully apply them to image a variety of biological processes and diseases.1.We first designed and synthesized a novel hepatocyte-targeting ratiometric fluorescent probe（GCP）to detect H₂O₂.In this probe,benzopyran-diethylaminocoumarin is selected as fluorophore and galactose as hepatocyte-targetable group.The probe itself has near-infrared emission characteristics（706 nm）.After reaction with H₂O₂,a large emission shift from 706 to 482 nm（224 nm）is observed.In addition,the probe GCP has a good hepatocyte-targeting ability and can be used to monitor the upregulation of H₂O₂ in Hep G2 cells during drug-induced liver injury.Most importantly,the probe GCP can target the liver of zebrafish to accurately detect H₂O₂in the liver,providing information for the diagnosis of drug-induced liver injury.2.Based on the research on the diagnosis of drug-induced liver injury,we continued to study the remediation of different hepatoprotective medicines on drug-induced liver injury.In order to make the probe have longer emission wavelength,benzopyran-quinolizine-coumarin is selected as the fluorophore.And a novel liver-targeted near-infrared fluorescent probe（Gal-NIR）is synthesized for monitor ONOO^-.The probe has high sensitivity and selectivity for ONOO^-.In addition,Gal-NIR can specifically target the hepatocyte,which can be used to evaluate the fluctuation of ONOO^-during APAP-induced liver injury in mice and the repair effect of hepatoprotective medicines.3.On the basis of the previous works,we found that benzopyran-coumarin is an excellent fluorophore,but it is not easily linked to the recognition group,which makes it impossible to detect other biomarkers.Here,we adjust the amino group of benzopyrane to make it an optically controllable“switch”.By connecting allyl formate to benzopyran-coumarin,a near infrared ratiometric fluorescent probe（CP-CO）is constructed to detect CO.Due to the spirolactone structure of the probe,CP-CO itself showes strong coumarin emission（490 nm）.After reacted with CO and Pd Cl₂,a notable enhancement of emission intensity at 690 nm can be found,which results in an obvious red-shift of emission（200 nm）.Moreover,the probe can be used to ratiometric monitor exogenous and endogenous CO level in Hep G2 cells.Most importantly,CP-CO can visualize the upregulation of CO under LPS-induced oxidative stress in zebrafish model.4.A rhodamine-dexylactam based fluorescent probe（d RB-OPD）is designed and synthesized for fast and selective detection NO.The probe shows fast response to NO within 40 seconds with 170-fold fluorescence enhancement.In addition,the probe shows high selectivity towards NO over dehydroascorbic acid（DHA）,ascorbic acid（AA）,and methylglyoxal（MGO）.In particular,the probe can avoid the serious interference of cysteine in the detection of NO.Moreover,d RB-OPD has the ability to monitor NO in cells.5.Based on the previous work,we found that rhodamine is indeed an excellent fluorophore,but its wavelength is relatively short,which is not suitable for in vivo imaging in mice.Therefore,we synthesize a Si-rhodamine fluorophore by substituting O in xanthene with Si,and make it possess near infrared emissin.A tumor-targeting near infrared fluorescent probe（Bio-Si R）has been developed to image ATP in real-time by connecting diethylenetriamine recognition group.When Bio-Si R reacts with ATP,a turn-on fluorescence at 675 nm（NIR region）is observed clearly,which is suitable for its application in vivo.In addition,with the assistance of biotin,the probe successfully images endogenous ATP in real time in tumor-bearing mice.6.Through the previous work,we found that a single biomarker could not provide enough information to reveal complex biological processes.Therefore,we design a dual-response fluorescent probe（Lyso-NRB）to simultaneously monitor ATP and polarity changes in autophagy.The probe itself is non-fluorescent.After the decrease of polarity,Lyso-NRB exhibits significant green emission due to the unique ICT effect.Upon the addition of ATP,the probe can react with ATP to rapidly open the spirocyclic of rhodamine and a strong red emission can be observed.In addition,we successfully apply it to image lysosomal polarity and ATP fluctuations during H₂O₂ or starvation induced autophagy in living cells.