Design,Synthesis And Applications Of Ratiometric Fluorescent Probes Based On Hemicyanine And Flavonol Fluorophores

In organisms,sulfur dioxide?SO₂?could be produced from enzymatic reactions including decomposition of sulfur-containing amino acids,oxidation of hydrogen sulfide?H₂S?,etc.Studies showed that SO₂ at low concentrations could play important roles in physiological or pathological processes like blood pressure regulation,inflammatory response and so on.Like nitric oxide,SO₂ is also a gaseous signal transmitter in biological systems.SO₂ dissolves very easily in water and forms its derivatives:bisulfite?HSO₃^-?and sulfite?SO₃²-?anions.So the biological functions of SO₂ can be attributed to its derivatives.On the other hand,SO₂ and its derivatives have been applied widely in industries and our daily life.NaHSO₃ and Na₂SO₃ were used as food antioxidants and antibacterial agents;SO₂ dissolved in wine could serve as bactericide.However,the physiological roles of SO₂ and its derivatives have not been well studied yet due to the limitations of existing analytical methods.Therefore,it is highly urgent to develop real-time and in situ analytical methods with high sensitivity and high resolution.Fluorescence analysis method has attracted extensive attention in biology,pharmacy and medical science on account of its good biocompatibility and user-friendly control.Fluorescent probes based on small organic molecules develop rapidly in recent years on account of their short exploiting period,good photostability,low cytotoxicity,simple operation and intuitive signals.A few fluorescent probes for SO₂ derivatives have been reported.Most reported fluorescent probes of SO₂ derivatives just depended on fluorescence intensity changes?increase or decrease?at one emission wavelength.Detection based on one signal is susceptible to various environmental factors including the test systems,the probe concentrations and the instrument parameters.Ratiometric fluorescent probes possess two fluorescence emission peaks.The fluorescence intensity at one or two emission wavelengths will change upon reaction with SO₂ derivatives.Thus,high detection accuracy could be achieved by eliminating the interferences from environmental factors via cross-reference of the two emission wavelengths.Ratiometric fluorescent probes possess one or two fluorescent groups.The main modulating principles for the former are Intramolecular Charge Transfer?ICT?and Excited State Intramolecular Proton Transfer?ESIPT?,and the main modulating principles for probes based on two fluorophores include Fluorescence Resonance energy Transfer?FRET?and Through Bond Energy Transfer?TBET?.Flavonols are the typical representatives of ESIPT fluorescent dyes.Flavonol fluorophores have attracted wide attention due to the large Stokes shifts,good biological membrane permeability and low cytotoxicity.Most fluorescent probes based on flavonols work by modulating the "on-off" states of the ESIPT processes.FRET processes need the co-participation of two fluorophores,one as the energy donor and the other as the energy receptor.Hemicyanine fluorophores that usually have high fluorescence quantum yields,long emission wavelengths and easy structure modifications,are good candidates as FRET acceptors.The dissertation committed to the design,synthesis and applications of.ratiometric fluorescent probes.Four probes for SO₂ derivatives and one probe for H₂S were designed and synthesized.These probes used FRET or ESIPT as the main signal modulating priciples,and used hemicyanine or flavonol strucutures as the main singal reporters.The fluorescence properties and biological applications were also investigated.Chapter 1,the basic concepts,the fluorescence signals as well as its regulation principles were introduced briefly.The advances of fluorescence probes for SO₂ derivatives were summarized.The advances of fluorescent probes based on hemicyanine or flavonol structures were also summarized.Chapter 2,a new FRET platform was designed and synthesized using a hemicyanine fluorophore as energy and analyte "two-in-one" receptor.The dye was also applied in ratiometric detection of SO₂ derivatives.The FRET platform has high energy transfer efficiency with significant ratio characteristics.The probe could target mitochondria and differentiate normal hepatocytes?L-02?from cancerous hepatocytes?HepG2?due to their different endogenous bisulfite levels.Chapter 3,a new ratiometric and colorimetric probe?HCy-NBD?was synthesized for detection of SO₂ derivatives.The new FRET template was used in the probe design with a hemicyanine fluorophore as the acceptor and NBD fluorophore as the donor.Detection with high sensitivity,fast response as well as naked-eye recognition was achieved.Chapter 4,a flavonol fluorophore as the energy donor was introduced into the former FRET template,thus linking the ESIPT process with the FRET process in one compound.The ESIPT property of the donor could result in large Stokes shift;the acceptor could also have large pseudo Stokes shift due to the ESIPT and FRET processes.Thus,probes based on the ESIPT-FRET template would have large?pseudo?Stokes shifts both before and after addition of the analytes.Probe L-HF1 also used hemicyanine as the energy receptor,which has high reaction specificity toward SO₂ derivatives to improve the sensitivity and selectivity.Compound L-HF2 was obtained by transferring the free-OH of probe L-HF1 into an ester group.No FRET fluorescence signal changes were detected because of the inhibited ESIPT process.This further supported the importance of ESIPT for FRET.Similarly,this probe was applied to image bisulfite in cells and demonstrated good ratio characteristics with low cytotoxicity.Chapter 5,to reduce the overlap between the emission band of the donor and that of the acceptor,and increase the resolution of the two emission peaks as a result,probe L-HF3 was designed and synthesized based on the ESIPT assisted FRET template.Due to the long wavelength or near-infrared emission property of the receptor fluorophore TCF,the probe has lower fluorescence background and smaller spectral overlap,thus resulting in a greater degree of ratio changes and more reliable detection data.At the same time,probe L-HF3 could detect in situ produced SO₂ and its derivatives from a synthetic SO₂ donor.Chapter 6,a H₂S fluorescent probe?HF-PBA?was designed and synthesized using flavonol as the fluorophore,disulfide and ester group as the reactive sites.The detection was based on the regulation of the "on-off" states of the ESIPT process.The probe itself emitted weak fluorescence peaked at 400 nm due to inhibition of the ESIPT process.H₂S attacked disulfide bond firstly which subsequently induced a two-step reaction to release the free flavonol fluorophore.As a result,a strong emission band from the keto form and a relative weak emission from the enol form appeared.The probe has high reactivity,high sensitivity and low cytotoxicity,and can be applied to intracellular hydrogen sulfide imaging.