Development Of Fluorescent Probes Based On Hemicyanines And Their Applications

Hemicyanines,a main member of the cyanine family,are commonly used to develop fluorescent sensors owing to their desirable properties such as high absorption coefficient,relatively long absorption and emission wavelength,high fluorescence quantum yield,large Stokes shift,and so on.However,because of the lack of conjugated functional groups,it is difficult to construct fluorescent probes based on hemicyanines.To develop the diversification of hemicyanine fluorophores,a new strategy must be exploited.Non-N-alkylated hemicyanine(one of the indole nitrogen atoms is not alkylated)has a protonatable amino group within the chromophore core,and modulation of protonation/deprotonation of the indole nitrogen atom of the dye would provide an alternative strategy to modulate the optical properties of the dye.On the other hand,it was reported that the pKa values of the imine nitrogen of hemicyanine Schiff base shifted by modulating the microenvironment or electron-withdrawing ability of the substituent proximal to the imine group,which will cause a significant change of the spectral properties of the dye.The shift of equilibrium between Schiff base(SB)and protonated Schiff base(PSB)may provide a new strategy to modulate spectral properties of the hemicyanines.Based on the modulation of equilibrium between SB and PSB of hemicyanine in aqueous solution by molecular recognition reaction,the work of this dissertation focused on design and synthesis of fluorescent probes for quantitative determination of vicinal dithiol-containing proteins(VDPs),selenocysteine(Sec)and thiophenol.The main works are as follows:(1)The microenvironment proximal to the imine group of the hemicyanine probe MCAs was modulated by molecular recognition reaction,which caused the shift of pKa value of MCAs.Based on this sensing mechanism,we have developed a hemicyanine-based probe for VDPs.The imine pKa of MCAs is 4.8,and it exists predominantly in the SB form at physiological pH.Due to the absence of a resonating positive charge,MCAs absorbs at a relatively short wavelength and is essentially non-fluorescent.After treating with reduced bovine serum albumin(rBSA,selected as the model protein),MCAs was brought from aqueous media to the binding pockets of the protein,causing a large increase in pKa value of MCAs(pKa = 7.1).Thus,an increase in the protonated Schiff base(PSB)form of MCAs was observed at the physiological pH conditions,which affords a bathochromic shift and a significant fluorescence turn-on response.MCAs features high selectivity and sensitivity toward VDPs over other interfering substance.Furthermore,this probe has been demonstrated to be capable of imaging endogenous VDPs in living cells.(2)It was reported that the stronger the electron-withdrawing ability of the substituent,the lower the pKa value of the imine nitrogen.Based on this sensing mechanism,we construct a red fluorescent probe MC-Sec for qualitatively and quantitatively detecting selenol in living cells by incorporating 2,4-dinitrobenzenesulfonamide moiety into the structure of hemicyanine Schiff base.The pKa of the imine nitrogen of MC-Sec is 6.40,and it exists predominantly in the SB form at physiological pH condition.Due to the absence of a resonating positive charge,MCAs is essentially non-fluorescent.In the presence of Sec,the 2,4-dinitrobenzenesulfonamide group of MC-Sec was readily cleaved via a nucleophilic reaction,leading to the generation of free MCP whose pKa shifts to 9.04.At pH 7.4,the MCP predominantly present as the PSB form,which leads to a bathochromically shifted chromophore and a significant fluorescence turn-on response.MC-Sec can selectively report Sec without interference from biologically relevant analytes under physiological conditions.In addition,MC-Sec has been demonstrated to be capable of imaging both exogenous and endogenous Sec in living cells.(3)On the basis of previous works,we develop a near-infrared fluorescent probe MCSH for thiophenols based on the mechanism that the electron-withdrawing ability of the substituent will lower the pKa value of the imine nitrogen and a bathochromically shifted chromophore will occur by incorporating bridge ring structure into the hemicyanines.The maximum emission of MCSH is 625 nm and the pKa value of MC-Sec is 4.11.In the neutral condition,it exists predominantly in the SB form.Due to the selective removal of the strong electron-withdrawing 2,4-dinitrobenzene-sulfonyl group by thiophenols,free MCPD generated.The pKa of the imine nitrogen shifts from 4.11 to 8.11.The MCPD is predominantly present as the PSB form at pH 7.4,which leads to a bathochromically shifted chromophore and a significant increase in fluorescence intensity simultaneously in the neutral condition.This probe shows a rapid,highly selective,and sensitive detection process for thiophenols.The potential applications of this new NIR fluorescent probe were demonstrated by the quantitative detection of thiophenol in real water samples and fluorescent imaging of thiophenol in living cells.(4)Except for the above three experiments,we designed and synthesized a novel NO donor,N-nitrosated rhodamine 6G acid(NOG)by the nitrosation of two N atoms in rhodamine 6G(R6G).It exisits as its spirocyclic form and is colorless and non-fluorescent in aqueous solution.After UV irradiation at 365 nm,NO and rhodamine 6G acid(ring-opened form)were released from NOG,accompaning by a drastic fluorescence turn-on at 547 nm,which was convenient to monitor the kinetics or the dose of NO release in a real-time fashion with spectroscopic methods.In addition,the NO release from NOG was not interfered by biothiols.Furthermore,this NO donor enabled time-and site-controlled NO release in living cells.