Fluorescence Imaging Probes For Thiols Based On Michael Addition

Thiols,such as cysteine(Cys),homocysteine(Hcy)and glutathione(GSH),play crucial roles in maintaining biological processes.The endogenous concentrations of these thiols suggest the functional state of the corresponding enzymes and proteins.The abnormal levels of cellular thiols correlate with a number of diseases.Accordingly,the detection and monitoring of these sulfur-containing molecules in biological samples are very important.Among the various detection techniques,fluorescence detection techniques have been proved to be the most appealing methods promoted by their high sensitivity,simplicity,low detection limit and fairly simple technical implementation.Due to the biological importance of thiols,the development of fluorescent probes for thiols has been an active research area in recent few years.In this thesis,five kinds of novel compounds were synthesized as thiol-reactive fluorescent probes,based on the thiols' strong nucleophilicity.The detection of thiols was achieved by studying the changes of color and fluorescence caused by Michael addition of the probes to thiols.Detailed as follows:1.A new near-infrared(NIR)sensitive Cys/Hcy probe(2-1)based on squaraine was rationally designed and synthesized.2-1 featured NIR fluorescent emission,excellent selectivity,rapid signal responses and low detection limit for Cys/Hcy over other amino acids including GSH.Moreover,confocal laser scanning microscopy experiments indicated that 2-1 could be used for Cys/Hcy imaging in living cells.2.A turn-on fluorescent imaging probe(3-1)with triphenylamine as the fluorophore and maleimide as the bonding group selective for thiols detection was synthesized.3-1 was a novel thiol-reactive fluorescent probe based on Michael addition reaction for selectively detecting thiols over other relevant biological species.The thiol adduct was characterized using NMR and mass spectroscopy and detection mechanism was further confirmed.For the practical application of the sensor,3-1 could be used to image thiols in live cells with turn-on fluorescence emission.3.A 1,8-naphthalimide-based probe 4-1 bearing maleimide group was synthesized to detect thiols with high selectivity and sensitivity.After thiols introduction,4-1 containing system showed significant fluorescence enhancement,but other amino acids did not generate any change.The study of time-dependent fluorescence response showed that the reaction was complete within 120 s for Cys,Hcy and GSH,indicating that 4-1 reacts rapidly with thiols under the experimental conditions.Most importantly,confocal laser fluorescence scanning microscopic experiments demonstrated that the probe can readily be used to detect the intracellular thiols in living HepG2 cells.4.Two isomers of 1,8-Naphthalimide derivatives were synthesized and characterized to examine the recognition ability of different substitution position of maleimide groups on the 1,8-Naphthalimide thiols over various amino acids.The results showed that the fluorescence of 1,8-Naphthalimide on 5-1 could be regenerate by Cys,whereas fluorescence of 5-7 had none change upon addition Cys.Hence,5-1 could act as an off-on fluorescent probe for selectively detecting cysteine over other relevant biological species based on Michael addition reaction with a detection limit of 0.064 ?M and could be used for detection of Cys in living cells.5.A novel Dual-Fluorophore rearrangment type of fluorescent probe for selective detection of Cys/Hcy in neutral buffer was synthesized and characterized.The key design feature of fluorescent probe 6-1 is the integration of two potential fluorophore,naphthalimide and coumarin,which was designed to react with Cys/Hcy and generate distinct photophysical properties.The probe exhibited blue emission in neutra buffer,and the nucleophilic attack of Cys/Hcy toward the naphthopyrones moiety of 6-1 will interrupt the ?-conjugation,inducing both a dramatic bathochromic shift(125 nm)and a huge ratiometric value response(Cys,142-fold,Hcy,133-fold,I560/I435)in the fluorescence spectra.Further study of time-dependent fluorescence response indicated that 6-1 reacts with Cys faster than Hcy,but there was only a slight increase in the fluorescence for GSH.With these unique properties of 6-1,the probe was successfully applied to image Cys/Hcy in live cells and in vivo.