Design,Synthesis And Bio-Imaging Of Sulfur Dioxide Fluorescent Probe

Exploring the biomolecules and their physiological roles in living system is one of the hot issues in chemical biology research.Powerful imaging tools are needed for in-situ monitoring of molecular metabolism of biological signals.Nowadays,fluorescent probes combined with bioimaging techniques have become one of the most powerful tools for real-time imaging biomolecules in living systems due to their visual and non-destructive advantages.Sulfur dioxide(SO2),a common gaseous pollutant,which poses a great threat to environmental pollution and human health,has been found to mediate a series of physiological process,such as myocardial injury,swelling and deformation.Increasing studies suggested that SO2 might serve as a new functionalized gaseous signaling molecule in living system.Moreover,it should note that endogenous SO2 and its derivatives play an important role in regulating various physiological processes in cardiovascular systems.However,abnormal SO2 levels and metabolism processes are closely related to many diseases such as cancer and neurological diseases.In this thesis,we are focused on the development of highly sensitive and selective fluorescent probes for imaging SO2 in cells,tissues and living bodies.With these successful probes in hand,we have achieved the following goals such as reversible imaging SO2 in living cells and zebrafish with ratiometric manners,efficiently distinguishing detection of H2S/SO2 in living cells and mice models,revealing the relationship between heat stroke and lysosome SO2 in intestinal tissues,tracking of SO2 Metabolism in living cells and tumors mice models.The specific research of this thesis are as follows:1.An ICT(intramolecular charge transfer)based fluorescent probe 2-1 has been successfully designed and developed by conjugation of naphthopyran-benzothiazolium system.The probe 2-1 displayed ratiometric fluorescent manners for SO2 based on the?-conjugation interruption reaction along with the cancelation of an ICT process Additionally,the addition product of probe 2-1 with bisulfite can be applied to the design of regenerative chemodosimeters based on the H2O2 promoted elimination of bisulfite and recovery of 2-1.The cell and zebrafish imaging experiment have demonstrated that probe 2-1 could reversible imaging SO2/H2O2 with ratiometric manners.Since cells possess an elaborate regulation system to maintain their redox balance and the large or significant redox state changes can be buffered by the redox-active molecules,we speculate that probe 2-1 could also be used for response of intracellular redox homeostasis in biological system upon oxidative stress induced by H2O2(hydrogen peroxide)2.From chemistry perspective,H2S(hydrogen sulphide)and SO2 showed similar chemical properties.Therefore,the two sulfur-contained compounds might interfere the detection of each other.In order to better understand the physiological roles of SO2 and H2S,a novel fluorescent probe 3-1 by integrating 4-azide-1,8-naphthalic anhydride(azide as a H2S specific site)and spiropyran(as a photocontrolled site for SO2)was obtained Originally,the 3-1 was capable of specifically monitoring H2S by azide reduced reaction Upon the irradiation of alternate ultraviolet and visible light,both the structure and fluorescent emission of spiropyran moiety in 3-1 can be reversibly tuned.However,in the presence of SO2,the alternation will be interrupted by the reaction between the released double bond and SO2.Particularly,it should be pointed out that the UV-activated 3-1 was only reactive to SO2 and inert to H2S.We speculate that the phenolic hydroxyl group in the merocyanine moiety might weaken the reactivity of the C=C double bond.Therefore,3-1 shows a high selectivity for SO2(stronger nucleophilic)over H2S.Relying on these merits,3-1 has been successfully applied to in vivo imaging in living cells and mice with high selectivity and sensitivity3.Taking advantage of the unique regulation of UV light on spiropyran group,we designed and synthesized a lysosomal targeted SO2 fluorescent probe 4-1.The strategy of UV-activation can efficiently control the reactivity of probe 4-1,which improve the targeting performance and eliminate the false signals,and finally result in precisely imaging SO2 in lysosome compared with other no light-controlled sensors.In particular,the UV-activated 4-1 contains a special C=C double bonds,which is a proposed recognition site and in suit attacked by SO2.In this regard,this in suit UV-activated strategy would allow us accurate monitor SO2 in lysosomes with ratiometric manners based on FRET(Forster resonance energy transfer),which largely avoided the interference of false positive signals during the transit of probes in cells.Eventually,probe 4-1 has been successfully applied to image increasing SO2 values in lysosomes during heat shock for the first time.Moreover,we also confirmed that the increased SO2 could protect the small intestine against damage induced by heat shock through regulating oxidative stress in cells and mice.4.A novel dual-site fluorescent probe 5-1 was rationally designed and exploited for the simultaneous detection of GSH(glutathione)and its metabolite SO2 via an ICT-FRET synergetic mechanism.The coumarin-cyanoacetic acid moiety could not only served as energy donors of FRET-1 process,but also act as the GSH reaction site.Besides,the benzopyrylium unit in 5-1 served as both energy acceptor and highly selective SO2 reaction site.After the probe 5-1 reacted with SO2,the FRET-1 process of 5-1 was blocked and the red fluorescence was quenched upon excited at 405 nm as the benzopyrylium unit was interrupted.The presence of GSH would destroy the?-conjunction between coumarin and cyanoacetic acid.Simultaneously,the inhibited ICT process triggers the FRET-2 process,which finally results in an enhancement of red emission with excitation wavelength at 405 nm.Particularly,the further addition of SO2 would breaks the conjugated system of benzopyrylium unit,which induces the prohibition of the FRET-2 process and blue-shift of fluorescence emission.As a whole,we speculate probe 5-1 displayed totally reversed fluorescence responses towards GSH(enhanced red emission)and SO2(annihilated red fluorescence)with highly selectivity and sensitivity Particularly,the probe 5-1 displayed completely different fluorescent signals(blue-shift)with SO2 in the presence of GSH,thereby allowing imaging the metabolism process of GSH to SO2 in two independent channels without spectral cross interference.Thanks to these favorable performances,5-1 has been successfully used for the visualization of enzymatic conversion of intracellular GSH to SO2 in different cell lines and tumor-bearing nude mice.