The New Applications Of Biocompatible Reactions In Regulating Or Imaging Live Cells

Biocompatible reactions are chemical reactions that can proceed in mild biological conditions with high reactivity and specificity.Biocompatible reactions have played important roles in the investigation of living systems.The development of new biocompatible reactions as well as the exploration on new applications of biocompatible reactions for better understanding complex living systems is of great research interests for chemical biologists.This Ph.D dissertation presented our efforts to apply biocompatible reactions to the imaging of biological species in living cells and to the regulation of live cells through changing their microenvironment.Our attmepts to develop biocompatible reactions based on photocycloaddition reactions were also presented.Studies on the construction of small molecular imaging probes based on bio-compatible reactions to visualize cysteine(Cys)or other thiol-species leading to the reduction condition inside living cells was presented in the first and second part respectively.The condensation reaction of 2-cyanobenzothiazole and 2-aminoethanethiol is regarded as a biocompatible reaction with fast kinetic in neutral water solution at room temperature.The first part of this dissertation described the construction of the fluorescent probe CBTOA for imaging of endogenous Cys in living cells.The fluorescence enhancement is attributed to Cys-mediated removal of the acrylate group in CBTOA,followed by the fast biocompatible reaction between the resulting CBTOH and Cys to build the negative and hydrophilic luciferin in situ,leading to intracellular retention.We have demonstrated that CBTOA can be used to detect Cys levels in living cells under ROS-induced oxidative stress and in mouse liver tissues ex vivo.Moreover,CBTOA has also been successfully applied to report Cys levels in human mesenchymal stem cells(hMSCs)during adipogenic differentiation.The second part of this thesis dissertation described the construction of trimodal probe with fluorescence/19F-MRS/1H-MRI for reducing environment detection.The biothiol-mediated disulfide cleavage resulted in fluorescence turn-on,along with 19F-MRS signal enhancement and 1H-MRI contrast reduction.Combined the high sensitivity of optical imaging with high resolution of MRI imaging,we have demonstrated that the probe can be used to image biothiol levels both in living cancer cells and animals.The third part of this dissertation described photo-modulation of the 3D microenvironment of stem cells via the photo induced biocompatible reaction between tetrazole and alkene,also called 'photo-click reaction'.Our lab has sucessfully constructed photoresponsive supramolecular hydrogel systems by combining different diaryl tetrazoles and short peptides.Based on previous screening of the hydrogelators with different tetrazoles and short peptides,we found an ideal gelator TPI-GGF,which could apply for long time encapulation and culture of stem cells with high viability.Moreover,by using the intermolecular photo-click reaction between tetrazole and olefin linked with bio-ligand RGD,the active bio-ligand RGD were selectivily patterned in hydrogel matrix to guide stem cells differentiation.In this part,we combined photo-uncaging of caged RGD with the photo-ligation of the olefin bearing RGD with the tetrazole moiety on the hydrogelator.As a result,the differentiation of hMSCs encapsulated in the photo-modulated hydrogel matrix was significantly promoted for both osteogenesis and adipogenesis compared with control cells.The forth part of this dissertation described our eforts to develop new biocompatible reactions triggered by visible light irradiation.Based on our previous work on the photocycloadditon of o-diones with C=C containing compounds in organic solvents,we tested which of these reactions were able to proceed smoothly in aqueous solution.We found two[4+2]photocycloadditons of 9,10-phenanthrenequinone derivate PQ-1 or 1,10-phenanthroline-5,6-dione PN with specific C=C containing molecules proceeded rapidly in water to give[4+2]photocycloadducts with fluorescence turn-on effect.Preliminary tests using the photocycloaddition to label BSA protein demonstrated some potential biological applications of these two reactions.