Thioether-Bonded Fluorescent Probes for Deciphering Thiol-Mediated Exchange Reactions on the Cell Surface

Study on the processes of the thiol-mediated disulfide exchange reactions on the cell surface is not only important to our understanding of extracellular natural bioreduction processes but to the development of novel strategies for the intracellular delivery of synthetic bioactive molecules. However, disulfide-bonded probes have their intrinsic inferiority in exploring the detailed exchange pathway because of the bidirectional reactivity of disulfide bonds toward reactive thiols. In this work, we developed thioether-bonded fluorescent probes that enable us to explore thiol-mediated thioether (and disulfide) exchange reactions on the cell surface through fluorescence recovery and/or cell imaging. We demonstrated that our thioether-bonded probes can be efficiently cleaved through thiol-thioether exchanges with exofacial protein thiols and/or glutathione (GSH) efflux. The exchanges mainly take place on the cell surface, and GSH efflux-mediated exchange reactions can take place without the requirement of pre-exchanges of the probes with cell surface-associated protein thiols. On the basis of our founder methodology, for the first time we demonstrated the interplay of exofacial protein thiols and GSH efflux on the cleavage of external thioether-bonded compounds. Moreover, given that an understanding of the process of GSH efflux and the mechanism on which it relies is crucial to our understanding of the cellular redox homeostasis and the mechanism of multidrug resistance, we expect that our thioether-bonded probes and strategies would greatly benefit the fundamental study of GSH efflux in living cells.