Development Of Fluorescent Probes For The Specific Detection Of Hypobromous Acid And Their Application In Bioimaging

Hypobromous acid(HOBr),an important reactive oxygen species(ROS)in living systems,is involved in many physiological and pathological processes.However,abnormally high levels of HOBr has been implicated in several human diseases,such as arthritis,cardiovascular disease,and some cancers.Thus,efficient methods for monitoring HOBr in living systems are significant for biological research and clinical diagnoses.Fluorescent probes are powerful chemical tools for sensing and imaging of ROS production in living systems because of its similicity,high sensitivity,high spatial resolution,and noninvasiveness.However,the design of probes for the sensitive and in particular selective detection of HOBr still remains a great challenge because of its high reactivity,short life time and similar chemistry with that of HOCl.The paper mainly divides into three chapters,which are listed as following:Chapter 1.The research progress of HOBr fluorescent probes were summarized.Based on this,the research ideas of this paper were proposed.Chapter 2.A FRET-based rhodamine-coumarin ratiometric fluorescent probe for imaging HOBr based on the bromination-induced spirocyclization of rhodamine dyes.In our recent studies,we observed that HOBr can induce the electrophilic bromination of xanthene ring of rhodamine B to afford the colorless and non-fluorescent spirocyclic derivatives.Based on the above results,a FRET-based ratiometric fluorescent probe for HOBr was proposed.The probe Cou-Rh B is comprised of a coumarin donor and a rhodamine acceptor linked by a rigid spacer.In the absence of HOBr,the rhodamine acceptor of the probe exists in an open,colored,and fluorescent form.Spectral overlap between coumarin emission and rhodamine absorption is large,the FRET process is switched on and Cou-Rh B displays strong emission of the rhodamine acceptor upon excitation at the coumarin donor.However,upon treatment of probe Cou-Rh B with HOBr,the electrophilic bromination of xanthene ring occurs,which gives brominated rhodamines and thereby shifts the equilibrium of such fluorophores from the highly absorbing and fluorescent zwitterion form to the colorless and non-fluorescent spirolactone form.Thus,spectral overlap between coumarin emission and rhodamine absorption is minimized,FRET is suppressed,and mainly the emission of the coumarin is observed.The above reaction affords a large emission wavelength shift(ca.89 nm),and ratiometric sensing of HOBr can be realized by measuring the ratio of coumarin-to rhodamine-type intensities(I₄₉₁/I₅₈₀).The new fluorescent probe possesses outstanding selectivity toward HOBr and has been proved to be capable of ratiometric fluorescent imaging of HOBr in living cells and zebrafish.Chapter 3.A fluorescein-based fluorescent probe for the specific detection of HOBr based on the strong electrophilicity of HOBr.In this chapter,we designed and synthesized a fluorescein-based fluorescent probe Lyso-Y for detecting HOBr in lysosome.The probe Lyso-Y is a spirohydrazide of 4',5'-dimethyl fluorescein,and the lysosome-targetable group morpholine ring is introduced at its 6-position via an amide bond.Lyso-Y is present as the nonfluorescent spirohydrazide form in aqueous solution.Upon treatment with HOBr,the spirohydrazide of Lyso-Y is oxidazed to afford the parent fluorescein dye,which can further react with HOBr at p H 5.0 to afford the cleavage of its xanthene ring to give a new molecule with strong fluorescence emission(?_ex/?_em=388/554 nm).The above reaction leads to a significant increase in fluorescence emission at 554 nm.The probe can be used to detect HOBr with high sensitivity(detection limit:4.5 n M)and high specificity.Preliminary results indicate that Lyso-Y can be applied for fluorescence imaging of lysosomal HOBr in living cells.