Development Of Fluorescent Probes For Hydrogen Peroxide Based On Tandem Payne/Dakin Reaction And Their Applications In Bioimaging

Hydrogen peroxide（H₂O₂）,as an important reactive oxygen species（ROS）,is mainly generated in mitochondria by the activation of nicotinamide adenine dinucleotide phosphate（NADPH）oxidase complex,and can diffuse across cellular membranes to regulate a large variety of physiological processes from the immune response to cell signaling.However,abnormal levels of H₂O₂ can lead to many diseases,including cancer,diabetes,metabolic disease,Alzheimer’s disease,heart disease,Parkinson disease and Huntington’s disease.Consequently,evaluation of the levels of hydrogen peroxide in biological systems may aid early diagnosis of some diseases.At present,there are many analytical methods for detecting H₂O₂.Among them,fluorescence spectroscopy is attractive due to its ease of use,high sensitivity,noninvasiveness and little interference,and can be applied for bioimaging in living systems.Based on the tandem Payne/Dakin reaction,two fluorescent probes for the selective detection of H₂O₂ were developed,and their photophysical properties and applications were investigated in detail.The main contents were as follows:Chapter 1.Different fluorescence detection mechanisms for selective detection of H₂O₂were summarized.Based on this,the main research ideas of this thesis were proposed.Chapter 2.A coumarin-based fluorescent probe for H₂O₂ based on the Payne/Dakin tandem reaction was proposed.Here,we report 7-diethylamino-3-formyl-coumarin（Cou-CHO）as a turn-on fluorescent probe for H₂O₂.In aqueous polar solvents,Cou-CHO is weakly emissive due to the formation of the twisted intramolecular charge transfer（TICT）state upon photoexcitation.Upon mixing with H₂O₂ in the presence of trichloroacetonitrile（CCl₃CN）,trichloroperoxyacetamidic acid（TCPAA）is formed from the reaction of CCl₃CN with H₂O₂（Payne reaction）,which then affords a Dakin oxidation via nucleophilic attack of the aldehyde on the 3-position of Cou-CHO by its anion form,and subsequent hydrolysis of the intermediate gives the corresponding Cou-OH as the final product.This tandem reaction converts the electron-withdrawing aldehyde moiety of Cou-CHO to the electron-donating hydroxyl group,thus hindering formation of the TICT state and thereby resulting in a significant fluorescence enhancement at 502 nm.The proposed probe exhibits excellent selectivity toward H₂O₂ over other ROS,reactive nitrogen species（RNS）and related biological species.The practical utility of Cou-CHO in biological contexts has been demonstrated by imaging basal and endogenous H₂O₂ in living Hep G2 cells.Chapter 3.Based on the tandem Payne/Dakin reaction,we constructed a methylene blue-based near-infrared fluorescent probe M1 for selective detection of H₂O₂.This chapter we designed a near-infared fluorescent probe M1 with salicylaldehyde as the recognition moiety and methylene blue（MB）as the fluorophore.In probe M1,theÏ€-conjugation system of MB was interrupted due to the acetylation of its N atom.While,as salicylaldehyde is a fluorescent molecule,probe M1 only gives the fluorescence of its salicylaldehyde moiety(λ_em=488 nm).Upon mixing with H₂O₂ in the presence of trichloroacetonitrile（CCl₃CN）,the salicylaldehyde moisty of M1 was converted to the corresponding catechol via a tandem Payne/Dakin reaction,which was unstable and can be oxidized to the orthoquinone.The orthoquinone moiety subsequently undergoes elimination and hydrolyzation to form unstable leucomethylene blue（LMB）,which was oxidized to afford MB as the final product(λ_em=688 nm).The above reaction affords dramatic fluorescence decrease at 488 nm and enhancement at 688 nm simultaneously.Therefore,the probe M1 realized the dual-channel fluorescence detection of H₂O₂.The proposed probe exhibits excellent selectivity toward H₂O₂ over other ROS,reactive nitrogen species（RNS）and related biological species.In addition,the probe M1 has successfully applied for imaging endogenous H₂O₂ induced by cinnamaldehyde in RAW264.7 cells.