Construction And Performance Of Endoplasmic Reticulum-targeted Hypochlorous Acid,hyponitric Acid And Near Infrared ONOO~– Fluorescent Probe

As we all know,many chemical substances,including metal ions,anions,reactive oxygen species?ROS?,reactive nitrogen?RNS?,reactive sulfur?RSS?,etc.,are involved in a variety of physiological and pathological processes in the body,and the action process of some of the chemical substances mainly occurs In subcellular organelles,for example,increased endoplasmic reticulum stress caused by reactive oxygen species may be related to heart disease,stroke,neurodegenerative diseases,cancer,etc.Therefore,detecting the levels of chemicals in subcellular organelles such as the endoplasmic reticulum is of great scientific significance for understanding its role in physiology and pathology.At present,fluorescent probes for detecting various chemical substances have received extensive attention and there are many review reports.In view of the importance of the endoplasmic reticulum and the strong tissue penetration of near-infrared fluorescent molecules,this thesis designed and synthesized endoplasmic reticulum targeted detection of HClO,HNO and near-infrared detection ONOO^–fluorescent probes and their performance Research and discussion.Based on the naphthalimide fluorophore and the dimethylthiocarbamate response group,a fluorescent probe R1 with an intramolecular charge transfer?ICT?effect was designed and synthesized.Probe R1 can specifically recognize HClO,and other common reactive oxygen species,reactive nitrogen species,anions,and amino acids do not interfere with the recognition of the probe.The probe has high sensitivity for HClO recognition,the detection limit is as low as 82 n M,and it reaches the response platform within 3 minutes.Confocal imaging experiments show that probe R1 can specifically detect HClO in Ec1 living cells and has a good localization effect on the endoplasmic reticulum.Based on the naphthalimide fluorophore,a two-photon fluorescent probe R2targeting the endoplasmic reticulum to recognize hyponitric acid?HNO?was synthesized.HNO can react with the arylphosphine on the fluorescent probe molecule to form azalide,and then attack the adjacent ester group.The ester group hydrolyzes and releases the hydroxyl group.The ICT process of the fluorescent molecule can occur,so that the fluorescence enhancement is observed at 555 nm.The probe R2 has the advantages of good selectivity,fast response speed?reaching the response platform within 2 minutes?,low detection limit?32 n M?and naked eye recognition.Confocal imaging experiments show that probe R2 can be used for single-and two-photon imaging of exogenous HNO in MCF-7 cells and imaging of endogenous HNO in cells.It also shows that probe R2 has good biocompatibility and cell membrane penetration.In addition,probe R2 has also been successfully used for the detection of NO/H₂S crosstalk in live MCF-7 cells.This is the first two-photon fluorescence probe to visualize NO/H₂S crosstalk in the endoplasmic reticulum and monitor HNO in real time.A fluorescent probe R3 targeting endoplasmic reticulum to detect HClO was designed and synthesized.Since HClO has strong oxidizing properties for double bonds,it can destroy the large?-conjugated structure of probe R3,so that an obvious fluorescence enhancement can be observed at 557 nm,accompanied by a significant change in the color of the solution?from dark blue to light blue?.The probe R3 can be used for HClO"naked eye recognition"and has high sensitivity for the detection of HClO.When the content of HClO is 0-2 equivalents,the fluorescence intensity of the probe solution has a good linear relationship with the concentration of HClO,the linear correlation coefficient R²=0.99216,indicating that probe R3 can be used for quantitative detection of HClO.In addition,the probe R3 has good biocompatibility and cell membrane permeability,and has been successfully applied to the specific imaging of exogenous and endogenous HClO in MCF-7 living cells.Co-localization experiments show that probe R3 has good endoplasmic reticulum targeting and can achieve fluorescence imaging of HClO in the endoplasmic reticulum.A near-infrared ONOO^–fluorescent probe R4 with ICT effect was designed and synthesized based on the dicyanoisophorone fluorophore.The probe R4 can specifically recognize ONOO^–,and the common active oxygen,active nitrogen,anions,and amino acids in the organism do not interfere with the recognition.Probe R4 is highly sensitive to ONOO^–identification.After identification,the color of the solution changes from yellow to purple,and ONOO^–can be detected with“naked eyes”.In addition,probe R4 was successfully applied to exogenous ONOO^–imaging in MCF-7cells,with good biocompatibility and cell membrane permeability.