Synthesis Of Triphenylamine-based Hypochlorous Acid Fluorescent Probe And Its Application In Biological Imaging

Hypochlorous acid/hypochlorite(HClO/ClO?)is one of the most important reactive oxygen species(ROS)in the organism,which is closely related to many physiological activities.In vivo,HClO/ClO?is mainly produced by the reaction of H₂O₂with chloride ions catalyzed by myeloperoxidase.In vivo,it plays a significant role in the human immune system through preventing the invasion of pathogens and regulating cellular apoptosis.Under normal physiological conditions,the concentration of ClO?is usually between 5 and25?M.However,excessive ClO?in organisms can cause oxidative damage to biological molecules such as nucleic acids,proteins and lipids,leading to a series of diseases such as atherosclerosis,neurodegeneration,Alzheimer's disease and cancer.Therefore,developing a fast and efficient method for detecting ClO?in living organisms is quite meaningful for studies of the physiological function.In the past few years,fluorescence imaging has attracted more and more attention due to its advantages such as high sensitivity,fast response time,high selectivity,low cost and convenient operation.In recent years,lots of fluorescent probes for HClO has been reported.These probes are generally based on the following fluorescent probe recognition mechanisms,such as photoinduced electron transfer(PET),intramolecular charge transfer(ICT),twisted intramolecular charge transfer(TICT),fluorescence resonance energy transfer(FRET),excited state intramolecular proton transfer(ESIPT),and aggregation-induced emission(AIE).Combined with literature reports and previous work of our research group,we designed and synthesized three types of HClO/ClO?fluorescent probes based on the mechanism of TICT+AIE,PET and ICT respectively,and characterized their molecular structures by H-NMR,C-NMR and high resolution mass spectrometry.Moreover,the photophysical properties of the compounds were verified by spectroscopic method,and molecular orbital analysis of the compounds was performed by theoretical calculation.Subsequently,fluorescence titration and laser confocal scanning imaging were performed for detection of HClO/ClO?.The main content is as follows:1.We developed a TICT+AIE fluorescent probe(named TPB-CN)for detection of hypochlorite,which consists of a diaminomaleonitrile(DMN)as the electron accepter,triphenylamine(TPA)as the electron donor,and a phenyl group as?-bridge.The experimental results show that with the increase of solvent polarity,the maximum emission wavelength of probe TPB-CN is gradually redshifted.In addition,the effect of solvent polarity on fluorescence emission was verified by the Lippert Mataga equation,which further confirmed the TICT characteristics of the probe.In order to verify the AIE properties of the probe,the fluorescence changes of TPB-CN probe in H₂O/Et OH mixed solutions with different water content were studied.The results showed that the fluorescence intensity of the TICT state of the probe decreases gradually with the increase of water content.When the water content increases to 70%,a new fluorescence peak appears at 558 nm,indicating that TPB-CN forms an aggregate and exhibits AIE properties.Fluorescence titration experiment showed that TPB-CN displayed high sensitivity,good selectivity,and quick response ability(<2 s)toward ClO?.What's more,probe TPB-CN possessed capability of detecting ClO?in living cells and mouse.2.We prepared four Schiff base PET probes named TPA-NO₂,TPA-2NO₂,TPB-NO₂and TPB-2NO₂,and studied their detecting performances to ClO?.These probes are composed of triphenylamine(TPA)derivatives as fluorophore,nitroaniline moieties as receptor,and a cleavable C=N bond as a linker.Before the reation with ClO?,the fluorescence of the probes was quenched due to the PET effect from nitroaniline group to triphenylamine(TPA)derivatives.After the reaction with ClO?,the cleavage of C=N bond broke the PET process and the fluorescence was turned on.Among the four probes,TPB-NO₂displayed the most obvious fluorescence changes and fastest response towards ClO?.Moreover,TPB-NO₂was also successfully applied to detect ClO?in living cells and zebrafish,indicating the feasibility of our strategy in bioimaging.3.We described a novel dual-site fluorescent probe CN-N for discriminately detection low and high concentration of ClO?with multi response fluorescence signals by the two different reactive C=C double bond in probe.We envisioned that the C=C double bond of malononitrile group in probe was quite reactivity than another C=C group between isophorone and triphenylamine as the strong electron-withdrawing effect of malononitrile.In the absence of ClO?,the probe underwent an ICT-I process and displayed a red fluorescence emission at 650 nm.In the presence of low concentration of ClO^?(0-40?M),the ICT-I process of probe was inhibited,which induced a49 nm blue-shift of fluorescence emission from 650 nm to 500 nm,and the malononitrile group of probe was oxidized to ketones by ClO?.It's worth noting that along with the higher concentration of ClO^?(40-140?M)adding to the detection system,the probe showed a further blue-shift of fluorescence emission from 500 nm to 464 nm,and the C=C double bond between isophorone and triphenylamine was oxidized to aldehydes by ClO^?.As a result,the ICT-II process from triphenylamine to isophorone was also disturbed.The results showed that probe could detection of different concentration of ClO^?with two different fluorescence responses,which could help us understand the complex oxidation characteristics of ClO^?in biological samples.Importantly,the probe has been successfully applied to imaging low and high concentration of ClO^?in living cells for the first time.In summary,we prepared and synthesized some probes based on the characteristics of hypochlorous acid detection,and characterized the molecular structure of these probes,and studied their applications in hypochlorous acid fluorescence detection and biological imaging.The results showed that the probes TPB-CN,TPB-NO₂and CN-N all showed good fluorescence response to hypochlorous acid.It further proves the feasibility of our design strategy and provides a new idea and scheme for the development of more probes in the future.