Design,Synthesis And Application Of Organic Fluorescent Probe And Photocontrolled Releaser For Carbon Monoxide

Carbon monoxide?CO?,an extremely important gasotransmitter,plays an indispensable regulatory role in a variety of physiopathological events,such as redox balance regulation,cytoprotective function,vasodilation,etc.The unnatural level fluctuation of endogenous CO is as tightly bound to the occurrence and worsening of clinical diseases,including nerve degeneration,hypertension,heart failure,inflammation,etc.Meanwhile,exogenous CO has displayed satisfactory therapeutic effect in inflammation,cardiovascular disease,atherosclerosis,severe sepsis,severe malaria,organ transplantation,tumor and other disease models,thus making it to be an emerging therapeutic gas molecule.The physiological effects of CO are determined by its dose and spatial location.Therefore,the specific recognition of CO and its controllable supplying are of great significance for understanding and effectively utilizing its physiological and pathological effects.Fluorescence detection and photocontrolled releasing have been widely applied to track and carry active molecules in cells,tissues and in vivo in view of their unique spatiotemporal controllability and noninvasive capacity.Especially,the near-infrared and two-photon imaging methods are more appropriate for in situ visualization analysis in vivo in consideration of their low interference of spontaneous fluorescence,deep tissue penetration and low phototoxicity.So far,various CO fluorescent probes and photocontrolled releaser have been developed.However,there are still many shortcomings in the construction strategies and bio-applications of these probes and releasers.For example,the concentration fluctuation of endogenous CO in cells under given conditions is less discussed;it is impossible to achieve near-infrared CO photocontrolled releasing in the endoplasmic reticulum and mitochondria;two-photon CO photocontrolled releaser have not been reported.Hence,a novel CO fluorescent probe and a series of CO photocontrolled releasers have been developed to achieve endogenous CO concentration fluctuation detection,endoplasmic reticulum and mitochondrial localized near-infrared CO photocontrolled releasing and oxidative stress-mediated two-photon CO photocontrolled releasing,and to discuss the physiological effects of CO in different physiological and pathological processes.The main results of this dissertation are shown as follows:1.By utilizing azobenzene-cyclopalladium moiety as recognition group,borondipyrromethene?BODIPY?as fluorophore,a novel fluorescent probe,ACP-2,was designed and synthesized for CO detection with high specificity and good sensitivity.With the aid of ACP-2,the first direct visual evidence of the up-regulation of endogenous CO level under hypoxia was provided.Meanwhile,the increment of endogenous CO level during Oxygen-glucose deprivation reperfusion?OGD/R?was also imaged.This work shows the up-regulation fluctuation of CO in cells under given physiological conditions,and provides a new type of CO recognition group,azobenzene-cyclopalladium group,which may provide a strategy for CO probe construction for other bioanalytical applications.2.A near-infrared photocontrolled CO releaser,ERC,was designed and synthesized for CO releasing in endoplasmic reticulum.The symmetrical phenol groups of ERC facilitate the interaction of ERC with the lipid membranes of the endoplasmic reticulum,thus enabling ERC to aggregate in endoplasmic reticulum.Meanwhile,upon irradiation,ERC can quickly undergo photolysis reaction to produce CO gas.After photolysis,the length of the molecular conjugate system becomes shorter and the spectrum obviously blue-shifts,which makes the excitation and emission spectra distinguishable before and after photolysis,thus allowing fluorescence tracking of the photolysis process.By employing peroxynitrite anion?ONOO^–?as a biomarker,the physiological effects of CO in the process of drug-induced liver injury were explored at the cellular and in vivo levels.The results showed that the CO released by ERC can relieve the endoplasmic reticulum stress induced by acetaminophen?APAP?and inhibit the up-regulation of ONOO^-caused by APAP.This work shows that CO has potential effects in alleviating drug-induced liver toxicity.3.A near-infrared photocontrolled CO releaser,Mito C,was designed and synthesized for CO releasing in mitochondria.Mito C was capable of photocontrolled releasing CO in physiological buffer,cells and in vivo,and the photolysis process can be tracked by spectrum changes.Meanwhile,triphenylphosphate positive ions enables Mito C to target mitochondria,which allows photocontrolled releasing of CO in mitochondria.By employing adenine nucleoside triphosphate?ATP?as biomarker of energy metabolism,the regulation effect of CO on energy metabolism was evaluated.The results displayed that CO released by Mito C could significantly up regulate ATP levels in cells and diabetic mice.In addition,two-photon imaging results exhibited that the energy metabolism level of diabetic mice was lower than that of normal mice.Therefore,the energy metabolism regulation effect of CO is expected to be used to correct metabolic abnormalities in diabetic mice,which has potential application value in the treatment of diabetes.4.A oxidative stress activated two-photon CO photoreleaser,FB,was designed and synthesized by utilizing phenylborate as hydrogen peroxide?H₂O₂?recognition group.In the presence of H₂O₂,the excitation state intramolecular proton transfer?ESIPT?effect of flavonol recovered and the fluorescence increased.Meanwhile,the generated flavonol was capable of releasing CO under irradiation.Therefore,FB can implement both H₂O₂ tracing and H₂O₂-mediated photo-driven CO releasing.With the aid of FB,it was found that the level of H₂O₂ was significantly up-regulated after angiotensin II administration,and the vasodilatory effect of CO was visualized.The superior performance of FB will make it to be a promising tool in oxidative stress warning and CO photocontrolled releasing.