Design Of Novel Fluorescent Probes For Analysis Of Bioactive Molecules Under Hypoxic Stress

Hypoxia is one of the environmental effects brought by environmental pollution.Environmental effects are related to the survival and development of humans and organisms.Therefore,we should attach great importance to the study of its mechanism of action.Cell is the basic unit of the first action after the environment affects the organism.Environmental hypoxia can lead to changes in various intracellular bioactive molecular in the organism,which in turn affects the health of the organism.Environment imaging analysis technology is a comprehensive analytical technology integrating environmental factors and optical analysis methods.That is,optical imaging method is applied to solve the problems in environmental field.It is a non-invasive imaging analysis technique that can detect the distribution of endogenous bioactive moleculars in organisms through fluorescence imaging analysis.Thus,it will contribute to explore how environmental factors affect organisms,which in further reveals the relationship between abnormal changes in the environment and human health.Molecular fluorescent probe is an important tool of environment imaging analysis techniques.In this dissertation we focused on the development novel fluorescent probe for the detection of bioactive moleculars under environmental hypoxia.We studied the typical mammals on land,elucidated environmental hypoxia on the influence of concentration and distribution of active molecules in the organisms,explored the role of environmental hypoxia,revealed the environmental directive significance of typical active molecules in the organisms,and provided scientific basis and technical support for further study marine aquatics organisms.These fluorescent probes owned satisfied optical response performance,which could lead to changes in fluorescent signals when response to target and achieve the detection of target.The detail contents of this dissertation are listed as follows:(1)In this work,a mitochondrial-specific near-infrared fluorescent probe Mito-SH for the evaluation of sulfane sulfur in oxygen-glucose deprived cell models and in acute ischemia mice models was developed.The probe Mito-SH consisted of three parts: the lipophilic triphenylphosphonium cation and mercaptobenzoic acid were respectively modified to the hydroxyl group of BODIPY fluorophore.The lipophilic triphenylphosphonium cation could guide the probe into mitochondria.The mercapto(-SH)response unit could selectively respond to the reactive sulfur atom in sulfane sulfur to trigger remarkable near infrared region(NIR)fluorescence emission changes of azo-BODIPY fluorophore.The probe exhibited high selectivity,good photostability and low cytotoxicity when used in the tests.Owing to these excellent properties,MitoSH had been applied for imaging of sulfane sulfur dynamic changes in cells under oxygen-glucose deprivation.Sulfane sulfur could behave cytoprotective roles against oxidative damage caused by oxygen-glucose deprivation.Moreover,Mito-SH had been successfully utilized to imaging sulfane sulfur in acute ischemia mice models.Acute ischemia could lead to a burst produce of reactive oxygen species in the hippocampus,and sulfane sulfur functioned physiological protection via directly eliminating reactive oxygen species.All these results featured that our probe Mito-SH could serve as a potential tool for exploring functions of sulfane sulfur in cells and in vivo.The successful application of Mito-SH in cells under pathological hypoxia lays a scientific foundation for the subsequent application of cells under environmental hypoxia.(2)In this work,a near-infrared fluorescent probe BD-diSeH for detection of sulfane sulfur under environmental hypoxia stress was designed and synthesized.The probe BD-diSeH was composed of two moieties: the strong nucleophilic phenylselenol group(-SeH)was integrated into the azo-BODIPY fluorophore via an ester bridge.BD-diSeH exhibited excellent selectivity and high sensitivity for the detection of sulfane sulfur.This new developed probe could be used for tracing endogenous sulfane sulfur changes under environmental hypoxia stress.The probe was used to imaging the distribution of sulfane sulfur in 3D-multicellular spheroid.Finally,the probe was applied for imaging sulfane sulfur in mice and zebrafish under environmental hypoxia.Therefore,the probe could successfully detect the concentration and distribution of sulfane sulfur in the cells under environmental hypoxia,which was helpful to elucidate the effect of environmental hypoxia on organisms.(3)In this work,a mitochondria-targeting selenium-containing near-infrared fluorescent probe was applied for the detection of sulfane sulfur under environmental hypoxia stress to clarify the relationship of sulfane sulfur and reactive oxygen species under hypoxic condition.The distribution of sulfane sulfur in monolayer cells and three-dimensional multicellular spheroid were measured using this fluorescent probe to clarify sulfane sulfur could prevent cells from oxidative damage when suffering from hypoxia stress both in living cells and in vivo.The protective mechanism against hypoxia was to inhibit caspase-dependent apoptosis through scavenging reactive oxygen species pathway in mitochondria.The probe could detect and determine sulfane sulfur level in ex vivo-dissected organs of hypoxic mice modal including heart,liver,spleen,lung,kidney and brain.The probe was applied to monitor the changes of sulfane sulfur and evaluate the biological effects of sulfane sulfur on eliminating the overproduction of reactive oxygen species in acute ischemia models of mice.The probe also could be used to detect sulfane sulfur in zebrafish under hypoxic condition.The new probe offers us a valuable tool for further explore physiological and pathological bio-roles of sulfane sulfur in cells and in vivo.(4)In this work,a suitable near-infrared fluorescent probe for combined-response of superoxide anion and hydrogen polysulfides under hypoxia stress was designed and synthesized.The probe was composed of two units: 4-nitrobenzyl alcohol was integrated to the middle position of cyanine fluorophore.4-nitrobenzyl alcohol was acted as response unit for hydrogen polysulfides.A heptamethine cyanine dye was both acted as fluorophore and as response unit for superoxide anion.HCy-ONO should first react with superoxide anion through a hydrogen abstraction to form Cy-ONO with conjugated system and release low fluorescence.Subsequently,Cy-ONO continued a reduce reaction of nitro group to amino group by hydrogen polysulfides with strong electrophile ability to release the cyanine fluorophore with strong fluorescence.The probe was sensitive,selective and stable,allowing for imaging superoxide anion and hydrogen polysulfides in living cells.Meanwhile,the level changes of superoxide anion and hydrogen polysulfides were captured in living cells both under continuous hypoxic condition and intermittent hypoxic condition.The relationship of superoxide anion and hydrogen polysulfides in acute peritonitis model of mice was investigated for discerning the inflamed tissue from normal tissue.In addition,our probe was applied for imaging superoxide anion and hydrogen polysulfides in tumor.Therefore,the probe with wide applications provides a unique method for further understanding the physiological activities of superoxide anion and hydrogen polysulfides.Therefore,HCy-ONO provides a new method for the study of redox state under hypoxic condition.(5)In this work,a activatable “Hairpin” fluorescent probe for tumor imaging by labeling thioredoxin reductase based on Michael-type reaction in living cells and in vivo was designed and developed.The probe was composed of three parts: a good inhibitor of thioredoxin reductase(2a)was selected as response unit;6-(benzo[d]thiazol-2?-yl)-2-(methylamino)naphthalene was chosen as the fluorescent manipulator and a linear alkyl chain was selected as the linker.Upon reduction by thioredoxin reductase,the fold-YLS was stretched to form an unfold-YLS,and then turned on the fluorescence.The probe was successfully applied for evaluating thioredoxin reductase activity in different cell lines.YLS could efficiently inhibit the thioredoxin reductase activity,which was associated with the increased reactive oxygen species level and induced cell apoptosis.In addition,YLS in combination with an in vivo fluorescence imaging system could visualize tumor by labelling thioredoxin reductase in xenografted nude mice.This work will provide a powerful strategy to develop fluorescent probe for understanding the physiological and pathological functions of thioredoxin reductase and the cellular redox signaling processes and to develop theranostic agents for cancer diagnosis and therapy.