Design Synthesis And Biological Applications Of Fluorescent Probes Based On Oxazine And Hemiocyanine

Fluorescent molecular probes have become more and more effective tools for detecting various analytes in organisms because of their non-invasiveness,high sensitivity and real-time visual monitoring.When the probe molecule interacts with various cells,tissues,and analytes in the biological organism,it outputs fluorescent signal of absorbs and emits,enabling the detection of various active species.At the same time,there are more and more small-molecule fluorescent probe dyes,which are specifically designed as intracellular fluorescent markers,which are used to study the physiological activities of cells and the trace release process of intracellular drugs.Among them,near-infrared fluorescent probes have been increasingly used and designed in recent years due to their long wavelength absorption and emission,the ability to reduce spontaneous background fluorescence during the test,high fluorescence quantum yield and light stability,and good biocompatibility.The lysosome is an important organelle in the cytoplasm of each eukaryotic cell,and lysosome is a commanding and controlling center of cell metabolism,and plays an important role in the cell's digestive system.Designing and developing new near-infrared lysosomal targeting probes can visualize the various activities of lysosomes involved in cell physiology.Sulfur dioxide content is generally considered as one of the important indicators for assessing the level of air pollution.It is also a pollutant that causes severe weather such as acid rain.With the common use of chemical material burning in our lives,sulfur dioxide will be inhaled by the body and dissolved in human body fluids,and its derivatives sulphite(SO₃^2-)and bisulphite?HSO₃^-?,which will cause great toxic effects on our human body.Bisulfite is present in food additives in daily life.Excessive inhaled bisulfite can also cause great harm to human health.Therefore,the development of new lysosomal targeting and near-infrared fluorescent probes for detecting bisulphite is not only useful for the understanding of their biological role in cells,but also for the diagnosis of life phenomena and diseases.In this paper,three types of fluorescent probes were designed and synthesized from oxazine and stupid-halogen cyanine as the fluorophore,and their optical properties and biological properties were further studied.The details are as follows:1.Seven near-infrared probes 1a-g based on phenoxazines linked to different nitrogen-containing groups via amide condensation were synthesized.The optical properties of different nitrogen-containing groups for lysosomal targeting of probe 1a-g were evaluated.The 1b-d three probes exhibited a fluorescence emission response as a function of pH.They all exhibited the highest fluorescence emission at around 675 nm with pK_a of 3.99,5.57,and 2.46,respectively.The electron donating ability of the group increases,and the degree of quenching of the fluorescence intensity also increases.Cellular experiments demonstrated that probes 1c and 1d selectively labeled lysosomes of HeLa cancer cells and normal Ges-1 cells.Therefore,the aromatic N,N-diethylanilino and quinoline groups contained in 1c and 1d are considered to be novel lysosomal targeting groups.2.A series of six benzoxazine-based near-infrared pH probes 2a-f were designed and synthesized.Under the mechanism of photoinduced electron transfer based on fluorophore reaction?FBR-PET?,the optical properties as well as the influence of cell lysosome location of probes 2a-f were compared.The pH titration experiments showed that the three probes 2a-c exhibited near-infrared maximum fluorescence emission at 700 nm,and exhibited nearly "ON-OFF" emission response with increasing pH,with p K_a 5.03,4.92,4.90,respectively.Probes 2d-f exhibited near-infrared maximum fluorescence emission at the maximum emission of 691-717 nm with increasing p H?1.0-4.0?,but probes 2d-f did not show significant "ON-OFF" fluorescence emission.In response,the p K_a was 2.26,2.23,and 2.26,respectively.Cell experiments showed that probes 2a-c were lysosomal targeting probes in both cancer cells?HeLa?and normal cells?Ges-1?.Probe 2a is also very selective and reversible for protons.Fluorine imaging can be used to monitor the pH-induced increase in chloroquine.The above experimental results indicate that the oligomeric glycol chains have the ability to improve the localization of lysosomes for probes 2a-c that are suitable for pK_a values.3.A bisulphite probe 3c based on a stupid phthalocyanine was designed and synthesized,and its optical response to sodium bisulphite and its application in HeLa cells were studied in detail.The titration experiments on bisulfite showed that the probe 3c had fluorescence response to HSO₃^-colorimetric and OFF-ON type.At pH 5.0-8.0,the fluorescence emission of the probe 3c increases with increasing pH.To further investigate the detection application of the probe in a physiological environment,all the tests were performed in PBS with pH=7.4 containing 10%of DMSO.When NaHSO₃ was not added,the probe 3c showed very weak emission at 460 nm.When 8.0 equivalents of NaHSO₃were added,the fluorescence emission was significantly enhanced,and the fluorescence intensity at 460 nm was enhanced by 468 times.The response time was 180 seconds,and when the NaHSO₃ concentration was in the range of 0.0-0.3 equivalents,there was a linear relationship between the fluorescence intensity of the probe 3c and the NaHSO3 concentration,and the detection limit was as low as 13.9 nM.HeLa cell imaging experiments show that probe 3c can be used to selectively detect exogenous bisulfite in mitochondria...