CN113896739A - A kind of reactive rhodamine B derivative fluorescent probe and its preparation method and application - Google Patents
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Abstract
The invention discloses a reactive rhodamine B derivative fluorescent probe and a preparation method and application thereof, and the rhodamine B derivative is reformed to synthesize the fluorescent probe capable of simultaneously aiming at H2O2And ClO‑The fluorescent molecular probe of the invention is subjected to H2O2And ClO‑Different optical effects can be generated during stimulation, and H can be effectively positioned2O2And ClO‑Production in cells for the detection of hydrogen peroxide and hypochlorite in inflammatory cells.
Description
Technical Field
The invention particularly relates to a reactive rhodamine B derivative fluorescent probe and a preparation method and application thereof, belonging to the technical field of cell detection and analysis.
Background
Active oxygen is involved in a variety of physiological and pathological processes, hydrogen peroxide (H)2O2) With hypochlorous acid (ClO)-) Is two important active oxygen species in organisms, and is widely present in various organisms. Under normal physiological environmental conditions, H2O2And ClO-Through respective regulation and control, the concentration is always kept in a relatively warm concentration state.
The nuclear factor kappa B (NF-kappa B) protein family can be selectively combined with B cell kappa-light chain enhancer to regulate the expression of a plurality of genes. NF- κ B is found in almost all animal cells and is involved in the response of cells to external stimuli, such as cytokines, radiation, heavy metals, viruses, etc. NF- κ B plays a critical role in the inflammatory response, immune response, etc. of cells, where they secrete large numbers of reactive oxygen species, such as H2O2，ClO-And ONOO-And the like. However, when the NF-. kappa.B pathway is stimulated, cells produce inflammatory responses that cause oxidative stress, which leads to H2O2And ClO-Abnormal expression, damage to cells and various common clinical diseases. Therefore, detection of intracellular ROS is crucial to prevent the development of disease.
In the last decade, fluorescence methods have attracted much attention due to their advantages of high sensitivity, fast response time, high selectivity, low cost, simple operation, etc., and more importantly, the use of fluorescent probes allows visualization of in vitro and in vivo detection processes. The patent with the application number of CN201611141760.X discloses a preparation method of a nano fluorescent probe for detecting active oxides and the fluorescent probe, and particularly discloses a method for preparing a nano fluorescent probe for detecting active oxides by using 2, 2' -dithiosalicylic acid as a ligand, 1, 10-phenanthroline as an auxiliary ligand, adding rhodamine 6G, and performing polymerization reaction by using cadmium ions as central metal ions to obtain a nano rod-shaped disulfide coordination polymer as the nano fluorescent probe for detecting the active oxides, wherein the fluorescent probe is only used for detecting H2O2. At present, H2O2And ClO-The development of probes in each field has made remarkable progress, however, the probe can not simultaneously aim at H2O2And ClO-Different corresponding reactivity probes are made, therefore, a need exists for a near infrared H with specific subcellular organelle labeling capability2O2And ClO-And (3) a probe.
Disclosure of Invention
The invention aims to provide a reactive rhodamine B derivative fluorescent probe and a preparation method and application thereof2O2And ClO-The fluorescent molecular probe of the reacted near-infrared light can generate different optical effects aiming at two active oxygen so as to better identify H2O2And ClO-。
The technical scheme of the invention is as follows:
the invention aims to provide a reactive rhodamine B derivative fluorescent probe, which has the following chemical structural formula:
the invention also aims to provide a preparation method of the reactive rhodamine B derivative fluorescent probe, which comprises the following steps:
(1) dissolving 15.6-20.6 mmol of 2- (4-dibutylamino-2-hydroxybenzoyl) benzoic acid and 15.6-20.6 mmol of 1, 6-dihydroxy naphthalene in a mixed solution of methanesulfonic acid and trifluoroacetic acid, fully reacting for 24-30 h at 80-85 ℃, standing to normal temperature, and adding 100-120 mL of deionized water for mixing;
(2) filtering the mixed solution, washing the mixed solution with deionized water for multiple times to obtain a crude product, and purifying the crude product by silica gel column chromatography to obtain a compound A;
wherein the reaction formula of the compound A is shown as follows:
(3) dissolving 1-1.2 mmol of the compound A and 1.2-1.5 mmol of 4-bromomethylbenzeneboronic acid pinacol ester in 10-12 mL of dry acetonitrile, adding 0.5-0.7 mmol of cesium carbonate, carrying out a light-shielding reflux reaction at 90-95 ℃ for 3-4 h, and purifying by silica gel column chromatography after the reaction is finished to obtain a product fluorescent probe;
wherein, the reaction formula of the product fluorescent probe is as follows:
furthermore, the volume ratio of the 1, 6-dihydroxynaphthalene dissolved in the mixed solution of the methanesulfonic acid and the trifluoroacetic acid is 40-60 mL, wherein the volume ratio of the methanesulfonic acid to the trifluoroacetic acid is 1: 1.
The invention also aims to provide an application of the reactive rhodamine B derivative fluorescent probe in detecting hydrogen peroxide and hypochlorite of inflammatory cells.
The invention also aims to provide an application of the reactive rhodamine B derivative fluorescent probe in the index detection of cell activity, cytotoxicity and apoptosis or specific subcellular organelle labeling.
Compared with the prior art, the invention has the beneficial effects that: the reactive rhodamine B derivative fluorescent probe provided by the invention can be simultaneously used with H2O2And ClO-The fluorescent probe of the invention can react with ClO and generate different optical effects aiming at two active oxygen-The reaction takes place rapidly, strong fluorescence is produced, but the fluorescence gradually disappears in a short time; and H2O2The reaction speed is relatively slow, and the fluorescence is gradually enhanced, so that the probe can better identify H2O2And ClO-And can better detect the active oxygen in the cells.
Reference numerals
FIG. 1 shows fluorescent probe P1 prepared according to example 1 of the present invention with different concentrations H2O2An optical property profile of the reaction;
FIG. 2 shows a fluorescent probe P1 prepared according to example 1 of the present invention and 1.2mM H2O2Optical property analysis graphs of the reaction at different times;
FIG. 3 shows a fluorescent probe P1 prepared according to example 1 of the present invention and 1.2mM ClO-Optical property analysis graphs of the reaction at different times;
FIG. 4 is a graph showing fluorescence absorption in comparison with the selectivity change of the fluorescent probe P1 prepared in example 1 according to the present invention;
FIG. 5 is a schematic diagram of the cytotoxicity analysis of fluorescent probe P1 prepared according to example 1 of the present invention at different concentrations;
FIG. 6 is a photograph of a live cell image of fluorescent probe P1 prepared according to example 1 of the present invention in normal raw264.7 cells;
FIG. 7 is a photograph showing live cell images of fluorescent probe P1 prepared according to example 1 of the present invention in raw264.7 cells after 1mg/L LPS treatment.
Detailed Description
The invention will be further described with reference to the accompanying drawings and preferred embodiments, which are given for illustration only and are not intended to limit the scope of the invention.
Materials, reagents and the like used in the following examples were commercially available and all commercial reagents were further purified unless otherwise specified;
drying solvents used in the reaction are all dried by using a molecular sieve 4A type (sodium-A type molecular sieve) or a molecular sieve 3A type (potassium-A type molecular sieve);
argon is used as protective gas in the inert atmosphere;
1h spectra were recorded on a JEOL ECZ600S (600MHz) spectrometer using CDCl3Or CD3OD as a solvent;
parts per million of the front field chemical shift is reported from the internal TMS (trimethylsilane) reference data;
the coupling constant (J) is expressed in Hertz (Hz), the spin-singlet is expressed in s (singlet), d (doublet), t (triplet), and m (multiplet);
the column chromatography adopts a thick-wall glass column and silica gel (300-400 meshes); performing Thin Layer Chromatography (TLC) with commercially available 0.25mm silica gel plate, and displaying with ultraviolet lamp;
obtaining an ultraviolet absorption spectrum in the solution by using an Shimadzu UV-1900 ultraviolet-visible-near infrared spectrophotometer; the fluorescence spectrum is measured by a Spectrofluorometer FS5 fluorescence spectrometer; recording the mass spectrum on a ThermoFisher high performance liquid chromatography-mass spectrometer;
cell fluorescence imaging was performed using a Zeiss confocal laser microscope.
Example 1
A preparation method of a reactive rhodamine B derivative fluorescent probe comprises the following steps:
(1) dissolving 15.6mmol of 2- (4-dibutylamino-2-hydroxybenzoyl) benzoic acid and 15.6mmol of 1, 6-dihydroxynaphthalene in 40mL of mixed solution of methanesulfonic acid and trifluoroacetic acid, fully reacting for 24h at 80 ℃, standing to normal temperature, and adding 100mL of deionized water for mixing; wherein the mixed solution consists of 20mL of methanesulfonic acid and 20mL of trifluoroacetic acid;
(2) filtering the mixed solution, washing the mixed solution for multiple times by using deionized water to obtain a crude product, and purifying the crude product by silica gel column chromatography to obtain a compound A;
(3) dissolving 1mmol of the compound A and 1.2mmol of 4-bromomethylbenzeneboronic acid pinacol ester in 10mL of dry acetonitrile, adding 0.5mmol of cesium carbonate, refluxing and reacting at 90 ℃ in the dark for 3h, and purifying by silica gel column chromatography after the reaction is finished to obtain a product, namely the fluorescent probe P1.
Example 2
A preparation method of a reactive rhodamine B derivative fluorescent probe comprises the following steps:
(1) dissolving 20.6mmol of 2- (4-dibutylamino-2-hydroxybenzoyl) benzoic acid and 20.6mmol of 1, 6-dihydroxynaphthalene in 60mL of mixed solution of methanesulfonic acid and trifluoroacetic acid, fully reacting at 85 ℃ for 30h, standing to normal temperature, and adding 120mL of deionized water for mixing, wherein the mixed solution consists of 30mL of methanesulfonic acid and 30mL of trifluoroacetic acid;
(2) filtering the mixed solution, washing the mixed solution for multiple times by using deionized water to obtain a crude product, and purifying the crude product by silica gel column chromatography to obtain a compound A;
(3) dissolving 1.2mmol of the compound A and 1.5mmol of 4-bromomethylbenzeneboronic acid pinacol ester in 12mL of dry acetonitrile, adding 0.7mmol of cesium carbonate, refluxing and reacting at 95 ℃ in the dark for 4 hours, and purifying by silica gel column chromatography after the reaction is finished to obtain a product, namely the fluorescent probe P2.
Example 3
A preparation method of a reactive rhodamine B derivative fluorescent probe comprises the following steps:
(1) dissolving 18.6mmol of 2- (4-dibutylamino-2-hydroxybenzoyl) benzoic acid and 18.6mmol of 1, 6-dihydroxynaphthalene in 50mL of mixed solution of methanesulfonic acid and trifluoroacetic acid, fully reacting at 82 ℃ for 26h, standing to normal temperature, and adding 110mL of deionized water for mixing, wherein the mixed solution consists of 25mL of methanesulfonic acid and 25mL of trifluoroacetic acid;
(2) filtering the mixed solution, washing the mixed solution for multiple times by using deionized water to obtain a crude product, and purifying the crude product by silica gel column chromatography to obtain a compound A;
(3) dissolving 1.1mmol of the compound A and 1.4mmol of 4-bromomethylbenzeneboronic acid pinacol ester in 10mL of dry acetonitrile, adding 0.6mmol of cesium carbonate, carrying out reflux reaction at 92 ℃ in the dark for 3h, and purifying by silica gel column chromatography after the reaction is finished to obtain a product, namely the fluorescent probe P3.
Example 4 spectroscopic measurement of fluorescent Probe P1 obtained in example 1
Dissolving P1 in DMSO to prepare 10mM working mother liquor for later use, adding a 3mL PBS and acetonitrile mixed solution into a 3.5mL cuvette, adding 7:3 acetonitrile into PBS, uniformly mixing 3uL P1(10mM) and 3uL 10% pluronic, adding into the cuvette, and respectively measuring the ultraviolet spectrum and the fluorescence spectrum of P1; ROSs (H) of different concentration gradients were added to the cuvette2O2、ClO-、ONOO-) Ion (K) commonly found in living body+、Ca2+、Mg2+、Cl-、Br-、I-、NO2 -、HCO3 -、CO3 2-、H2PO4 -、PO4 3-) And biological thiols (TBHP, Cys, GSH), ultraviolet spectral characteristics and fluorescence spectral characteristics of P1 after reaction with these substances were measured; the fluorescence excitation and emission slits used in the experiment were both 2nm, scanned 2 times.
Referring to FIG. 1, in FIG. 1(A), fluorescent probes P1 and H2O2(0-1.6mM) graph showing the change in UV absorption after 30min of reactionAs shown, with H2O2The ultraviolet absorption of the fluorescent probe P1 at 549nm is gradually enhanced when the concentration is increased, and when the H is higher2O2After the content reaches 120 times of the concentration of the fluorescent probe P1, H is continuously added2O2The ultraviolet absorption of the fluorescent probe P1 at 549nm is not increased continuously; FIG. 1(B) shows fluorescent probes P1 and H2O2(0-1.6mM) reaction time 30min, and the fluorescence absorbance change is shown as H2O2The fluorescence of the fluorescent probe P1 at 642nm is gradually increased when the concentration is increased, and when the concentration is increased, the fluorescence is increased2O2After the content reaches 120 times of the concentration of the fluorescent probe P1, H is continuously added2O2The fluorescence of the fluorescent probe P1 at 642nm does not continue to increase; the results demonstrate that fluorescent probe P1 can react with H2O2The reaction is carried out, and the upper limit of the reaction is 120 times of that of P1;
referring to FIG. 2, in FIG. 2(A), the fluorescent probe P1 and 1.2mM H are shown2O2Graph showing the change of ultraviolet absorption within 30min of the reaction, from which H was added2O2The time of the probe is gradually increased, the ultraviolet absorption of the fluorescent probe P1 at 549nm is gradually enhanced, and the ultraviolet absorption does not continuously increase after 24 min; FIG. 2(B) shows the fluorescent probe P1 and 1.2mM H2O2Shift plot of fluorescence absorption within 30min of reaction with addition of H2O2The time of the probe is gradually increased, the fluorescence of the fluorescent probe P1 at 642nm is gradually enhanced, and the fluorescence is not continuously enhanced after 24 min; the results demonstrate that fluorescent probes P1 and H2O2The reaction of (2) takes 24min to reach the optimal luminescence state;
referring to FIG. 3, in FIG. 3(A), the fluorescent probe P1 and 1.2mM ClO are used-Graph showing the change in ultraviolet absorption within 60min of the reaction, from which it was found that the fluorescent probe P1 was added with ClO-Then, ultraviolet absorption at 292nm and 586nm is increased instantly, and the ultraviolet absorption begins to be gradually weakened along with the change of time; FIG. 3(B) shows the fluorescent probe P1 and 1.2mM ClO-The fluorescence absorption in the reaction time of 60min was changed to a graph, and it was found that the ClO was added to the fluorescent probe P1-Then, the fluorescence intensity at 640-660nm is increased instantly, and the fluorescence intensity begins to gradually change along with the change of timeGradually weakening; the results demonstrate that the fluorescent probe P1 reacts with ClO-The reaction of (3) is completed instantaneously and the light absorption intensity is gradually decreased with time.
Referring to FIG. 4, FIG. 4(A) is a graph comparing the fluorescence absorbance of the selective change of the fluorescent probe P1 (immediately after the addition of the drug), and FIG. 4(B) is a graph comparing the fluorescence absorbance of the selective change of the fluorescent probe P1; as can be seen, the fluorescent probe P1 was added to ClO after the addition of the drug-Has good selectivity, and the fluorescent probe P1 is added with the medicine for 30min, but is on the contrary to H2O2Has good selectivity.
In conclusion, fluorescent molecular probe P1 has the function of detecting H simultaneously2O2And ClO-Can be better identified by different chemical effects and different optical effects2O2And ClO-。
EXAMPLE 5 CCK-8 toxicity testing of fluorescent Probe P1 prepared according to example 1
Gently blowing down adherent Raw264.7 cells by using a culture medium, centrifuging at low speed (900rpm/min, 25 ℃, 5min), removing the old culture medium, adding a new culture medium, uniformly blowing to prepare a cell suspension, and then spreading the cells into a 96-well plate; the experiment is divided into three groups, namely a blank group, a control group and an experiment group; wherein the blank group comprises a culture medium, a CCK-8 solution, no cell and no probe; the control group had cells, CCK-8 solution, no probe; the experimental group comprises cells, CCK-8 solution and probes; the number of cells plated in each well is 5 multiplied by 103, 200 mu L of culture medium is added in each well, after culturing for 24h and the cells are firmly attached to the wall, fluorescent probes (2 mu M, 5 mu M, 8 mu M, 10 mu M, 15 mu M and 20 mu M) with different concentrations are added in each well; incubating for 20min, adding 20 μ L CCK-8 reagent, reacting at 37 deg.C for 3h, mixing on oscillator, placing in microplate reader, measuring absorbance at 450nm wavelength, and setting 4 repeated controls under each condition;
calculating the cell survival rate: cell viability (%) - [ a (experimental) -a (blank) ]/[ a (control) -a (blank) ] × 100%.
Referring to fig. 5 for analysis of toxicity of fluorescent probe P1 on cells at different concentrations, it can be seen that the survival rate of cells shows significant difference after cells are stimulated for 24h by adding P1 at different concentrations, and the fluorescent probe P1 at 0-8 μ M shows substantially no toxicity on cells, and generates weak toxicity on cells after exceeding 10 μ M.
Example 6 cellular imaging of fluorescent Probe P1 prepared according to example 1
Raw264.7 cells were plated into confocal laser dishes (5X 10)3Individual cell), after 18h of culture, removing the culture medium, washing with HBSS twice, removing the residual culture medium, adding 10 mu M fluorescent probe P1 into a laser confocal dish, and incubating and dyeing for 20min in a dark place; after dyeing is finished, cleaning with HBSS, removing residual dye liquor, adding HBSS, performing imaging acquisition, taking a picture, and adding H with different concentrations into a dish2O2And then image acquisition is carried out.
Referring to FIG. 6 photograph of live cell image of fluorescent probe P1 in normal raw264.7 cell, it can be seen that the cell showed red fluorescence with time increasing and gradually increased fluorescence after fluorescent probe P1 was added, and the outer edge H was added2O2Thereafter, the cell fluorescence is further enhanced; the results demonstrate that the fluorescent probe P1 can detect H in living cells2O2；
Referring to FIG. 7, which is a photograph showing the live cells imaged in raw264.7 cells after 1mg/L LPS treatment with fluorescent probe P1, it can be seen that the cells showed strong red fluorescence after fluorescent probe P1 was added, and the fluorescence gradually increased with the increase of time, and the outer edge H was added2O2Thereafter, the cell fluorescence is further enhanced; the results demonstrate that fluorescent probe P1 can detect H in inflammation-induced living cells2O2。
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
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| CN113264954A (en) * | 2021-05-28 | 2021-08-17 | 中国科学院新疆理化技术研究所 | Fluorescent probe molecule for detecting hydrogen peroxide and preparation method thereof |
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| CN117986288A (en) * | 2024-03-04 | 2024-05-07 | 南京工业大学 | Hydrogen peroxide (H) based on silicon rhodamine2O2) Synthesis and application of detection fluorescent probe |
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