CN116425737A - Fluorescent molecular probe for detecting hydroxyl free radicals and preparation method and application thereof - Google Patents
Fluorescent molecular probe for detecting hydroxyl free radicals and preparation method and application thereof Download PDFInfo
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- 239000003068 molecular probe Substances 0.000 title claims abstract description 34
- 125000002887 hydroxy group Chemical group [H]O* 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 19
- NBEFMISJJNGCIZ-UHFFFAOYSA-N 3,5-dimethylbenzaldehyde Chemical compound CC1=CC(C)=CC(C=O)=C1 NBEFMISJJNGCIZ-UHFFFAOYSA-N 0.000 claims abstract description 12
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims abstract description 12
- 238000003384 imaging method Methods 0.000 claims abstract description 8
- VRVRGVPWCUEOGV-UHFFFAOYSA-N 2-aminothiophenol Chemical compound NC1=CC=CC=C1S VRVRGVPWCUEOGV-UHFFFAOYSA-N 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 5
- 238000006243 chemical reaction Methods 0.000 claims description 11
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- BSKHPKMHTQYZBB-UHFFFAOYSA-N 2-methylpyridine Chemical class CC1=CC=CC=N1 BSKHPKMHTQYZBB-UHFFFAOYSA-N 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 4
- BTNNPSLJPBRMLZ-UHFFFAOYSA-N benfotiamine Chemical compound C=1C=CC=CC=1C(=O)SC(CCOP(O)(O)=O)=C(C)N(C=O)CC1=CN=C(C)N=C1N BTNNPSLJPBRMLZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000010992 reflux Methods 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 3
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- -1 hydroxyl radicals Chemical class 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 12
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- 238000001727 in vivo Methods 0.000 description 3
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- DMDRLTJRQCKPLQ-UHFFFAOYSA-N 2-(2-aminophenyl)benzenethiol Chemical compound NC1=CC=CC=C1C1=CC=CC=C1S DMDRLTJRQCKPLQ-UHFFFAOYSA-N 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
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- 230000009471 action Effects 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
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Abstract
A fluorescent molecular probe for detecting hydroxyl free radicals, a preparation method and application thereof are provided, wherein the fluorescent molecular probe has the following structural formula:
by adopting 3, 5-dimethylbenzaldehyde, 2-amino thiophenol, 2-picolinate and piperidine as raw materials, the method has the advantages of simple synthesis steps, easily available raw materials, simple detection method, low cost, small light absorption or self light intensity of the prepared fluorescent molecular probe biological tissue, strong penetrating capacity to the biological tissue, high imaging signal-to-noise ratio and high imaging resolution, and good water solubility, and is more beneficial to monitoring hydroxyl free radicals in living cells.
Description
Technical Field
The invention relates to a fluorescent molecular probe for detecting hydroxyl free radicals, and a preparation method and application thereof, belonging to the technical fields of small organic molecular fluorescent probes and biosensing.
Background
Reactive oxygen species (Reactive oxygen species, ROS) mainly include hydroxyl radicals (. OH), singlet oxygen [ (] 1 O 2 ) Superoxide anion (O) 2 ·- ) Hydrogen peroxide (H) 2 O 2 ) Hypochlorous acid (HClO) and peroxy radicals (·oor), which have been shown to play an important role not only in the immune nerve and cardiovascular system, but also to be involved in a variety of physiological and pathological processes. Wherein the hydroxyl radical is one of the strongest oxidants in the aqueous solution, and can react with various organic compounds through electron transfer, electrophilic addition, dehydrogenation reaction and the like to degrade them into CO finally 2 、H 2 O, and other harmless substances. Hydroxyl radicals are also one of the most harmful cellular ROS in vivo and can cause serious damage to biological macromolecules such as nucleic acids, carbohydrates, lipids, proteins, etc. Therefore, the development of a method with high selectivity and high sensitivity to monitor the processes of hydroxyl radical generation, distribution, metabolism and the like has important significance for further understanding the biological roles of the hydroxyl radicals.
However, since hydroxyl radicals have a short lifetime, high reactivity and low concentration in cells, although Electron Spin Resonance (ESR) spectroscopy, high performance liquid chromatography, spectrophotometry, electrochemical sensing, and the like have been developed for their in vitro detection, they are not suitable for in vivo hydroxyl radical monitoring. In contrast, fluorescence imaging has great significance in developing a novel fluorescent molecular probe capable of specifically recognizing hydroxyl radicals due to excellent sensitivity and selectivity, easy operation, noninvasive properties and real-time sensing capability.
Disclosure of Invention
Aiming at the problem that the mode cannot be suitable for detecting in-vivo hydroxyl radicals, the invention provides a fluorescent molecular probe for detecting hydroxyl radicals, and a preparation method and application thereof, and can be used for detecting hydroxyl radicals in living cells.
The invention adopts the technical means for solving the problems that: a fluorescent molecular probe for detecting hydroxyl free radicals has the structural formula shown as follows:
a preparation method of a fluorescent molecular probe for detecting hydroxyl free radicals comprises the following steps:
(1) 3, 5-dimethylbenzaldehyde, 2-aminothiophenol, 30% H 2 O 2 Dissolving 37% HCl in anhydrous alcohol solution such as anhydrous methanol, anhydrous ethanol, etc., stirring at normal temperature until the reaction is complete, pouring pure water, adjusting pH to near neutral, suction filtering, collecting and washing filter cake, and vacuum drying to obtain pale yellow product BTM; wherein the dosage ratio of the 3, 5-dimethylbenzaldehyde to the 2-amino thiophenol is as follows: the dosage ratio of the 3, 5-dimethylbenzaldehyde to the anhydrous alcoholic solution is 1:1-1:2 mmol/mmol: 1:8-1:15mmol/mL,3, 5-dimethylbenzaldehyde with 30% H 2 O 2 The dosage ratio of (2) is as follows: the dosage ratio of 3, 5-dimethylbenzaldehyde to 37% HCl is 1:0.8-1:1.2 mmol/mL: 1.5:1-2.5:1mmol/mL;
(2) Dissolving BTM, 2-picoline salt and piperidine in anhydrous alcoholic solution, heating and refluxing at 90 ℃ until the reaction is finished, removing solvent by rotary evaporation, and purifying by column chromatography to obtain red fluorescent molecular probe BTMP; wherein the dosage ratio of BTM to 2-picoline salt is: 1:1.2-1:2mmol/mmol, the dosage ratio of BTM to piperidine is: 1:1.8-1:2.5mmol/mmol, the dosage ratio of BTM to anhydrous alcoholic solution is: 1:8-1:20mmol/mL.
The reaction scheme is as follows:
the application of the fluorescent molecular probe for detecting the hydroxyl radical is used for quantitatively detecting the hydroxyl radical in a solution and imaging the endogenous hydroxyl radical in living cells.
The fluorescent molecular probe BTMP for detecting the hydroxyl radical provided by the invention has no fluorescence, and a product after the probe responds to the hydroxyl radical has red fluorescence, and simultaneously has an excited-state intramolecular proton transfer (ESIPT) effect, and can realize the specific detection of the hydroxyl radical by adopting a fluorescence detector, wherein the detection principle is as follows:
the beneficial effects of the invention are as follows:
1. the fluorescent molecular probe provided by the invention can perform specific action with hydroxyl free radicals to generate change of fluorescence spectrum, and is not interfered by other active oxygen species of a biological system in the detection process, so that the quantitative and specific recognition of the hydroxyl free radicals is realized.
2. The fluorescent molecular probe provided by the invention has the advantages of simple synthesis steps, readily available raw materials, simple detection method and low cost, and the prepared fluorescent molecular probe has the advantages of light absorption of biological tissues or small self light intensity, strong penetrating capacity to the biological tissues, high imaging signal-to-noise ratio and high imaging resolution, and good water solubility, and is more beneficial to monitoring hydroxyl free radicals in living cells.
Drawings
FIG. 1 is a graph showing the fluorescence intensity of the fluorescent molecular probe according to the concentration of hydroxyl radicals;
FIG. 2 shows a linear relationship between fluorescence intensity and hydroxyl radical concentration of a two fluorescent molecular probe of the example;
FIG. 3 is a plot of selectivity of three fluorescent molecular probes for hydroxyl radicals;
FIG. 4 is a diagram of fluorescence confocal imaging of four fluorescent molecular probes in HeLa cells, wherein: a) After 20min of treatment of the cells with the probe, the cells were treated with LPS; b) After 20min of treatment with the probe, the cells were treated with a buffer solution of PBS (0.01 m, ph=7.4).
Detailed Description
The invention is further described below with reference to the accompanying drawings.
Example 1
Synthesis of the Compound BTM
3, 5-dimethylbenzaldehyde (268.36 mg,2.0 mmol) was added to a 50mL round bottom flask and the mixture was used20mL of absolute ethanol was dissolved, and 2-aminophenylthiophenol (300 mg,2.4 mmol) was added to the reaction system, 30% H 2 O 2 (2 ml) and 37% HCl (1 ml), and the reaction was stirred at room temperature. TLC monitors the reaction until the reaction is complete, the reaction system is poured into pure water, the pH is regulated to be near neutral by NaOH solution, a large amount of precipitate is separated out, the filter cake is filtered, collected and washed, and the pale yellow product 446.86mg is obtained by vacuum drying, and the yield is 88.2%. 1 H NMR(500MHz,CDCl 3 ):δ9.88(s,1H),8.36(s,1H),8.18(d,J＝8.2Hz,1H),8.06(s,1H),8.02(d,J＝8.0Hz,1H),7.77(s,1H),7.53-7.50(m,2H),2.42(s,3H)。
Synthesis of Compound BTMP
In a 25mL round bottom flask, compound BTM (253.32 mg,1.0 mmol) and 10mL ethanol were added and after complete dissolution, 2-methylpyridine salt (352.61 mg,1.5 mmol) and 0.5mmol piperidine were added and heated to reflux at 90 ℃. TLC monitoring was completed, after cooling at room temperature, the solvent was removed by rotary evaporation, and purification by column chromatography gave 278.02mg of the red product in 59.1% yield. 1 H NMR(500MHz,DMSO-d 6 ):δ8.84(d,J＝7.5Hz,1H),8.18(d,J＝7.0Hz,1H),8.10(t,J＝8.0Hz,1H),8.02(d,J＝8.0Hz,1H),7.85(s,1H),7.66-7.63(m,2H),7.53(t,J＝6.5Hz,1H),7.51(t,J＝7.0Hz,1H),7.29(d,J＝7.6Hz,1H),7.26(s,1H),6.59-6.57(m,2H),4.35(s,3H),2.44(s,3H).HRMS-ESI(C 22 H 19 IN 2 S)m/z:calc.for[M-I] + :343.1263；found,343.1405。
Example two
This example is a fluorescence response experiment of a fluorescent molecular probe to hydroxyl radicals
This example uses mild EDTA-Fe 2+ The complex is subjected to Fenton reaction in an aqueous phase and a biological system to generate OH. Firstly, the probe is combined with EDTA-Fe 2+ The complex was mixed in PBS buffer (0.01 m, ph=7.4) and then H was added 2 O 2 Such that the final concentration of OH is 0, 2, 5, 10, 15, 20, 25, 50, 100. Mu.M, respectively, and the final concentration of probe is 10. Mu.M. And (3) reacting the system to be detected for 20 minutes at room temperature for full response, and then performing fluorescence detection. As a result, as shown in FIG. 1, it can be seen that the probe itself is non-fluorescent at 667nm, along with OHThe concentration is increased, the fluorescence is gradually enhanced, and the fluorescence intensity is in good linear relation (R) with 667nm in the concentration range of 0-15 mu M 2 0.99448) (see fig. 2), the lowest limit of detection (LOD) of OH in solution was found to be 51.2nM using standard calculation methods (S/n=3). Indicating that the probe can respond well to OH in solution.
Example III
This example is a hydroxyl radical selectivity assay for fluorescent molecular probes
To investigate the selectivity of fluorescent molecular probes for different reactive oxygen species, we selected a number of well known reactive oxygen species, H 2 O 2 、 1 O 2 、O 2 - 、ROO·、NO·、ONOO - 、ClO - And the aqueous solution was prepared and added to the probe solution at a concentration of 30. Mu.M, 10. Mu.M probe and 10. Mu.M OH. After measuring the response of the fluorescence intensity of the probe to different active oxygen compounds and comparing the response with the control group of OH, the probe is found to have obvious fluorescence enhancement at 667nm only for OH, and the fluorescence change of other small molecules tested is weak (see figure 3). It was confirmed that the probe had excellent selectivity to OH.
Example IV
This example is an imaging experiment of fluorescent molecular probes on endogenous hydroxyl radicals in living cells
HeLa cells were seeded into 96-well cell culture plates at 37℃and 5% CO 2 The culture was carried out overnight under the conditions. Cells were divided into two groups, each of which was incubated with probe (20. Mu.M) for 20min, the experimental group was treated with 10. Mu.g/mL LPS (which stimulated ROS formation including hydroxyl radicals), and the control group was treated with PBS buffer (0.01M, pH=7.4), after 60min, cell imaging was performed. As a result, as shown in FIG. 4, it can be seen that the experimental group (FIG. 4 a) showed a clear red fluorescence, while the control group (FIG. 4 b) showed no clear red fluorescence, indicating that the probe was able to respond to the endogenous hydroxyl radical in the living cells.
The above embodiments are only for illustrating the present invention, not for limiting the present invention, and various changes and modifications may be made by one skilled in the relevant art without departing from the spirit and scope of the present invention, so that all equivalent technical solutions shall fall within the scope of the present invention, which is defined by the claims.
Claims (9)
1. A fluorescent molecular probe for detecting hydroxyl radicals, which is characterized in that: the structural formula is as follows:
2. a method for preparing a fluorescent molecular probe for detecting hydroxyl radicals according to claim 1, which is characterized in that: the method comprises the following steps:
(1) 3, 5-dimethylbenzaldehyde, 2-amino thiophenol and H 2 O 2 Dissolving HCl in anhydrous alcoholic solution, stirring at normal temperature until the reaction is complete, pouring pure water, adjusting pH to near neutral, purifying and drying to obtain light yellow product BTM;
(2) And dissolving BTM, 2-picoline salt and piperidine in an anhydrous alcoholic solution, heating and refluxing until the reaction is finished, and purifying to obtain the red fluorescent molecular probe BTMP.
3. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: in the step (1), the dosage ratio of 3, 5-dimethylbenzaldehyde to 2-amino thiophenol is as follows: the dosage ratio of the 3, 5-dimethylbenzaldehyde to the anhydrous alcoholic solution is 1:1-1:2 mmol/mmol: 1:8-1:15mmol/mL,3, 5-dimethylbenzaldehyde and H 2 O 2 The dosage ratio of (2) is as follows: 1:0.8-1:1.2mmol/mL (when H) 2 O 2 At a concentration of 30%), the ratio of 3, 5-dimethylbenzaldehyde to HCl used was: 1.5:1-2.5:1mmol/mL (when HCl concentration is 37%).
4. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: in the step (2), the dosage ratio of BTM to 2-picoline salt is as follows: 1:1.2-1:2mmol/mmol, the dosage ratio of BTM to piperidine is: 1:1.8-1:2.5mmol/mmol, the dosage ratio of BTM to anhydrous alcoholic solution is: 1:8-1:20mmol/mL.
5. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: the anhydrous alcoholic solution is anhydrous methanol or anhydrous ethanol.
6. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: in the step (1), the purification and drying modes are as follows: the filter cake is filtered off with suction, collected and washed and then dried in vacuo.
7. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: in the step (2), the heating reflux temperature is 90 ℃.
8. The method for preparing the fluorescent molecular probe for detecting hydroxyl radicals according to claim 2, wherein the method comprises the following steps: in the step (2), the purification mode is as follows: the solvent was removed by rotary evaporation and then purified by column chromatography.
9. Use of a fluorescent molecular probe for detecting hydroxyl radicals according to claim 1, characterized in that: the method is used for quantitatively detecting the hydroxyl free radicals in the solution and imaging the endogenous hydroxyl free radicals in living cells.
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| CN202310345791.0A CN116425737A (en) | 2023-04-03 | 2023-04-03 | Fluorescent molecular probe for detecting hydroxyl free radicals and preparation method and application thereof |
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