Synthesis and application of difunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals

Abstract

The invention discloses a difunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate (ATP) and hydroxyl radicals (. OH). The chemical structural formula of the difunctional probe is as follows: # imgabs0 #. According to the bifunctional fluorescent probe, a rhodamine derivative and a coumarin derivative are combined through a simple condensation reaction, and ATP and. OH are detected and distinguished through specific interaction of a nitrogen-containing group and ATP and aromatic hydroxylation of. OH. When the probe reacts with ATP (adenosine triphosphate), red fluorescence of 590 nm is emitted under the excitation wavelength of 520 nm; when acting with. OH, green fluorescence of 496 nm is emitted under the excitation wavelength of 420 nm. The probe for detecting ATP and. OH has the advantages of good selectivity, high sensitivity and the like, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and specifically relates to the synthesis and application of a bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl free radicals. The probe combines two fluorophores through a simple condensation reaction and can rapidly and selectively detect adenosine triphosphate and hydroxyl free radicals from various biologically active substances. The green fluorescent channel selectively detects hydroxyl free radicals, and the infrared fluorescent channel selectively detects adenosine triphosphate. It has the advantages of good selectivity, high detection sensitivity, and visual detection. Background Art

[0002] Abnormal mitochondrial oxidative stress and impaired energy metabolism are closely related to the occurrence and onset of a variety of diseases (Nat.Med., 2014, 20, 555−560). The study of key active species associated with these diseases not only provides an understanding of the pathogenesis of the disease, but also helps to develop targeted and therapeutic drugs. Hydroxyl free radicals (·OH) are a type of reactive oxygen species with a very short life span that can damage almost all types of macromolecules: DNA, nucleic acids (mutation), proteins and lipids (lipid peroxidation), etc. It is considered to be the most toxic reactive oxygen species. Excessive ·OH can cause irreversible damage to mitochondria (Chem. Res.Toxicol., 2008, 21, 172-188). At the same time, ATP is an important messenger that regulates mitochondrial energy metabolism. It is worth mentioning that ATP has overlapping roles with ·OH. In the mitochondrial respiratory chain, it produces energy along with the production of free radicals. However, higher metabolic rates induce more ·OH production. Surprisingly, insufficient ATP production can in turn lead to an increase in ·OH, which is due to changes in mitochondrial membrane potential. On the other hand, excessive levels of ·OH may damage proteins or DNA, further altering ATP synthesis (Nat. Metab., 2019, 1, 975−984; Cell Res., 2011, 21, 817−834.). Therefore, it is extremely important to simultaneously monitor the real-time dynamic changes of ·OH and ATP levels in mitochondria.

[0003] Currently, fluorescent probe imaging analysis is generally favored due to its rapid response and high sensitivity, as well as spatial resolution and satisfactory biocompatibility (Angew. Chem. Int. Ed., 2017, 56, 16611–16615; Anal. Chem., 2016, 76, 166–181). Many fluorescent probes for detecting ATP and ·OH have been reported, but most of them detect only one of them (Anal. Chem., 2017, 89, 1749−1756). At present, the simultaneous detection of ATP and ·OH remains a huge challenge. Summary of the invention

[0004] In view of the above situation, in order to overcome some deficiencies of the prior art, the present invention aims to provide a dual-function fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl free radicals. The probe can rapidly and selectively detect adenosine triphosphate and hydroxyl free radicals from various biologically active substances under specific detection conditions.

[0005] The present invention also aims to provide a method for synthesizing and applying the above-mentioned bifunctional fluorescent probe with simple preparation method, high sensitivity, low detection limit and low cost.

[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize a bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals, and to prepare a device for quantitatively analyzing adenosine triphosphate and hydroxyl radicals in the environment and simultaneously distinguishing and imaging adenosine triphosphate and hydroxyl radicals in living cells. The chemical structure of the bifunctional probe is as follows: .

[0007] A method for preparing a bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals comprises the following steps: Step 1. Synthesis of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid Add methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoate to a methanol solution, then add sodium hydroxide, react overnight at 40 degrees Celsius, and after the reaction is complete, decompress the reaction solution and spin dry it in a vacuum, and separate and purify the crude product by column chromatography to obtain 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid; Step 2. Synthesis of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid is added to ethanol, and then malononitrile is added, and the mixture is stirred at room temperature overnight. After the reaction is complete, the mixture is filtered, and the solid is vacuum-dried to obtain 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid; Step 3. Synthesis of the bifunctional fluorescent probe 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthenyl]-3-one is added and stirred for 5 minutes, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is separated and purified by column chromatography.

[0008] The method for using the dual-function fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals of the present invention is as follows: unless otherwise specified, the dual-function fluorescent probe is usually dissolved in a N,N -Dimethylformamide (DMF) was used for analysis and detection in an environment with an organic phase and an aqueous phase volume ratio of 4:6. The organic phase was methanol and the aqueous phase was a phosphate buffer solution (PBS) with a pH = 7.4.

[0009] The specific characteristics of the dual-function fluorescent probe of the present invention for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals are as follows: the dual-function fluorescent probe is dissolved in DMF, and the dual-function probe is dissolved in an organic phase and an aqueous phase (4:6, v / v) solution. After 30 minutes with adenosine triphosphate, a red fluorescence of 590 nm is emitted at an excitation wavelength of 520 nm; the probe responds to hydroxyl radicals and emits a green fluorescence of 496 nm at an excitation wavelength of 420 nm. Therefore, the detection of specific analytes with specific excitation and fluorescence emission signals is achieved. When both substances are present, the two can be well distinguished by using different excitation and fluorescence emission signals. The above-mentioned dual-function fluorescent probe realizes the simultaneous distinguishing and detecting of adenosine triphosphate and hydroxyl radicals under different detection conditions, and has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen. The detection limits of adenosine triphosphate and hydroxyl radicals are as low as 63.6 μM and 21.3 nM, respectively. Therefore, the dual-function fluorescent probe disclosed in the present invention can achieve highly sensitive distinguishing and quantitative detection of the two. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the bifunctional fluorescent probe of the present invention.

[0011] Figure 2 The dual-function fluorescent probe of the present invention responds to ultraviolet and fluorescence spectra of adenosine triphosphate and hydroxyl free radicals.

[0012] Figure 3 The dual-function fluorescent probe of the present invention responds to the fluorescence quantitative spectrum of adenosine triphosphate and hydroxyl free radicals.

[0013] Figure 4 The dual-function fluorescent probe of the present invention responds to the selective spectrum of adenosine triphosphate and hydroxyl free radicals. DETAILED DESCRIPTION

[0014] The present invention is further described with reference to the following figures.

[0015] The synthesis route of the bifunctional fluorescent probe of the present invention is shown in the figure below:

[0016] Example 1. Synthesis of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid 300.0 mg (942.29 μmol) of methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoate was added to 20 mL of methanol solution, followed by 376.9 mg (9.42 mmol) of sodium hydroxide. The mixture was reacted at 40 degrees Celsius overnight. After the reaction was complete, the reaction solution was dried under reduced pressure. The crude product was separated and purified by column chromatography to obtain 216 mg of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid with a yield of 75.32%.

[0017] Example 2. Synthesis of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid 150.0 mg (492.86 μmol) of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid was added to 8 mL of ethanol, and then 65.1 mg (985.72 μmol) of malononitrile was added. The mixture was stirred at room temperature overnight. After the reaction was complete, the mixture was filtered and the solid was dried under vacuum to obtain 130 mg of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid with a yield of 74.85%.

[0018] Example 3. Synthesis of the bifunctional fluorescent probe 50.0 mg (141.89 μmol) of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid was added to 6 mL of anhydrous dichloromethane, and then 3.5 mg (28.38 μmol) of 4-dimethylaminopyridine (DMAP) was added. The mixture was reacted at room temperature for 5 min. Then, 74.9 mg (141.89 μmol) of 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthenyl]-3-one was added and stirred for 5 min. Then, 54.4 mg (283.77 The mixture was stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by rotary evaporation and purified by column chromatography to obtain 50.0 mg of the bifunctional fluorescent probe with a yield of 40.88%.

[0019] Example 4. Dual-functional fluorescent probe for simultaneous detection of ATP and ·OH in vitro The spectral property experiment of the bifunctional fluorescent probe described in the present invention: the bifunctional probe is dissolved in DMF to prepare a probe solution with a concentration of 1 mM, and a 100 mM ATP analysis mother solution and a 10 mM ·OH analysis mother solution are prepared. The specific test method is: take 20 μL of 1 mM probe solution, add 200 μL of 100 mM ATP solution, and finally add 780 μL of methanol and 1000 μL of PBS buffer. All tests maintain the volume ratio of organic phase to aqueous phase at 4:6 (the total volume of each test sample is 2 mL). For example, when the test concentration of ATP fluorescence intensity is required to be 10 mM, the sample preparation is: take 20 μL of 1 mM probe solution, 200 μL of 100 mM ATP analysis solution, and then add 780 μL of methanol and 1000 μL of PBS buffer solution in a 2 mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured with an excitation wavelength of 520 nm. When detecting 100 μM·OH, the specific test method is: take 20 μL of 1 mM probe solution, 20 μL of 10 mM·OH analysis solution, add 780 μL of methanol and 1000 μL of PBS buffer solution in a 2 mL sample tube, shake at room temperature for 30 minutes, and measure its fluorescence emission intensity with an excitation wavelength of 420 nm. This dual-function probe can distinguish and detect two biologically active substances, ATP and ·OH, with different excitation wavelengths and fluorescence emission signals. It has high sensitivity, and the detection limits are as low as 63.6 μM and 21.3 nM, respectively, which is very suitable for imaging / quantitative analysis of endogenous ATP and ·OH in living cells.

[0020] The present invention provides a bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals. The rhodamine derivative and the coumarin derivative are connected by a simple condensation reaction, and the specific interaction between the nitrogen-containing group and ATP and the aromatic hydroxylation of OH are used to detect and distinguish ATP and OH. When the probe reacts with ATP, it emits a red fluorescence of 590 nm at an excitation wavelength of 520 nm; when it acts with OH, it emits a green fluorescence of 496 nm at an excitation wavelength of 420 nm. The fluorescence discrimination is obvious, the selectivity is good, and the water solubility of the product after the reaction is good, the response speed is fast, and the like. It has great practical application value in the fields of biochemistry, analysis and detection. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be regarded as a limitation of the present invention. After reading the above content, it will be obvious to those skilled in the art for various modifications and substitutions of the present invention. Therefore, fluorescent probes with similar technical features as described herein all fall within the scope of protection of this patent.

Claims

1. A bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals, characterized in that: The chemical structure of the bifunctional fluorescent probe is as follows: 。 2. The synthesis of the bifunctional fluorescent probe according to claim 1, characterized in that: The synthesis method of the bifunctional fluorescent probe comprises the following steps: Step 1. Synthesis of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid Add methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoate to a methanol solution, then add sodium hydroxide, react overnight at 40 degrees Celsius, and after the reaction is complete, decompress the reaction solution and spin dry it in a vacuum, and separate and purify the crude product by column chromatography to obtain 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid; Step 2. Synthesis of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid is added to ethanol, and then malononitrile is added, and the mixture is stirred at room temperature overnight. After the reaction is complete, the mixture is filtered, and the solid is vacuum-dried to obtain 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid; Step 3. Synthesis of the bifunctional fluorescent probe 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propionic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 minutes. Subsequently, 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthenyl]-3-one is added and stirred for 5 minutes, and then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight. After the reaction is complete, the reaction system is spin-dried, and the bifunctional fluorescent probe is separated and purified by column chromatography.

3. The method for synthesizing a bifunctional probe according to claim 2, wherein: The molar ratio of 3-(7-(2,2-dicyanovinyl)-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxaline-5(1H)-yl)propanoic acid and 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthenyl]-3-one in step 3 is 1:

1.

4. The use of the bifunctional fluorescent probe according to claim 1 for preparing a device, characterized in that: The prepared device is capable of quantitatively analyzing ATP and hydroxyl radicals in the environment, as well as simultaneously distinguishing and imaging ATP and hydroxyl radicals in cells, tissues, and living bodies.

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