Synthesis and Application of a Bifunctional Fluorescent Probe for Simultaneous Detection of Adenosine Triphosphate and Hydroxyl Radicals

Abstract

This invention discloses a bifunctional fluorescent probe that simultaneously distinguishes between adenosine triphosphate (ATP) and hydroxyl radicals (·OH). The chemical structure of the bifunctional probe is as follows: This bifunctional fluorescent probe combines a rhodamine derivative and a coumarin derivative through a simple condensation reaction, utilizing the specific interaction between the nitrogen-containing group and ATP, and the aromatic hydroxylation of ·OH, to detect and distinguish between ATP and ·OH. When the probe reacts with ATP, it emits red fluorescence at 590 nm under an excitation wavelength of 520 nm; when it reacts with ·OH, it emits green fluorescence at 496 nm under an excitation wavelength of 420 nm. This probe exhibits excellent selectivity and high sensitivity in detecting ATP and ·OH, and has great application prospects in analytical chemistry, life sciences, biomedicine, and other technical fields.

Description

TECHNICAL FIELD

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

[0002] Abnormal oxidative stress and impaired energy metabolism of mitochondria are closely related to the occurrence and onset of various diseases (Nat. Med., 2014, 20, 555−560). Research on key active species related to these diseases not only provides an understanding of the pathogenesis of diseases, but also helps to develop targeted and therapeutic drugs. Hydroxyl radicals (·OH) are a kind of active oxygen with extremely short lifetime, which can almost damage all types of macromolecules: DNA, nucleic acids (mutation), proteins and lipids (lipid peroxidation), etc. and are considered to be the most toxic active oxygen. Excessive ·OH can cause irreversible damage to mitochondria (Chem. Res. Toxicol., 2008, 21, 172-188). At the same time, ATP is an important messenger for regulating mitochondrial energy metabolism. It is worth mentioning that ATP and ·OH have overlapping effects. In the mitochondrial respiratory chain, it generates energy accompanied by the generation of free radicals. However, a higher metabolic rate induces more ·OH generation, and surprisingly, insufficient ATP production in turn also leads to an increase in ·OH due to changes in mitochondrial membrane potential. On the other hand, excessive levels of ·OH can damage proteins or DNA, further altering ATP synthesis (Nat. Metab., 2019, 1, 975−984; Cell Res., 2011, 21, 817−834.). Therefore, it is of great importance 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, 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 can only detect one of them (Anal. Chem., 2017, 89, 1749-1756). Currently, simultaneous and differential detection of ATP and ·OH is still a great challenge. SUMMARY

[0004] In view of the above, in order to overcome some shortcomings of the prior art, the purpose of the present application is to provide a bifunctional fluorescent probe for simultaneously and differentially detecting adenosine triphosphate and hydroxyl radicals. The probe can rapidly and selectively detect adenosine triphosphate and hydroxyl radicals from various biological active substances under specific detection conditions.

[0005] The purpose of the present application is also to provide a synthesis and application method of the above-mentioned bifunctional fluorescent probe, which is simple in preparation method, high in sensitivity, low in detection limit and low in cost.

[0006] The specific technical scheme adopted by the present application to solve the problem is a bifunctional fluorescent probe for simultaneously and differentially detecting adenosine triphosphate and hydroxyl radicals, and the application of the device for quantitatively analyzing adenosine triphosphate and hydroxyl radicals in the environment and simultaneously and differentially imaging adenosine triphosphate and hydroxyl radicals in living cells. The chemical structural formula of the bifunctional probe is as follows:

[0007] .

[0008] A synthesis of a bifunctional fluorescent probe for simultaneously and differentially detecting adenosine triphosphate and hydroxyl radicals, characterized by a preparation method of the bifunctional fluorescent probe, comprising the following steps:

[0009] Step 1. Synthesis of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid

[0010] Methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoate is added to a methanol solution, followed by sodium hydroxide, and reacted at 40°C overnight. After the reaction is complete, the reaction solution is vacuum rotary evaporated under reduced pressure, and the crude product is separated and purified by column chromatography to obtain 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid.

[0011] Step 2. Synthesis of 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid

[0012] 3-(7-Formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid was added into ethanol, then malononitrile was added, stirred at room temperature overnight, after the reaction was completed, filtered, and the solid was vacuum dried to obtain 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid;

[0013] Step 3. Synthesis of the bifunctional fluorescent probe

[0014] 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid was added into anhydrous dichloromethane, then 4-dimethylaminopyridine (DMAP) was added, reacted at room temperature for 5 min, then 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one was added and stirred for 5 min, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added, stirred at room temperature overnight, after the reaction was completed, the reaction system was dried, and column chromatography was used for purification and separation to obtain the bifunctional fluorescent probe.

[0015] The use method of the bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals according to the present application is as follows: without special instructions, the bifunctional probe is usually dissolved in N,N dimethylformamide (DMF) at room temperature, and is used for analysis and detection in an environment with a volume ratio of organic phase to aqueous phase of 4:6, wherein the organic phase is methanol, and the aqueous phase is a phosphate buffer solution (PBS) with pH = 7.4.

[0016] The specific features of the bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals are as follows: the bifunctional fluorescent probe is dissolved in DMF, the bifunctional probe is dissolved in an organic phase and an aqueous phase (4:6, v / v) solution, emits red fluorescence of 590 nm under an excitation wavelength of 520 nm after 30 minutes of adenosine triphosphate, and the probe responds to hydroxyl radicals, emits green fluorescence of 496 nm under an excitation wavelength of 420 nm. Therefore, specific analytes are detected by specific excitation and fluorescence emission signals, and when both substances exist, the two can also be well distinguished by using different excitation and fluorescence emission signals. The bifunctional fluorescent probe realizes simultaneous distinguishing and detecting adenosine triphosphate and hydroxyl radicals under different detection conditions, has no obvious response to other active oxygen, active sulfur, common amino acids, metal ions and active nitrogen, and the detection limits of adenosine triphosphate and hydroxyl radicals are as low as 63.6 μM and 21.3 nM, respectively. Therefore, the bifunctional fluorescent probe disclosed by the application can realize high-sensitivity distinguishing and quantitative detection of the two. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the bifunctional fluorescent probe.

[0018] Figure 2 The ultraviolet and fluorescence spectrum of the bifunctional fluorescent probe responding to adenosine triphosphate and hydroxyl radicals.

[0019] Figure 3 The fluorescence quantitative spectrum of the bifunctional fluorescent probe responding to adenosine triphosphate and hydroxyl radicals.

[0020] Figure 4 The selective spectrum of the bifunctional fluorescent probe responding to adenosine triphosphate and hydroxyl radicals. DETAILED DESCRIPTION

[0021] The application is further described in combination with the following drawings.

[0022] The synthesis route of the bifunctional fluorescent probe is shown in the following figure:

[0023]

[0024] Example 1. Synthesis of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydropyrrolo[1,2-a]quinoxalin-5(1H)-yl)propanoic acid

[0025] To 300.0 mg (942.29 μmol) of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid methyl ester in 20 mL of methanol, 376.9 mg (9.42 mmol) of sodium hydroxide was added and the reaction was allowed to proceed at 40 °C overnight. After the reaction was completed, the reaction mixture was concentrated under vacuum. The crude product was purified by column chromatography to obtain 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid in 216 mg yield, 75.32%.

[0026] Example 2. Synthesis of 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid

[0027] To 150.0 mg (492.86 μmol) of 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid in 8 mL of ethanol, 65.1 mg (985.72 μmol) of malononitrile was added and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the solid was filtered and dried under vacuum to obtain 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid in 130 mg yield, 74.85%.

[0028] Example 3. Synthesis of the bifunctional fluorescent probe

[0029] To 50.0 mg (141.89 μmol) of 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propionic acid in 6 mL of anhydrous dichloromethane, 3.5 mg (28.38 μmol) of 4-dimethylaminopyridine (DMAP) was added and the reaction was allowed to proceed 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'-xanthene]-3-one was added and stirred for 5 min. After that, 54.4 mg (283.77 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and the reaction was allowed to proceed at room temperature overnight. After the reaction was completed, the reaction mixture was concentrated under vacuum. The product was purified by column chromatography to obtain the bifunctional fluorescent probe in 50.0 mg yield, 40.88%.

[0030] Example 4. Simultaneous detection of ATP and ·OH in vitro by bifunctional fluorescent probe

[0031] The bifunctional fluorescent probe of the present application has the following spectral properties: the bifunctional probe is dissolved in DMF to prepare a probe solution with a concentration of 1 mM, and an ATP analysis stock solution with a concentration of 100 mM and a ·OH analysis stock solution with a concentration of 10 mM are prepared. The specific testing method is as follows: 20 μL of the 1 mM probe solution is taken, 200 μL of the 100 mM ATP solution is added, and finally 780 μL of methanol and 1000 μL of PBS buffer are added, and the volume ratio of the organic phase and the aqueous phase is maintained at 4:6 (the total volume of each test sample is 2 mL). For example, when the fluorescence intensity of ATP with a concentration of 10 mM is required to be tested, the sample is prepared as follows: 20 μL of the 1 mM probe solution, 200 μL of the 100 mM ATP analysis solution, 780 μL of methanol and 1000 μL of PBS buffer are added to a 2 mL sample tube, and the fluorescence emission intensity is measured at an excitation wavelength of 520 nm after shaking at room temperature for 30 minutes. When 100 μM ·OH is detected, the specific testing method is as follows: 20 μL of the 1 mM probe solution, 20 μL of the 10 mM ·OH analysis solution, 780 μL of methanol and 1000 μL of PBS buffer are added to a 2 mL sample tube, and the fluorescence emission intensity is measured at an excitation wavelength of 420 nm after shaking at room temperature for 30 minutes. The bifunctional probe realizes the simultaneous detection of ATP and ·OH, two kinds of biologically active substances, by using different excitation wavelengths and fluorescence emission signals, has high sensitivity, and the detection limits are as low as 63.6 μM and 21.3 nM, respectively, and is very suitable for imaging / quantitative analysis of endogenous ATP and ·OH in living cells.

[0032] The application provides a bifunctional fluorescent probe for simultaneously distinguishing and detecting adenosine triphosphate and hydroxyl radicals, which connects rhodamine derivatives and coumarin derivatives through a simple condensation reaction, and detects and distinguishes ATP and ·OH through the specific interaction of nitrogen-containing groups with ATP and the aromatic hydroxylation of ·OH. When the probe reacts with ATP, red fluorescence of 590 nm is emitted under an excitation wavelength of 520 nm; when the probe reacts with ·OH, green fluorescence of 496 nm is emitted under an excitation wavelength of 420 nm. The fluorescence distinction degree is obvious, the selectivity is good, the water solubility of the product after the reaction is good, the response speed is fast, and the like. The application has great practical application value in the fields of biochemistry, analysis and detection, and the like. Although the content of the application has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation on the application. After reading the above content, various modifications and substitutions of the application will be apparent to those skilled in the art. Therefore, the fluorescent probes with similar technical features described herein all fall within the protection scope of the patent.

Claims

1. A bifunctional fluorescent probe for simultaneously distinguishing detection of adenosine triphosphate and hydroxyl radicals, characterized in that, The chemical structural formula of the bifunctional fluorescent probe is as follows:

2. The synthesis of bifunctional fluorescent probe as claimed in claim 1, wherein, 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-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid Methyl 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoate is added into a methanol solution, then sodium hydroxide is added, and the reaction is carried out at 40 DEG C overnight; after the reaction is completed, the reaction solution is vacuum rotary evaporated under reduced pressure, and the crude product is separated and purified by column chromatography to obtain 3-(7-formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid; Step 2. Synthesis of 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid 3-(7-Formyl-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid is added into ethanol, then malononitrile is added, and the reaction is stirred at room temperature overnight; after the reaction is completed, filtration is performed, and the solid is vacuum rotary evaporated under reduced pressure to obtain 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid; Step 3. Synthesis of the bifunctional fluorescent probe 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid is added into anhydrous dichloromethane, then 4-dimethylaminopyridine (DMAP) is added, and the reaction is carried out at room temperature for 5 min; then 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one is added and stirred for 5 min, then 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, and the reaction is stirred at room temperature overnight; after the reaction is completed, the reaction system is rotary evaporated, and column chromatography is performed to separate and purify to obtain the bifunctional fluorescent probe.

3. The method of synthesis of bifunctional fluorescent probe as claimed in claim 2, wherein, The molar ratio of 3-(7-(2,2-dicyanoethenyl)-8-methoxy-2,3,3a,4-tetrahydro-pyrrolo[1,2-a]quinoxalin-5(1H)-yl) propanoic acid and 2-(2-((2-aminoethyl)amino)ethyl)-3',6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one in step 3 is 1:

1.

4. The use of the bifunctional fluorescent probe according to claim 1 for the preparation of a device, characterized in that, The prepared device can quantitatively analyze adenosine triphosphate and hydroxyl radicals in the environment, and simultaneously distinguish and image adenosine triphosphate and hydroxyl radicals in cells, tissues and living bodies.

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