Coumarin-rhodamine-based dual-channel fluorescent probe for simultaneously detecting carbon monoxide and adenosine triphosphate as well as preparation method and application of coumarin-rhodamine-based dual-channel fluorescent probe

Abstract

The invention discloses a coumarin-rhodamine-based dual-channel fluorescent probe for simultaneously detecting carbon monoxide and adenosine triphosphate as well as a preparation method and application of the coumarin-rhodamine-based dual-channel fluorescent probe. The fluorescent probe is N-(2-((2-(3 ', 6'-bis (diethylamino)-3-oxo-spiro [isoindoline-1, 9 '-xanthene]-2-yl) ethyl) amino) ethyl)-7-nitro-2-oxo-2H-chromene-3-formamide, and the structural formula of the fluorescent probe is shown in the description. According to the preparation method, 7-nitro-3-carboxylic acid coumarin is used as a raw material, and the 7-nitro-3-carboxylic acid coumarin and 2-[2-((2-aminoethyl) amino) ethyl]-3 '6'-bis (diethylamino) spiro [isoindoline-1, 9 '-xanthene]-3-ketone are subjected to an amidation reaction. The compound can react with carbon monoxide and adenosine triphosphate respectively. Therefore, the compound can be used as a coumarin-rhodamine-based dual-channel fluorescent probe for simultaneously detecting carbon monoxide and adenosine triphosphate.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes and relates to a coumarin-rhodamine-based dual-channel fluorescent probe for simultaneously detecting carbon monoxide and adenosine triphosphate, and a preparation method and application thereof. Background Art

[0002] Carbon monoxide (CO), an environmental pollutant, not only harms life and health but also performs many physiological functions in organisms. Low-level CO exposure can cause headaches, nausea, and exacerbation of some diseases. Acute CO poisoning can lead to anemia, neurological damage, and even death. In some animal studies, CO poisoning can cause compensatory hemodynamic changes, cardiac hypertrophy, or atherosclerosis. Adenosine triphosphate (ATP) is considered one of the most important biomolecules in organisms and is essential for cellular metabolism. Studies have found that ATP can serve as an energy source for many physiological processes, including respiration, enzyme catalysis, cell division, DNA synthesis, and energy transduction. In addition, ATP acts as an important signaling molecule to mediate cell motility. However, low levels of ATP are closely associated with a range of diseases, such as ischemia, hypoglycemia, and Parkinson's disease. Therefore, developing a method to simultaneously image ATP and CO in living systems is of great significance for pathological research and disease treatment.

[0003] To date, several methods for detecting ATP and CO have been extensively explored. Compared to other methods, fluorescent probes offer advantages such as high sensitivity, excellent selectivity, immediate response, simple instrumentation, real-time analysis, low cost, and high spatiotemporal resolution. However, there are no reports on the synthesis of a coumarin-rhodamine-based dual-channel fluorescent probe for the simultaneous detection of carbon monoxide and adenosine triphosphate using 7-nitro-3-carboxycoumarin and 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one as raw materials. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention aims to provide a coumarin-rhodamine-based dual-channel fluorescent probe for the simultaneous detection of carbon monoxide and adenosine triphosphate, which meets the requirements of use. Another technical problem to be solved by the present invention is to provide a method for preparing N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide. Another technical problem to be solved by the present invention is to provide a use of the above-mentioned N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0006] A coumarin-rhodamine-based dual-channel fluorescent probe for simultaneous detection of carbon monoxide and adenosine triphosphate is N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide, and its structural formula is:

[0007]

[0008] The method for preparing a coumarin-rhodamine-based dual-channel fluorescent probe for simultaneous detection of carbon monoxide and adenosine triphosphate comprises the following steps: 7-nitro-3-carboxylic acid coumarin undergoes an amidation reaction with 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one to obtain N-(2-((2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide.

[0009] (1) 0.25 mol of 7-nitro-3-carboxylic acid coumarin, 0.27-0.43 mol of 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one, 0.05-0.18 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 0.12-0.22 mol of 1-hydroxybenzotriazole, 35-43 μL of triethylamine, and 8-15 mL of anhydrous acetonitrile were sequentially added into a three-necked flask equipped with a stirrer and reacted at 35°C for 24 h under nitrogen protection;

[0010] (2) the reaction solution was distilled to recover the solvent, thereby obtaining a crude product of 3-((2-((4-methylphenyl)sulfonamido)ethyl)carbamoyl)-2-oxo-2H-chromen-7-yl-2-(diphenylphosphino)benzoate;

[0011] (3) The crude product of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-oxanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was chromatographed on a silica gel column (dichloromethane:methanol=20:1) to obtain brown powder N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-oxanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide.

[0012] The compound N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide can sensitively detect the concentration of carbon monoxide in the range of 0-200 μM, with a detection limit as low as 5.4×10 -8 mol / L. In addition, the compound can also sensitively detect the concentration of adenosine triphosphate in the range of 0-10mM, with a detection limit as low as 8.4×10 -6 mol / L.

[0013] Beneficial effects: Compared with the prior art, the present invention uses 7-nitro-3-carboxylic acid coumarin as a raw material, undergoes an amidation reaction with 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one to obtain N-(2-((2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide. This compound can be used as a coumarin-rhodamine-based dual-channel fluorescent probe for the simultaneous detection of carbon monoxide and adenosine triphosphate, and has many advantages, such as high sensitivity, good selectivity, low detection limit, and a wide pH range of use, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The fluorescence spectra of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide under the action of different concentrations of carbon monoxide at an excitation wavelength of 365 nm;

[0015] Figure 2 The fluorescence spectra of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide in the presence of different concentrations of adenosine triphosphate at an excitation wavelength of 520 nm;

[0016] Figure 3 The figure shows the fluorescence spectra of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide interacting with different analytes such as metal ions, anions, biological thiols and reactive oxygen species at an excitation wavelength of 365 nm.

[0017] Figure 4 The figure shows the fluorescence spectra of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide interacting with different analytes such as metal ions, anions, biological thiols, reactive oxygen species, and nucleotides at an excitation wavelength of 520 nm. DETAILED DESCRIPTION

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] Example 1

[0020] Preparation of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide, the reaction formula is as follows:

[0021]

[0022] The specific steps are as follows:

[0023] Preparation of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide:

[0024] 0.25 mol of 7-nitro-3-carboxylic acid coumarin, 0.3 mol of 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one, 0.1 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 0.3 mol of 1-Hydroxybenzotriazole, 40 μL triethylamine, and 10 mL anhydrous acetonitrile were added sequentially into a three-necked flask equipped with a stirrer, and the mixture was reacted at 35° C. for 24 h under nitrogen protection. The reaction solution was distilled to recover the solvent, and the crude product of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was obtained; N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was obtained. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=20:1) to obtain brown powder N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide). 1 H NMR (600MHz, DMSO-d6) δ: 8.91 (s, 1H), 8.68 (t, J=5.5Hz, 1H), 8.31 (s, 1H), 8.25-8.22 (m, 2H), 7.75 (d, J=7.8Hz, 1H), 7.52-7.47 (m, 2H), 7.02 (d, J=6.9Hz, 1H), 6.38-6.34 (m, 6H), 3.30 (t, J=7.1Hz, 10H), 3.25-3.20 (m, 2H), 3.10 (t, J=7.2Hz, 2H), 2.28 (s, 2H), 1.07 (t, J=7.0Hz, 12H). 13 C NMR (151MHz, DMSO-d6) δ: 166.81, 160.12, 159.11, 153.09, 153.06, 152.35, 149.41, 148.09, 145.01, 132.35, 131.22, 130.18, 128.04, 127. 95, 123.32, 123.26, 121.96, 121.81, 119.11, 111.32, 107.86, 104.70, 96.94, 63.74, 47.06, 46.32, 43.39, 38.61, 12.08. HRMS (m / z): [M+Na] + calcd for C 42H 44 N6NaO7 + , 745.3344; found, 745.3351.

[0025] Example 2

[0026] N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was prepared into 1×10 -5 mol / L PBS buffer solution (pH = 7.4, 10% EtOH, 10 mmol / L), carbon monoxide was dissolved in PBS buffer solution to a concentration of 0, 0.5×10 -5 , 2.5×10 -5 , 5.5×10 -5 , 1.2×10 -4 , 2×10 -4 mol / L solution. The fluorescence emission spectrum of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide in the presence of different concentrations of carbon monoxide at an excitation wavelength of 365 nm was measured using a fluorescence spectrophotometer. Figure 1 The results show that as the concentration of carbon monoxide in the solution gradually increases from 0 mol / L to 2×10 -4 mol / L, the fluorescence emission intensity of the compound at 448 nm gradually increased, indicating that the compound can be used as a fluorescent probe for sensitive detection of carbon monoxide.

[0027] Example 3

[0028] N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was prepared into 1×10 -5 mol / L PBS buffer solution (pH = 7.4, 10% EtOH, 10 mmol / L), adenosine triphosphate was dissolved in PBS buffer solution to a concentration of 0, 5.5×10 -5 , 1.2×10 -4 , 6.4×10 -4 , 4.5×10 -3 , 10×10 -3mol / L solution. The fluorescence emission spectrum of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide in the presence of different concentrations of adenosine triphosphate at an excitation wavelength of 520 nm was measured on a fluorescence spectrophotometer using a fluorescence spectrophotometer. Figure 2 The results show that as the concentration of ATP in the solution gradually increases from 0 mol / L to 10×10 -3 mol / L, the fluorescence emission intensity of the compound at 584 nm gradually increased, indicating that the compound can be used as a fluorescent probe for sensitive detection of ATP.

[0029] Example 4

[0030] N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was prepared into 1×10 -5 mol / L PBS buffer solution (pH = 7.4, 10% EtOH, 10 mmol / L), and different analytes such as metal ions, anions, biothiols and reactive oxygen species were dissolved in PBS buffer to a concentration of 1.5×10 -4 mol / L solution. The fluorescence emission spectrum of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide in the presence of different analytes such as metal ions, anions, biothiols, and reactive oxygen species at an excitation wavelength of 365 nm was measured using a fluorescence spectrophotometer. Figure 3 As shown. The compound reacts with carbon monoxide (CO) to produce an obvious fluorescence emission peak at 448nm, and the addition of metal ions (Mg 2+ , Fe 2+ , Zn 2+ , Ca 2 + , Cu 2+ , Mn 2+ , K + , Na + , A1 3+ ), anion (Br - , Cl - ,HPO4 2- , NO3 - , HS - , I - NO2- ), biothiols (Cys, Hcy, GSH) and reactive oxygen species (ClO - , ONOO - ) and other analytes, the fluorescence spectrum of the compound did not change significantly. This shows that the compound can be used as a fluorescent probe for the selective detection of carbon monoxide.

[0031] Example 5

[0032] N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was prepared into 1×10 -5 mol / L PBS buffer solution (pH = 7.4, 10% EtOH, 10 mmol / L), and different analytes such as metal ions, anions, biothiols and reactive oxygen species were dissolved in PBS buffer to a concentration of 1.5×10 -4 mol / L solution. The fluorescence emission spectrum of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide complex in the presence of different analytes such as metal ions, anions, biothiols, reactive oxygen species, and nucleotides at an excitation wavelength of 520 nm was measured using a fluorescence spectrophotometer. Figure 4 As shown. After the compound reacts with adenosine triphosphate (ATP), it produces an obvious fluorescence emission peak at 584nm, and the addition of metal ions (Mg 2+ , Fe 2+ , Zn 2+ , Ca 2+ , Cu 2+ , Mn 3+ , K + , Na + , Al 3+ ), anion (Br - , Cl - ,HPO4 2- , NO3 - ,HS - ,I - , NO2 - ), biothiols (Cys, Hcy, GSH), reactive oxygen species (ClO - , ONOO -) and nucleotides (ADP, AMP, GTP, CTP, UTP) and other analytes, the fluorescence spectrum of the compound did not change significantly. This shows that the compound can be used as a fluorescent probe for the selective detection of ATP.

Claims

1. A coumarin-rhodamine-based dual-channel fluorescent probe for simultaneous detection of carbon monoxide and adenosine triphosphate, characterized in that: Its structural formula is:

2. The method for preparing the coumarin-rhodamine based dual-channel fluorescent probe for simultaneous detection of carbon monoxide and adenosine triphosphate according to claim 1, characterized in that: The specific steps include: 7-Nitro-3-carboxylic acid coumarin undergoes amidation reaction with 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one to give N-(2-((2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide.

3. The method for preparing a coumarin-rhodamine based dual-channel fluorescent probe for simultaneous detection of carbon monoxide and adenosine triphosphate according to claim 2, characterized in that: 7-nitro-3-carboxylic acid coumarin undergoes an amidation reaction with 2-[2-((2-aminoethyl)amino)ethyl]-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthen]-3-one to obtain N-(2-((2-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-1,9'-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide. The specific preparation method includes: (1) 0.25 mol of 7-nitro-3-carboxylic acid coumarin, 0.27-0.43 mol of 2-[2-((2-aminoethyl)amino)ethyl s-3'6'-bis(diethylamino)spiro[isoindoline-1,9'-xanthene]-3-one, 0.05-0.18 mol of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 0.12-0.22 mol of 1-hydroxybenzotriazole, 35-43 μL of triethylamine, and 8-15 mL of anhydrous acetonitrile were sequentially added into a three-necked flask equipped with a stirrer and reacted at 35°C for 24 h under nitrogen protection; (2) The reaction solution was distilled to recover the solvent, thereby obtaining a crude product of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide; (3) The crude product of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-oxanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide was chromatographed on a silica gel column (dichloromethane:methanol=20:1) to obtain brown powder N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-oxanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide.

4. Use of N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthene]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide according to claim 1 in the detection of carbon monoxide and adenosine triphosphate.

5. The use according to claim 4, characterized in that N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide can selectively undergo a reduction reaction with carbon monoxide, and the fluorescence of its solution changes from colorless to bright blue. In addition, the compound can also selectively undergo hydrogen bonding with adenosine triphosphate, and the fluorescence of its solution changes from colorless to bright red.

6. The use according to claim 4, characterized in that The concentration of carbon monoxide in the solution was detected using N-(2-((2-(3′,6′-bis(diethylamino)-3-oxospiro[isoindoline-1,9′-xanthen]-2-yl)ethyl)amino)ethyl)-7-nitro-2-oxo-2H-chromene-3-carboxamide with a detection range of 0-200 μM and a detection limit as low as 5.4×10 -8 mol / L. In addition, the compound can also sensitively detect the concentration of adenosine triphosphate in the solution, with a detection range of 0-10mM and a detection limit as low as 8.4×10 -6 mol / L.

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