An adenosine triphosphate and nitric oxide dual-responsive near-infrared fluorescent probe and a preparation method and application thereof

Abstract

The application provides an adenosine triphosphate and nitric oxide dual-response near-infrared fluorescent probe and a preparation method and application thereof. The near-infrared fluorescent probe has the structure shown in the following formula I, is responsive to both adenosine triphosphate and nitric oxide, and has high sensitivity and selectivity.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis and detection technology, specifically relating to a near-infrared fluorescent probe that is dual-responsive to adenosine triphosphate and nitric oxide, and its preparation and application. Background Technology

[0002] Adenosine triphosphate (ATP) is the primary energy source for organisms and a crucial substance for regulating energy metabolism, participating in numerous life activities. ATP synthesis mainly occurs in the mitochondria of cells. Blood ATP concentration levels serve as an important indicator of human health; several diseases, such as Parkinson's disease, Alzheimer's disease, cardiovascular disease, colitis, and malignant tumors, are closely related to abnormal fluctuations in ATP concentration. Many established methods for detecting ATP, such as liquid chromatography and mass spectrometry, often lack spatiotemporal resolution or compatibility with live specimens. Therefore, there is an urgent need to develop molecular probes to analyze ATP fluctuations and replace instrumental analysis.

[0003] Nitric oxide (NO) is a biological messenger molecule widely distributed in various tissues of organisms and plays a crucial role in human physiology. Under normoxic conditions, NO is biosynthesized by nitric oxide synthase (NOS), which oxidizes L-arginine to L-citrulline. Abnormal levels of intracellular NO are closely associated with many biological dysfunctions, including tumor progression, acute and chronic diseases, and neurodegenerative diseases. Due to the various roles of NO in physiological and pathological processes, the identification, detection, and quantification of NO remain an active research area. Among various methods for tracking NO molecules, fluorescence imaging technology has become an effective molecular tool due to its high sensitivity and high spatiotemporal resolution.

[0004] ATP and NO are key bioanalytes related to oxidative stress and energy metabolism, respectively, and understanding their level fluctuations and synergistic effects in living organisms is crucial. However, the simultaneous use of two fluorescent probes inevitably leads to challenges such as significant invasiveness, different cellular uptake, and spectral disruption; therefore, developing single-molecule probes that respond to both substances is essential. Summary of the Invention

[0005] To address one of the aforementioned technical problems in the prior art, this invention provides a near-infrared fluorescent probe that is dual-responsive to adenosine triphosphate (ATP) and nitric oxide (NO), responding to ATP and NO with independent fluorescence signals, respectively. Furthermore, this invention also provides a method for preparing the near-infrared fluorescent probe and its application in the detection of ATP and NO.

[0006] The technical solution of the present invention is as follows:

[0007] On one hand, the present invention provides a near-infrared fluorescent probe having the structure shown in Formula I:

[0008]

[0009] On the other hand, the present invention provides a method for preparing a near-infrared fluorescent probe, the method comprising the following steps:

[0010] (1) Rhodamine B was reacted with diethylenetriamine to give intermediate compound 1;

[0011]

[0012] (2) React intermediate compound 1 with IR-780 dye to obtain the near-infrared fluorescent probe shown in Formula I;

[0013]

[0014] According to some embodiments of the present invention, in step (1), the molar ratio of rhodamine B to diethylenetriamine is 1:(2-10), for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10 or any value between them. In some preferred embodiments, in step (1), the molar ratio of rhodamine B to diethylenetriamine is 1:(2-5).

[0015] According to some embodiments of the present invention, in step (1), the reaction temperature is 60–80°C. According to some embodiments of the present invention, in step (1), the reaction time is 8–12 hours.

[0016] According to some embodiments of the present invention, in step (1), the reaction is carried out in a first organic solvent, which comprises a C1-C4 low alcohol, such as ethanol. In some embodiments, the amount of the first organic solvent used is 5-10 L of the first organic solvent per 1 mol of Rhodamine B.

[0017] According to some embodiments of the present invention, step (1) further includes: after the reaction is completed, removing the solvent in the reaction solution by vacuum distillation to obtain a crude product; dissolving the crude product in a haloalkane solvent such as dichloromethane and washing it with saturated brine to obtain an organic layer; after drying and removing the solvent from the organic layer, separating and purifying it by silica gel column chromatography to obtain the intermediate compound 1.

[0018] According to some embodiments of the present invention, in step (2), the molar ratio of IR-780 dye to the intermediate compound 1 is 1:(1 to 1.5).

[0019] According to some embodiments of the present invention, in step (2), the reaction temperature is 30–80°C. According to some embodiments of the present invention, in step (2), the reaction time is 6–24 hours, preferably 6–12 hours.

[0020] According to some embodiments of the present invention, in step (2), the reaction is carried out in a second organic solvent, which includes one or more of nitrile and amide organic solvents. In some embodiments, the second organic solvent includes acetonitrile and / or N,N-dimethylformamide. In the present invention, the second organic solvent is preferably a dry solvent, i.e., free of water. In some embodiments, the amount of the second organic solvent used is 10-20 L of the second organic solvent per 1 mol of IR-780 dye.

[0021] According to some embodiments of the present invention, in step (2), the reaction is carried out in the presence of an organic weak base. The organic weak base of the present invention includes aliphatic amines. In some embodiments, the organic weak amine includes triethylamine and / or N,N-diisopropylethylamine.

[0022] According to some embodiments of the present invention, in step (2), the molar ratio of IR-780 dye to the organic weak base is 1:(1-3).

[0023] According to some embodiments of the present invention, in step (2), the reaction is carried out in a protective gas, preferably in a nitrogen atmosphere.

[0024] In another aspect, the present invention provides the application of the above-described near-infrared fluorescent probe or the near-infrared fluorescent probe prepared by the above-described method in the detection of ATP and / or NO.

[0025] According to some embodiments of the present invention, the application includes:

[0026] (S1) The sample to be tested is brought into contact with the fluorescent probe to obtain a mixture;

[0027] (S2) Measure the fluorescence intensity of the mixture.

[0028] The fluorescent probe of the present invention can be used to detect test samples containing NO and its donors, wherein the NO donors include, but are not limited to, sodium N-diethylamino-N-oxonitrosamide.

[0029] In some embodiments, step (S2) includes measuring the fluorescence spectrum of the mixture in the range of 570-875 nm.

[0030] In some embodiments, step (S2) includes: measuring the fluorescence spectrum of the mixture at 570 nm-650 nm and / or 740 nm-875 nm.

[0031] In some embodiments, when detecting ATP in the test sample, step (S2) includes: measuring the fluorescence spectrum of the mixture in the range of 570 nm to 650 nm. In some embodiments, when detecting ATP in the test sample, step (S2) includes: measuring the fluorescence intensity of the mixture in the range of 580 nm to 590 nm. In some embodiments, when detecting ATP in the test sample, step (S2) includes: measuring the fluorescence intensity of the mixture at 586 nm.

[0032] In some embodiments, when detecting NO in the sample to be tested, step (S2) includes: measuring the fluorescence spectrum of the mixture in the range of 740 nm to 875 nm. In some embodiments, when detecting NO in the sample to be tested, step (S2) includes: measuring the fluorescence intensity of the mixture at 810 nm to 820 nm and 760 nm to 770 nm. In some embodiments, when detecting NO in the sample to be tested, step (S2) includes: measuring the fluorescence intensity of the mixture at 816 nm and 762 nm.

[0033] In some embodiments, step (S1) includes contacting the sample to be tested with a dispersion of the fluorescent probe to obtain a mixture. In some embodiments, the solvent used for the dispersion of the fluorescent probe is a mixture of ethanol and PBS buffer. Preferably, the pH of the PBS buffer is 7.0-8.0, more preferably 7.2-7.6.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The fluorescent probe of the present invention, as a single-molecule probe, has the ability to detect two analytes, ATP and NO, and generates independent responses to ATP and NO without spectral crosstalk.

[0036] (2) The fluorescent probe of the present invention exhibits good sensitivity and selectivity in response to ATP and NO, with a detection limit as low as 1.3 μM for ATP and as low as 0.26 μM for NO. Furthermore, the fluorescent probe of the present invention responds to NO with ratiometric fluorescence, which is less affected by external environmental factors and results in higher detection accuracy.

[0037] (3) The preparation method of the fluorescent probe of the present invention is simple and the conditions are mild, making it suitable for industrial application. Attached Figure Description

[0038] Figure 1 This is a schematic diagram illustrating the principle of the near-infrared fluorescent probe of the present invention for recognizing ATP and NO.

[0039] Figure 2 This is a synthesis route diagram of the near-infrared fluorescent probe of the present invention.

[0040] Figure 3 The results of detecting ATP after the fluorescent probe prepared in Example 2 of the present invention was co-incubated with ATP for different times are shown in Figure A). Figure A) shows the fluorescence spectrum of the fluorescent probe after incubation with ATP for different times (0-30 min); Figure B) shows the fluorescence intensity of the fluorescent probe at 586 nm as a function of time.

[0041] Figure 4 The results of the fluorescent probe prepared in Example 2 of this invention for detecting different concentrations of ATP are shown in Figure A). Figure A) shows the fluorescence spectrum of the fluorescent probe after incubation with different concentrations of ATP (0-10mM) for 30 min; Figure B) shows the change of fluorescence intensity of the fluorescent probe at 586nm with the ATP level.

[0042] Figure 5 The fluorescent spectrum of the fluorescent probe prepared in Example 2 of this invention for the selective detection of ATP is shown.

[0043] Figure 6 The following are the detection results of the fluorescent probe prepared in Example 2 of the present invention and NO response at different times. In Figure A), the fluorescence spectrum of the fluorescent probe and NO response after 0 to 360 s is shown; and in Figure B), the logarithm of the ratio of fluorescence intensity of the fluorescent probe at 816 nm and 762 nm changes with time.

[0044] Figure 7 The results of the fluorescent probe prepared in Example 2 of this invention for detecting different concentrations of NO are shown in Figure A). Figure A) shows the fluorescence spectrum of the fluorescent probe after responding to different concentrations of NO (0-100 μM) for 5 min. Figure B) shows the logarithm of the ratio of fluorescence intensity of the fluorescent probe at 816 nm and 762 nm as a function of NO level.

[0045] Figure 8 The results show the selectivity of the fluorescent probe for NO detection obtained in Example 2 of this invention.

[0046] Figure 9 This is the 1H NMR spectrum of compound 1 of the present invention.

[0047] Figure 10 The image shows the hydrogen NMR spectrum of the near-infrared fluorescent probe prepared in Example 2 of this invention.

[0048] Figure 11 This is the mass spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0050] This invention provides a near-infrared fluorescent probe that is dual-responsive to ATP and NO, as shown in Formula I:

[0051]

[0052] The principle of the fluorescent probe of this invention for recognizing ATP and NO is as follows: Figure 1 As shown. The fluorescent probe of this invention is designed by linking rhodamine B and the near-infrared anthocyanin dye IR-780 to a backbone of diethylenetriamine. Diethylenetriamine is not only the linking group of the backbone, but also a responsive group that recognizes ATP and NO. The amidation reaction of the terminal amino group of diethylenetriamine with rhodamine B causes rhodamine B to close its ring (no fluorescence), and ATP induces the spironolactone of rhodamine B to open its ring, thereby turning on fluorescence (λ). ex =550nm, λ em =586nm). The other amino group of diethylenetriamine reacts with the near-infrared dye IR-780 through a substitution reaction to form an aromatic secondary amine, which serves as the NO recognition group. As it undergoes an N-nitrosation reaction with NO, the fluorescence of the probe increases at 816nm, while the fluorescence at 762nm decreases, indicating a ratiometric response to NO.

[0053] The synthetic route of the ATP and NO dual-responsive near-infrared fluorescent probe of the present invention is as follows: Figure 2 As shown.

[0054] As one specific embodiment, the preparation method of the fluorescent probe of the present invention includes the following steps:

[0055] 1) Take Rhodamine B and diethylenetriamine separately at a molar ratio of 1:5-10, and then take ethanol at a ratio of 5-10 L of ethanol per mol of Rhodamine B. First, dissolve Rhodamine B in ethanol, then add diethylenetriamine. After the addition is complete, heat to 60-80℃ and reflux for 8-12 h. Cool the reaction to room temperature and remove the solvent by vacuum evaporation. Dissolve the crude product in dichloromethane, wash three times with saturated brine, dry the organic layer with anhydrous sodium sulfate, and then remove the solvent by vacuum evaporation. The obtained solid is purified by silica gel column chromatography (200-300 mesh) using dichloromethane and methanol (volume ratio).

[0056] V 二氯甲烷 :V 甲醇 Compound 1 was prepared by mixing a mixture of 20:1 to 5:1.

[0057] 2) Dissolve compound 1 obtained in step 1) and the near-infrared dye IR-780 in a dry organic solvent, and pass a strong nitrogen stream through the reaction apparatus; then add a weak organic base dropwise to the solution; after the addition is complete, maintain a nitrogen atmosphere and heat the mixture to 30–80°C and stir overnight. After cooling, separate and purify the crude product by silica gel column chromatography (200–300 mesh), using dichloromethane and methanol (volume ratio V) as the eluent. 二氯甲烷 :V 甲醇 A near-infrared fluorescent probe that is responsive to both ATP and NO was prepared by mixing a mixture of 200:1 to 50:1.

[0058] The dry organic solvents are ultra-dry acetonitrile and ultra-dry N,N-dimethylformamide; the organic weak bases are triethylamine and N,N-diisopropylethylamine; 1 mol of near-infrared dye IR-780 requires 10-20 L of dry organic solvent; the molar ratio of near-infrared dye IR-780 to compound 1 is 1:1-1.5, and the molar ratio of near-infrared dye IR-780 to organic weak base is 1:1-3.

[0059] As one specific embodiment, the application of the fluorescent probe of the present invention in ATP detection includes the following steps:

[0060] 1) First, prepare the following solutions:

[0061] Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0062] Prepare a 10 μM dispersion solution of the fluorescent probe: the solvent used is a mixture of 10% ethanol and the above-mentioned PBS buffer, V 乙醇 V PBS =1:9;

[0063] Dissolve ATP in deionized water to prepare an ATP stock solution with a molar concentration of 1M.

[0064] 2) Detection of ATP:

[0065] i) Take 10 μM of the fluorescent probe in 10% ethanol-PBS buffer dispersion, add ATP stock solution, incubate at 37℃ for 0–30 min, and measure and record the fluorescence spectrum at 586 nm.

[0066] ii) Take a series of fluorescent probes in 10 μM 10% ethanol-PBS buffer dispersions, add different volumes of 1M ATP stock solution, and incubate the resulting mixed solutions at 37℃ for 30 min. Measure the fluorescence spectra of the solutions after the fluorescent probes react with different concentrations of ATP, and construct linear fitting curves based on the relationship between the intensity of the fluorescence emission peak and the ATP concentration.

[0067] iii) Take a series of fluorescent probes in 10 μM 10% ethanol-PBS buffer dispersions, and add K to each. + Na + Ca 2 + Mg 2+ Zn 2+ Cu 2+ Fe 3+ H2PO2 2- PO4 3- HPO4 2- H2PO4 - SO4 2- CO3 2- Aqueous solutions of GSH, Cys, AMP, ADP, and ATP were prepared and incubated at 37°C for 30 min. The fluorescence spectra at 586 nm were measured and recorded to verify the selectivity of the probe molecules for ATP.

[0068] As one specific implementation, the application of the fluorescent probe of the present invention in NO detection includes the following steps:

[0069] 1) First, prepare the following solutions:

[0070] Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0071] Prepare a 10 μM dispersion solution of the fluorescent probe: the solvent used is a mixture of 50% ethanol and the above-mentioned PBS buffer, V 乙醇 V PBS =1:1;

[0072] DEA·NONOate sodium hydrate was dissolved in PBS buffer (10 mM, pH 7.4) to prepare a NO stock solution with a molar concentration of 10 mM.

[0073] 2) Detect NO:

[0074] i) Take 10 μM of the fluorescent probe in 50% ethanol-PBS buffer dispersion, add NO stock solution, and measure and record the fluorescence intensity at 816 nm and 762 nm respectively.

[0075] ii) Take a series of fluorescent probes in 10 μM 50% ethanol-PBS buffer dispersions, add different volumes of 10 mM NO stock solution, and let the resulting mixed solutions stand at room temperature for 5 min. Measure the fluorescence spectra of the solutions after the fluorescent probes react with different concentrations of NO, and plot a linear fitting curve based on the logarithm of the ratio of fluorescence intensity at 816 nm and 762 nm to the NO concentration.

[0076] iii) Take a series of fluorescent probes in 10 μM 50% ethanol-PBS buffered dispersions, and add K to each. + Na + Ca 2 + Mg 2+ Zn 2+ Cu 2+ Fe 3+ NO3 - NO2 - GSH, Cys, H2S, ONOO - Aqueous solutions of OH, H2O2, HClO, and NO were prepared and placed at room temperature for 5 minutes. The fluorescence intensity at 816 nm and 762 nm was measured and recorded. The selectivity of the probe molecule for NO was verified by the logarithm of the fluorescence intensity ratio.

[0077] Unless otherwise specified, all reagents used in the following experiments of this invention are commercially available products or reagents prepared according to conventional methods. Unless otherwise specified, all methods used in the experiments are conventional experimental methods. Unless otherwise specified, all instruments used in the experiments are commercially available.

[0078] Example 1: Synthesis of Compound 1

[0079] Rhodamine B (10 mmol) was dissolved in 50 mL of ethanol, and then diethylenetriamine (22 mmol) was added. After the addition was complete, the mixture was heated to 70 °C and refluxed for 12 h. The reaction mixture was cooled to room temperature, and the solvent was removed by evaporation under reduced pressure. The crude product was dissolved in dichloromethane and washed three times with saturated brine. The organic layer was dried over anhydrous sodium sulfate, and the solvent was removed by evaporation under reduced pressure. The resulting solid was purified by silica gel column chromatography (200–300 mesh) using dichloromethane and methanol (v / v). 二氯甲烷 :V 甲醇 Compound 1 (4.33 g, yield 82.1%) was prepared by mixing a mixture of 20:1 to 5:1.

[0080] The NMR and mass spectrometry characterization of compound 1 obtained in Example 1 is as follows: 1 H NMR(400MHz,Chloroform-d)δ7.72(d,J=7.4Hz,2H),7.32(dq,J=21.3,7.7Hz,2H),6.98(d,J=7.4Hz,2H),6.38(d,J=8.7Hz,2H),6.29(d,J=2.7Hz,2H) ,6.20(dd,J=9.0,2.6Hz,2H),3.24(p,J=7.9,7.4Hz,10H),2.98(t,J=5.3Hz ,2H),2.69(t,J=5.4Hz,2H),2.24(t,J=5.8Hz,2H),1.07(t,J=6.9Hz,12H).

[0081] 1H NMR spectrum as follows Figure 9 As shown.

[0082] Example 2: Synthesis of Compound I

[0083] Compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 (0.2 mmol) were dissolved in dry acetonitrile (2 mL), and a strong nitrogen stream was passed through the reaction apparatus. Then, N,N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution. After the addition was complete, the mixture was heated to 30 °C and stirred overnight under a nitrogen atmosphere. After cooling, the solvent was removed by vacuum evaporation. The crude product was purified by silica gel column chromatography (200–300 mesh) using dichloromethane and methanol (v / v). 二氯甲烷 :V 甲醇 A mixture of 200:1 to 50:1 was used to prepare a near-infrared fluorescent probe (compound of formula I) that is responsive to both ATP and NO (58.7 mg, yield 25.9%).

[0084] The NMR and mass spectrometry characterization of the fluorescent probe (compound of formula I) prepared in Example 2 is as follows: 1H NMR(400MHz,Chloroform-d)δ9.41(s,1H),7.86(dt,J=8.0,2.8Hz,1H),7.58-7.51(m,3H),7.28(t,J=7.7Hz,3H),7.23(s,1H),7.12(dd, J=5.8,2.9Hz,1H),7.06(td,J=7.3,2.5Hz,2H),6.88(dd,J=8.1,3.2Hz,2H),6.47(d,J=8.8Hz,2H),6.36(d,J=2.6Hz,2H),6.30(dd,J=8. 9,2.6Hz,2H),5.61(dd,J=13.0,9.5Hz,2H),3.89(p,J=7.4Hz,4H),3.55-3.45(m,2H),3.42(s,1H),3.41-3.24(m,J=7.0Hz,9H),2.48(t, J=6.4Hz,4H),1.81(dt,J=12.1,6.3Hz,2H),1.58(s,10H),1.31(t,J=7.3Hz,8H),1.22(s,6H),1.14(t,J=7.0Hz,12H),0.91-0.80(m,3H).

[0085] MS(ESI): calcd for [C 66 H 80 N7O2] + 1002.6368, found 1002.7694.

[0086] 1H NMR spectrum as follows Figure 10 As shown; mass spectrum as shown Figure 11 As shown.

[0087] Example 3: Synthesis of Compound I

[0088] Compound 1 (0.3 mmol) obtained in Example 1 and the near-infrared dye IR-780 (0.2 mmol) were dissolved in dry acetonitrile (2 mL), and a strong nitrogen stream was passed through the reaction apparatus. Triethylamine (0.6 mmol) was then added dropwise to the solution. After the addition was complete, the mixture was heated to 30 °C and stirred overnight under a nitrogen atmosphere. After cooling, the solvent was removed by vacuum evaporation. The crude product was purified by silica gel column chromatography (200–300 mesh) using dichloromethane and methanol (v / v). 二氯甲烷 :V 甲醇 A mixture of 200:1 to 50:1 was used to prepare a near-infrared fluorescent probe (compound of formula I) that is responsive to both ATP and NO.

[0089] Example 4: Synthesis of Compound I

[0090] Compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 (0.2 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and a strong nitrogen stream was passed through the reaction apparatus. Then, N,N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution. After the addition was complete, the mixture was heated to 30°C and stirred overnight under a nitrogen atmosphere. After cooling, the reaction solution was quickly poured into ice-cold ether, which formed a large amount of blue solid precipitate. The precipitate was filtered, and the crude product was purified by silica gel column chromatography (200–300 mesh) using dichloromethane and methanol (v / v). 二氯甲烷 :V 甲醇 A mixture of 200:1 to 50:1 was used to prepare a near-infrared fluorescent probe formula I that is responsive to both ATP and NO.

[0091] Example 5: Synthesis of Compound I

[0092] Compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 (0.2 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and a strong nitrogen stream was passed through the reaction apparatus. Then, N,N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution. After the addition was complete, the mixture was heated to 80 °C and stirred overnight under a nitrogen atmosphere. After cooling, the reaction solution was quickly poured into ice-cold ether, resulting in a large amount of blue solid precipitate. The precipitate was filtered, and the crude product was purified by silica gel column chromatography (200–300 mesh) using dichloromethane and methanol (v / v). 二氯甲烷 :V 甲醇 A mixture of 200:1 to 50:1 was used to prepare a near-infrared fluorescent probe (compound of formula I) that is responsive to both ATP and NO.

[0093] Example 6: Detection of the reaction kinetics of fluorescent probes to ATP

[0094] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0095] 2. Preparation of ATP stock solution: Dissolve ATP in deionized water to prepare an ATP stock solution with a molar concentration of 1M.

[0096] 3. Reaction kinetics test:

[0097] Mix 10% ethanol with the above PBS buffer according to V 乙醇 V PBS Using a 1:9 mixture as a solvent, the fluorescent probe prepared in Example 2 was prepared into a 10 μM 10% ethanol-PBS buffer dispersion. 2 mL of the 10 μM 10% ethanol-PBS buffer dispersion of the fluorescent probe was taken, and 20 μL of 1M ATP stock solution was added. The mixture was incubated at 37°C for 0–30 min. Fluorescence spectra were measured at different time points using 550 nm as the excitation wavelength, yielding fluorescence spectra of the fluorescent probe after incubation with ATP for different times (0–30 min). Figure 3 A). A fitting curve was plotted based on the fluorescence intensity of the fluorescent probe at 586 nm and the corresponding time, yielding a trend diagram of the fluorescence intensity of the fluorescent probe at 586 nm over time. Figure 3 B), from Figure 3 As shown in B, the response of the fluorescent probe to ATP reaches equilibrium and remains stable within 30 minutes.

[0098] Example 7: Response test of fluorescent probe to different concentrations of ATP

[0099] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0100] 2. Mix 10% ethanol with the above PBS buffer according to V 乙醇 V PBS Using the 1:9 mixed solution as a solvent, the fluorescent probe prepared in Example 2 was used to prepare a series of 10 μM 10% ethanol-PBS buffered dispersions.

[0101] 3. Preparation of ATP stock solution: Dissolve ATP in deionized water to prepare an ATP stock solution with a molar concentration of 1M.

[0102] 4. Response Testing:

[0103] A series of 2 mL solutions of fluorescent probe in 10 μM 10% ethanol-PBS buffer were prepared, and different volumes of ATP stock solution were added to each solution, resulting in ATP concentrations of 0–10 mM in each system. After incubation at 37 °C for 30 min, the fluorescence spectra of each system were measured under 550 nm excitation, yielding fluorescence spectra of the fluorescent probes responding to different concentrations of ATP (0–10 mM). Figure 4A). It can be observed that the fluorescence of the system gradually increases with the increase of ATP concentration. A graph is plotted with the fluorescence intensity at 586 nm as the ordinate and the ATP concentration as the abscissa, and the working curve of the fluorescent probe responding to ATP is obtained by fitting the graph. Figure 4 B). From Figure 4 As shown in Figure B, the fluorescence intensity of the system at 586 nm exhibits a good linear relationship with the concentration of ATP (0.5–8 mM) (R0). 2 =0.99285). Based on the slope of the fitted line, the detection limit of the fluorescent probe for ATP can be calculated to be approximately 1.3 μM using the formula 3σ / k (σ: standard deviation of the detection values ​​of 11 blank samples, k: slope of the fitted line).

[0104] The above experimental results demonstrate that the fluorescent probe of this invention has sufficient detection sensitivity in response to ATP.

[0105] Example 8: Selectivity test of fluorescent probe response to ATP

[0106] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0107] 2. Mix 10% ethanol with the above PBS buffer according to V 乙醇 V PBS Using the 1:9 mixed solution as a solvent, the fluorescent probe prepared in Example 2 was used to prepare a series of 10 μM 10% ethanol-PBS buffered dispersions.

[0108] 3. Preparation of ATP stock solution: Dissolve ATP in deionized water to prepare an ATP stock solution with a molar concentration of 1M.

[0109] 4. Selective testing:

[0110] Take nineteen 2 mL aliquots of 10 μM 10% ethanol-PBS buffered dispersion of the fluorescent probe, and then add K to each aliquot. + Na + Ca 2+ Mg 2+ Zn 2+ Cu 2+ Fe 3+ H2PO2 2- PO4 3- HPO4 2- H2PO4 - SO42- CO3 2- The final concentration of the interfering substances was determined to be K in aqueous solutions of GSH, Cys, AMP, ADP, and ATP. + Na + Ca 2+ Mg 2+ Zn 2+ Cu 2 + Fe 3+ H2PO2 2- PO4 3- HPO4 2- H2PO4 - SO4 2- CO3 2- GSH was 1 mM, Cys was 100 μM, and AMP, ADP, and ATP were all 10 mM. After incubating each solution at 37°C for 30 min, the fluorescence intensity at 586 nm was recorded on a fluorescence spectrometer. The ratio of the fluorescence intensity of each experimental group at 586 nm to that of the blank group is shown below. Figure 5 As shown, the experimental results demonstrate that the fluorescent probe can specifically identify and detect ATP, and the influence of other interfering substances is negligible, indicating that the fluorescent probe of the present invention has good selectivity for ATP.

[0111] Example 9: Reaction kinetics of NO by a fluorescent probe

[0112] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0113] 2. Mix 50% ethanol with the above PBS buffer according to V 乙醇 V PBS Using the 1:1 mixed solution as a solvent, the fluorescent probe prepared in Example 2 was prepared into a 10 μM 50% ethanol-PBS buffer dispersion solution.

[0114] 3. Preparation of NO stock solution: Dissolve DEA·NONOate sodium hydrate in PBS buffer solution (10mM, pH=7.4) to prepare NO stock solution with a molar concentration of 10mM.

[0115] 4. Reaction kinetics test:

[0116] Take 2 mL of 10 μM 50% ethanol-PBS buffered dispersion of the fluorescent probe, add 20 μL of 100 μM NO stock solution, and react at room temperature for 0–360 s. Using 725 nm as the excitation wavelength, measure the fluorescence spectra at different times within the wavelength range of 735–875 nm to obtain the fluorescence spectra of the fluorescent probe reacting with NO for different times (0–360 s). Figure 6 A). As the reaction time between the fluorescent probe and NO increases, the fluorescence emission at 762 nm decreases, while the fluorescence emission peak at 816 nm significantly increases, eventually transforming into a fluorescence spectrum with only the new emission peak. This indicates that the fluorescent probe has completely reacted with NO. A graph showing the time-varying trend of the reaction between the fluorescent probe and NO is obtained by plotting the logarithm of the ratio of fluorescence intensity at 816 nm and 762 nm on the ordinate and the reaction time on the abscissa. Figure 6 B), from Figure 6 As can be seen from B, the fluorescent probe responds rapidly to NO and reaches equilibrium in 270s.

[0117] Example 10: Response test of fluorescent probe to different concentrations of NO

[0118] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0119] 2. Mix 50% ethanol with the above PBS buffer according to V 乙醇 V PBS Using the 1:1 mixed solution as a solvent, the fluorescent probe prepared in Example 2 was used to prepare a series of 10 μM 50% ethanol-PBS buffered dispersions.

[0120] 3. Preparation of NO stock solution: Dissolve DEA·NONOate sodium hydrate in PBS buffer solution (10mM, pH=7.4) to prepare NO stock solution with a molar concentration of 10mM.

[0121] 4. Response Testing:

[0122] A series of 2 mL aliquots of 10 μM 50% ethanol-PBS buffered dispersions of the fluorescent probes prepared in Example 2 were taken, and different volumes of NO stock solution were added to each system, resulting in NO concentrations ranging from 0 to 100 μM. After incubation at room temperature for 5 min, the fluorescence spectra of each system were measured under 725 nm excitation, yielding fluorescence spectra of the fluorescent probes responding to different concentrations of NO (0–100 μM). Figure 7A). From Figure 7 As observed in Figure A, with the addition of NO, the fluorescence intensity of the probe at 762 nm gradually weakens, while the new fluorescence emission peak at 816 nm significantly increases. A working curve was plotted based on the logarithm of the ratio of fluorescence intensity at 816 nm and 762 nm to the corresponding NO concentration. Figure 7 B). From Figure 7 As shown in B, there is an excellent linear relationship between the logarithm of the fluorescence intensity ratio and the NO concentration (0–40 μM) (R0). 2 =0.99244). Based on the formula 3σ / k, the detection limit of the fluorescent probe for NO is approximately 0.26 μM. The above tests demonstrate that the fluorescent probe of this invention has high in vitro detection sensitivity for NO.

[0123] Example 11: Selectivity test of fluorescent probe response to NO

[0124] 1. Preparation of buffer solution: Weigh 8g sodium chloride, 0.2g potassium chloride, 1.44g dipotassium hydrogen phosphate and 0.24g sodium dihydrogen phosphate and dissolve them in 800mL of deionized water. Adjust the pH to 7.4 with 0.5M hydrochloric acid solution. Transfer to a 1L volumetric flask and make up to volume to obtain a 10mM PBS buffer solution (pH=7.4).

[0125] 2. Mix 50% ethanol with the above PBS buffer according to V 乙醇 V PBS Using the 1:1 mixed solution as a solvent, the fluorescent probe prepared in Example 2 was used to prepare a series of 10 μM 50% ethanol-PBS buffered dispersions.

[0126] 3. Preparation of NO stock solution: Dissolve DEA·NONOate sodium hydrate in PBS buffer solution (10mM, pH=7.4) to prepare NO stock solution with a molar concentration of 10mM.

[0127] 4. Selective testing:

[0128] Take eighteen portions, each 2 mL in volume, of a 10 μM 50% ethanol-PBS buffer dispersion of the fluorescent probe prepared in Example 2, and then add K to each portion of the solution. + Na + Ca 2+ Mg 2+ Zn 2+ Cu 2+ Fe 3+ NO3 - NO2 - GSH, Cys, H2S, ONOO -The final concentrations of interfering substances in aqueous solutions of ·OH, H2O2, HClO, and NO are respectively: K + Na + Ca 2+ Mg 2+ Zn 2+ Cu 2 + Fe 3+ NO3 - NO2 - Both GSH and Cys, H2S, and ONOO are 1mM. - ·OH, H₂O₂, HClO, and NO were all 100 μM. After standing at room temperature for 5 min, the fluorescence spectra of each group of solutions were measured, with an excitation wavelength of 725 nm. The logarithm of the ratio of fluorescence intensity at 816 nm and 762 nm for each group of solutions is shown below. Figure 8 As shown in the figure, it can be analyzed that the addition of several common metal cations, acid anions and active species does not significantly change the ratio fluorescence of the probe, indicating that the fluorescent probe of the present invention has superior specific recognition performance for NO.

[0129] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe that is dual-responsive to adenosine triphosphate and nitric oxide, having the structure shown in Formula I: Equation I.

2. A method for preparing a near-infrared fluorescent probe that is dual-responsive to adenosine triphosphate and nitric oxide, comprising the following steps: (1) Rhodamine B was reacted with diethylenetriamine to give intermediate compound 1; ； (2) React intermediate compound 1 with IR-780 dye to obtain the near-infrared fluorescent probe shown in Formula I; 。 3. The preparation method according to claim 2, characterized in that, In step (1), the molar ratio of Rhodamine B to diethylenetriamine is 1:(2~10); and / or, the reaction temperature is 60~80℃; and / or, the reaction time is 8~12 h.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of rhodamine B to diethylenetriamine is 1:(2~5).

5. The preparation method according to claim 2, characterized in that, In step (1), the reaction is carried out in a first organic solvent, which includes C1-C4 low alcohols; The C1-C4 lower alcohols include ethanol.

6. The preparation method according to any one of claims 2 to 5, characterized in that, In step (2), the molar ratio of IR-780 dye to intermediate compound 1 is 1:(1~1.5); and / or, In step (2), the reaction temperature is 30~80℃; and / or, In step (2), the reaction time is 6-24 hours.

7. The preparation method according to claim 6, characterized in that, In step (2), the reaction time is 6-12 hours.

8. The preparation method according to any one of claims 2 to 5, characterized in that, In step (2), the reaction is carried out in a second organic solvent, which includes one or more of nitrile and amide organic solvents.

9. The preparation method according to claim 8, characterized in that, The second organic solvent includes acetonitrile or N,N-dimethylformamide.

10. The preparation method according to any one of claims 2 to 5, characterized in that, In step (2), the reaction is carried out in the presence of a weak organic base.

11. The preparation method according to claim 10, characterized in that, The organic weak base includes fatty amines.

12. The preparation method according to claim 11, characterized in that, The organic weak base includes triethylamine or N,N-diisopropylethylamine.

13. The preparation method according to claim 10, characterized in that, The molar ratio of IR-780 dye to the organic weak base is 1:(1~3).

14. The use of the near-infrared fluorescent probe as described in claim 1 or the near-infrared fluorescent probe prepared by any one of claims 2 to 13 in the detection of adenosine triphosphate and / or nitric oxide.

15. The application according to claim 14, characterized in that, The applications include: (S1) The sample to be tested is brought into contact with the fluorescent probe to obtain a mixture; (S2) Measure the fluorescence intensity of the mixture; When detecting adenosine triphosphate in the sample to be tested, step (S2) includes: measuring the fluorescence spectrum of the mixture in the range of 570 nm to 650 nm; When detecting nitric oxide in the sample to be tested, step (S2) includes: measuring the fluorescence spectrum of the mixture in the range of 740 nm to 875 nm.

16. The application according to claim 15, characterized in that, In step (S1), the contact includes: mixing the sample to be tested with the dispersion of the fluorescent probe to obtain a mixture; The solvent used for the dispersion of the fluorescent probe is a mixture of ethanol and PBS buffer. The pH of the PBS buffer is 7.0-8.

0.

17. The application according to claim 16, characterized in that, The pH of the PBS buffer is 7.2-7.6.

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