Adenosine triphosphate and nitric oxide dual-response near-infrared fluorescent probe as well as preparation method and application thereof

Abstract

The invention provides an adenosine triphosphate and nitric oxide dual-response near-infrared fluorescent probe as well as a preparation method and application thereof. The near-infrared fluorescent probe disclosed by the invention has a structure as shown in the formula I, responds to two substances, namely adenosine triphosphate and nitric oxide, and is high in sensitivity and selectivity. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and specifically relates to a near-infrared fluorescent probe with dual response to adenosine triphosphate and nitric oxide, and a preparation and application thereof. Background Art

[0002] Adenosine triphosphate (ATP) is the main source of energy for organisms, an important substance for regulating energy metabolism, and is involved in many life activities. The synthesis of ATP mainly occurs in the mitochondria of cells. The ATP concentration level in the blood can be used as an important indicator of human health; some 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 mature methods are used to detect ATP, such as liquid chromatography, mass spectrometry, etc. These methods usually lack spatiotemporal resolution or compatibility with living specimens. Therefore, there is an urgent need to develop molecular probes for analyzing ATP fluctuations to replace instrumental analysis.

[0003] Nitric oxide (NO) is a biological messenger molecule that is widely distributed in various tissues in the body and plays a key role in human physiology. Under normoxic conditions, NO is biosynthesized by nitric oxide synthase (NOS) through the oxidation of l-arginine to l-citrulline. Abnormal levels of intracellular NO are closely related to 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 remains an active research area. Among the 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 biological analytes related to oxidative stress and energy metabolism, respectively, and are crucial for studying their level fluctuations and synergistic effects in living organisms. However, the simultaneous use of two fluorescent probes inevitably leads to challenges such as large invasive effects, different cellular uptake, and spectral interruption; therefore, it is very important to develop single-molecule probes that can respond to both substances. Summary of the invention

[0005] In order to solve one of the above technical problems existing in the prior art, the present invention provides a near-infrared fluorescent probe that responds to both ATP and NO through independent fluorescent signals. In addition, the present invention also provides a preparation method of the near-infrared fluorescent probe and its application in detecting ATP and NO.

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

[0007] In one aspect, the present invention provides a near-infrared fluorescent probe having a structure shown in the following formula I:

[0008]

[0009] In another aspect, the present invention provides a method for preparing a near-infrared fluorescent probe, the preparation method comprising the following steps:

[0010] (1) reacting rhodamine B with diethylenetriamine to obtain an intermediate compound 1;

[0011]

[0012] (2) reacting the intermediate compound 1 with IR-780 dye to obtain a near-infrared fluorescent probe represented by 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 therebetween. 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 to 80° C. According to some embodiments of the present invention, in step (1), the reaction time is 8 to 12 hours.

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

[0017] According to some embodiments of the present invention, step (1) further comprises: after the reaction is completed, removing the solvent in the reaction solution by reduced pressure distillation to obtain a crude product; dissolving the crude product with a halogenated alkane solvent such as dichloromethane, and washing with saturated brine to obtain an organic layer; after drying and removing the solvent from the organic layer, separating and purifying it using 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 the IR-780 dye to the intermediate compound 1 is 1:(1-1.5).

[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, and the second organic solvent 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., does not contain water. In some embodiments, the amount of the second organic solvent is: 10 to 20 L of the second organic solvent is used for every 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 comprises an aliphatic amine. In some embodiments, the organic weak amine comprises triethylamine and / or N,N-diisopropylethylamine.

[0022] According to some embodiments of the present invention, in step (2), the molar ratio of the 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 use of the above-mentioned near-infrared fluorescent probe or the near-infrared fluorescent probe obtained by the above-mentioned preparation method in detecting ATP and / or NO.

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

[0026] (S1) contacting the sample to be tested with the fluorescent probe to obtain a mixture;

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

[0028] The fluorescent probe of the present invention can be used to detect a sample containing NO and its donor, wherein the NO donor includes but is not limited to: N-diethylamino-N-oxynitrosamide sodium salt.

[0029] In some embodiments, step (S2) comprises: measuring the fluorescence spectrum of the mixture at 570-875 nm.

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

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

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

[0033] In some embodiments, in step (S1), the contacting comprises: mixing the sample to be tested with the dispersion of the fluorescent probe to obtain a mixed solution. In some embodiments, the solvent used for the dispersion of the fluorescent probe is a mixed solution 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 has the ability to detect two analytes, ATP and NO, as a single molecule probe, and produces independent responses to ATP and NO without spectral crosstalk.

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

[0037] (3) The preparation method of the fluorescent probe of the present invention is simple, the conditions are mild, and it is suitable for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0039] Figure 2 The figure is a synthetic route map of the near-infrared fluorescent probe of the present invention.

[0040] Figure 3 The results of ATP detection after the fluorescent probe prepared in Example 2 of the present invention was co-incubated with ATP for different time periods, wherein Figure A) is a fluorescence spectrum of the fluorescent probe after incubation with ATP for different time periods (0 to 30 min); Figure B) is a fluorescence intensity trend of the fluorescent probe at 586 nm over time.

[0041] Figure 4 The results of the detection of different concentrations of ATP by the fluorescent probe prepared in Example 2 of the present invention, wherein Figure A) is the fluorescence spectrum of the fluorescent probe after incubation with different concentrations of ATP (0-10 mM) for 30 minutes; Figure B) is the change of the fluorescence intensity of the fluorescent probe at 586 nm with the ATP level.

[0042] Figure 5 This is a fluorescence spectrum diagram of the fluorescent probe prepared in Example 2 of the present invention for detecting ATP selectivity.

[0043] Figure 6 These are the detection results of the fluorescent probe prepared in Example 2 of the present invention responding to NO at different times, wherein Figure A) is the fluorescence spectrum of the fluorescent probe responding to NO after 0 to 360 seconds; Figure B) is the logarithm of the fluorescence intensity ratio of the fluorescent probe at 816 nm and 762 nm changing with time.

[0044] Figure 7 The results of the detection of different concentrations of NO by the fluorescent probe prepared in Example 2 of the present invention, wherein Figure A) is the fluorescence spectrum of the fluorescent probe responding to different concentrations of NO (0-100 μM) for 5 minutes; Figure B) is the change in the logarithm of the fluorescence intensity ratio of the fluorescent probe at 816 nm and 762 nm with the NO level.

[0045] Figure 8 This is the result of the fluorescent probe prepared in Example 2 of the present invention detecting NO selectivity.

[0046] Fig. 9 It is the H NMR spectrum of compound 1 of the present invention.

[0047] Fig.10 This is the hydrogen nuclear magnetic spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention.

[0048] Fig.11 This is the mass spectrum of the near-infrared fluorescent probe prepared in Example 2 of the present invention. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0050] The present invention provides a near-infrared fluorescent probe that responds to both ATP and NO as shown in the following formula I:

[0051]

[0052] The recognition principle of the fluorescent probe of the present invention for ATP and NO is as follows Figure 1 As shown. The design of the fluorescent probe of the present invention is to connect the two skeletons of rhodamine B and near-infrared cyanine dye IR-780 through diethylenetriamine. Diethylenetriamine is not only a connecting group of the skeleton, but also a response group that recognizes ATP and NO. The amidation reaction of one end amino group of diethylenetriamine with rhodamine B makes rhodamine B closed (no fluorescence), and ATP induces the spironolactone ring of rhodamine B to open, thereby turning on fluorescence (λ ex =550nm,λ em =586nm). The other end amino group of diethylenetriamine and the near-infrared dye IR-780 dye form an aromatic secondary amine through substitution reaction as the recognition group of NO. As the N-nitrosation reaction occurs with NO, the fluorescence of the probe at 816nm increases, while the fluorescence at 762nm decreases, and the response to NO is ratiometric.

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

[0054] As a specific embodiment, the method for preparing the fluorescent probe of the present invention comprises the following steps:

[0055] 1) Take rhodamine B and diethylenetriamine at a molar ratio of 1:5-10, and then take ethanol at a ratio of 5-10L ethanol to 1mol of rhodamine B. First, dissolve rhodamine B with ethanol, then add diethylenetriamine. After the addition is complete, heat to 60-80°C and reflux for 8-12 hours; cool the reaction to room temperature, and remove the solvent by evaporation under reduced pressure; dissolve the crude product with dichloromethane, wash three times with saturated brine, dry the organic layer with anhydrous sodium sulfate, and remove the solvent by evaporation under reduced pressure; separate and purify the obtained solid by silica gel column chromatography (200-300 mesh), and the eluent is dichloromethane and methanol (volume ratio

[0056] V 二氯甲烷 :V 甲醇 =20:1~5:1) to obtain compound 1.

[0057] 2) Compound 1 obtained in step 1) above and near-infrared dye IR-780 dye are dissolved in a dry organic solvent, and a strong nitrogen flow is passed through the reaction device; then an organic weak base is added dropwise to the solution; after the addition is completed, the nitrogen atmosphere is maintained, and the mixture is heated to 30-80° C. and stirred overnight. After cooling, the obtained crude product is separated and purified by silica gel column chromatography (200-300 mesh), and the eluent is dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =200:1-50:1) to prepare a near-infrared fluorescent probe that responds to both ATP and NO;

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

[0059] As a 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] Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g potassium dihydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4);

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

[0063] Dissolve ATP in secondary water to prepare a 1M ATP stock solution.

[0064] 2) Detection of ATP:

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

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

[0067] iii) Take a series of 10 μM 10% ethanol-PBS buffer dispersion solutions of fluorescent probes and add K + 、Na + , Ca 2 + Mg 2+ 、Zn 2+ , Cu 2+ , Fe 3+ , H 2 PO 2 2- ,PO 4 3- , HPO 4 2- , H 2 PO 4 - 、SO 4 2- , CO 3 2- , GSH, Cys, AMP, ADP, and ATP aqueous solutions were incubated at 37°C for 30 min, and the fluorescence spectra at 586 nm were measured and recorded to verify the selectivity of the probe molecule for ATP.

[0068] As a specific embodiment, the application of the fluorescent probe of the present invention in NO detection comprises the following steps:

[0069] 1) First prepare the following solutions:

[0070] Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g potassium dihydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4);

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

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

[0073] 2) Detection of NO:

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

[0075] ii) Take a series of 10 μM 50% ethanol-PBS buffer dispersion solutions of fluorescent probes, add different volumes of 10 mM NO stock solution, and the resulting mixed solutions are placed at room temperature for 5 min. The fluorescence spectra of the solutions after the fluorescent probes react with different concentrations of NO are measured, and a linear fitting curve is prepared based on the relationship between the logarithm of the ratio of the fluorescence intensities at 816 nm and 762 nm and the NO concentration.

[0076] iii) Take a series of 10 μM 50% ethanol-PBS buffer dispersion solutions of fluorescent probes and add K + 、Na + , Ca 2 + Mg 2+ 、Zn 2+ , Cu 2+ , Fe 3+ 、NO 3 - 、NO 2 - , GSH, Cys, H 2 S.ONOO - , OH, H 2 O 2 The aqueous solutions of HClO and NO were placed at room temperature for 5 min, and the fluorescence intensities at 816 nm and 762 nm were measured and recorded respectively. The selectivity of the probe molecule for NO was verified based on the logarithm of the fluorescence intensity ratio.

[0077] The reagents used in the following experiments of the present invention, unless otherwise specified, are all conventional commercial products or reagents prepared by conventional methods, and the methods used in the experiments, unless otherwise specified, are all conventional experimental methods. The instruments used in the experiments, unless otherwise specified, can all be obtained through commercial channels.

[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 completed, the temperature was raised to 70°C and refluxed for 12 hours. The reaction 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 then the solvent was removed by evaporation under reduced pressure. The obtained solid was separated and purified by silica gel column chromatography (200-300 meshes). The eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =20:1~5:1) to obtain compound 1 (4.33 g, yield 82.1%).

[0080] The NMR and mass spectrometry characteristics of compound 1 prepared in Example 1 are 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] H NMR spectrum Fig. 9 shown.

[0082] Example 2: Synthesis of compounds of formula I

[0083] The compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 dye (0.2 mmol) were dissolved in dry acetonitrile (2 mL), and a strong nitrogen flow was passed through the reaction device; then N, N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution; after the addition was completed, the nitrogen atmosphere was maintained, and the mixture was heated to 30°C and stirred overnight. After cooling, the solvent was removed by evaporation under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (200-300 mesh), and the eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =200:1~50:1) to prepare a near-infrared fluorescent probe (compound of formula I) (58.7 mg, yield 25.9%) that responds to both ATP and NO.

[0084] The nuclear magnetic resonance and mass spectrometry characteristics of the fluorescent probe (compound of formula I) prepared in Example 2 are 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 N 7 O 2 ] + 1002.6368, found 1002.7694.

[0086] H NMR spectrum Fig.10 As shown; the mass spectrum is as Fig.11 shown.

[0087] Example 3: Synthesis of compounds of formula I

[0088] The compound 1 (0.3 mmol) obtained in Example 1 and the near-infrared dye IR-780 dye (0.2 mmol) were dissolved in dry acetonitrile (2 mL), and a strong nitrogen flow was passed through the reaction device; triethylamine (0.6 mmol) was then added dropwise to the solution; after the addition was completed, the nitrogen atmosphere was maintained, and the mixture was heated to 30°C and stirred overnight. After cooling, the solvent was removed by evaporation under reduced pressure, and the obtained crude product was separated and purified by silica gel column chromatography (200-300 mesh), and the eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =200:1~50:1) to prepare a mixture of ATP and NO dual-responsive near-infrared fluorescent probe (compound of formula I).

[0089] Example 4: Synthesis of compounds of formula I

[0090] The compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 dye (0.2 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and a strong nitrogen flow was passed through the reaction device; then N,N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution; after the addition was completed, the nitrogen atmosphere was maintained, and the mixture was heated to 30°C and stirred overnight. After cooling, the reaction solution was quickly poured into ice ether, and a large amount of blue solid precipitate was formed; the precipitate was filtered, and the obtained crude product was separated and purified by silica gel column chromatography (200-300 mesh), and the eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =200:1~50:1) to prepare a mixture of ATP and NO dual-responsive near-infrared fluorescent probe formula I.

[0091] Example 5: Synthesis of compounds of formula I

[0092] The compound 1 (0.24 mmol) obtained in Example 1 and the near-infrared dye IR-780 dye (0.2 mmol) were dissolved in dry N,N-dimethylformamide (2 mL), and a strong nitrogen flow was passed through the reaction device; then N,N-diisopropylethylamine (0.6 mmol) was added dropwise to the solution; after the addition was completed, the nitrogen atmosphere was maintained and the mixture was heated to 80°C and stirred overnight. After cooling, the reaction solution was quickly poured into ice ether to form a large amount of blue solid precipitation; the precipitate was filtered, and the obtained crude product was separated and purified by silica gel column chromatography (200-300 mesh), and the eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =200:1~50:1) to prepare a mixture of ATP and NO dual-responsive near-infrared fluorescent probe (compound of formula I).

[0093] Example 6: Fluorescent probe reaction kinetics test on ATP

[0094] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

[0095] 2. Prepare ATP stock solution: Dissolve ATP in secondary water to prepare 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 =1:9 mixed solution as solvent, the fluorescent probe prepared in Example 2 is configured into a 10μM 10% ethanol-PBS buffer dispersion solution. Take 2mL of 10μM 10% ethanol-PBS buffer dispersion solution of the fluorescent probe, add 20μL of 1M ATP stock solution, and incubate at 37℃ for 0-30min. With 550nm as the excitation wavelength, the fluorescence spectra at different times are measured respectively, and the fluorescence spectra of the fluorescent probe and ATP after incubation for different time (0-30min) are obtained ( Figure 3 A). According to the fluorescence intensity of the fluorescent probe at 586nm and the corresponding time, a fitting curve was drawn to obtain the fluorescence intensity of the fluorescent probe at 586nm versus time trend diagram ( Figure 3 B) from Figure 3 As can be seen in B, the response of the fluorescent probe to ATP reaches equilibrium within 30 min and remains stable.

[0098] Example 7: Fluorescent probe response test to different concentrations of ATP

[0099] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

[0100] 2. Mix 10% ethanol and the above PBS buffer according to V 乙醇 :V PBS =1:9 mixed solution was used as solvent, and the fluorescent probe prepared in Example 2 was configured into a series of 10 μM 10% ethanol-PBS buffer dispersion solutions.

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

[0102] 4. Response testing:

[0103] Take a series of 2mL of 10μM 10% ethanol-PBS buffer dispersion solutions of fluorescent probes, add different volumes of ATP stock solution, and finally the concentration of ATP in each system is 0-10mM. After incubation at 37℃ for 30min, measure the fluorescence spectrum of each system solution under 550nm excitation, and obtain the fluorescence spectrum of the fluorescent probe in response to different concentrations of ATP (0-10mM) ( Figure 4A). It can be observed that as the ATP concentration increases, the fluorescence of the system gradually increases. The fluorescence intensity at 586nm is used as the ordinate and the ATP concentration is used as the abscissa to obtain the working curve of the fluorescent probe responding to ATP ( Figure 4 B) From Figure 4 As can be seen from B, the fluorescence intensity of the system at 586 nm shows a good linear relationship with the concentration of ATP (0.5-8 mM) (R 2 =0.99285). Based on the slope of the fitted straight line, the detection limit of the fluorescent probe for ATP can be calculated to be about 1.3 μM using the formula 3σ / k (σ: standard deviation of the detection values ​​of 11 blank samples, k: slope of the fitted straight line).

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

[0105] Example 8: Selective testing of fluorescent probe response to ATP

[0106] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

[0107] 2. Mix 10% ethanol and the above PBS buffer according to V 乙醇 :V PBS =1:9 mixed solution was used as solvent, and the fluorescent probe prepared in Example 2 was configured into a series of 10 μM 10% ethanol-PBS buffer dispersion solutions.

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

[0109] 4. Selective testing:

[0110] Take 19 portions of 2 mL of 10 μM 10% ethanol-PBS buffer dispersion solution of the fluorescent probe, and add K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Cu 2+ , Fe 3+ , H 2 PO 2 2- ,PO 4 3- , HPO4 2- , H 2 PO 4 - 、SO 4 2- , CO 3 2- , GSH, Cys, AMP, ADP, ATP in aqueous solution, the final concentration of the interfering substance is determined as K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Cu 2 + , Fe 3+ , H 2 PO 2 2- ,PO 4 3- , HPO 4 2- , H 2 PO 4 - 、SO 4 2- , CO 3 2- The concentrations of 1 mM and 1 mM of GSH, 100 μM of Cys, and 10 mM of AMP, ADP, and ATP were incubated at 37°C for 30 min, and the fluorescence intensity at 586 nm was recorded on a fluorescence spectrometer. The ratio of the fluorescence intensity at 586 nm of each experimental group to the fluorescence intensity of the blank group is shown in Figure 5 As shown, the experimental results show that the fluorescent probe can specifically identify and detect ATP, and the influence of other interferents is negligible, indicating that the fluorescent probe of the present invention has good selectivity for ATP.

[0111] Example 9: Fluorescent probe reaction kinetics test on NO

[0112] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

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

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

[0115] 4. Reaction kinetics test:

[0116] Take 2mL of 10μM 50% ethanol-PBS buffer dispersion solution of the fluorescent probe, add 20μL of 100μM NO stock solution, and react at room temperature for 0 to 360s. With 725nm as the excitation wavelength, measure the fluorescence spectra at different times in the wavelength range of 735 to 875nm to obtain the fluorescence spectra of the fluorescent probe and NO at different reaction times (0 to 360s) ( Figure 6 A). As the reaction time of the fluorescent probe and NO increases, the fluorescence emission of the system at 762nm decreases. In correlation, another fluorescence emission peak, 816nm, is significantly enhanced, and finally completely transforms into a fluorescence spectrum with only the new emission peak, which means that the fluorescent probe reacts completely with NO. The logarithm of the fluorescence intensity ratio of the fluorescent probe at 816nm and 762nm is plotted as the ordinate and the reaction time is plotted as the abscissa to obtain the time variation trend diagram of the reaction of the fluorescent probe and NO ( Figure 6 B) from Figure 6 As can be seen in B, the fluorescent probe responds quickly to NO and reaches equilibrium at 270s.

[0117] Example 10: Fluorescent probe response test to different concentrations of NO

[0118] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

[0119] 2. Mix 50% ethanol and the above PBS buffer according to V 乙醇 :V PBS =1:1 mixed solution was used as solvent, and the fluorescent probe prepared in Example 2 was configured into a series of 10 μM 50% ethanol-PBS buffer dispersion solutions.

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

[0121] 4. Response testing:

[0122] A series of 2 mL of 10 μM 50% ethanol-PBS buffer dispersion solutions of the fluorescent probe prepared in Example 2 were taken, and different volumes of NO stock solution were added respectively, and the final NO concentration in each system was 0-100 μM. After being placed at room temperature for 5 minutes, the fluorescence spectra of each system solution under 725 nm excitation were measured to obtain the fluorescence spectra of the fluorescent probe in response to different concentrations of NO (0-100 μM) ( Figure 7 A) From Figure 7 It can be observed in A that with the addition of NO, the intensity of the fluorescence of the probe at 762nm gradually weakened, while the new fluorescence emission peak at 816nm was significantly enhanced. The working curve was drawn based on the logarithm of the ratio of the fluorescence intensity of the fluorescent probe at 816nm and 762nm and the corresponding NO concentration ( Figure 7 B) From Figure 7 As can be seen in B, there is an excellent linear relationship between the logarithm of the fluorescence intensity ratio and the NO concentration (0-40 μM) (R 2 =0.99244). According to the formula 3σ / k, the detection limit of the fluorescent probe for NO is about 0.26 μM. The above test shows that the fluorescent probe of the present invention has a high sensitivity for detecting NO in vitro.

[0123] Example 11: Selective testing of fluorescent probe response to NO

[0124] 1. Prepare buffer solution: weigh 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g dipotassium hydrogen phosphate and 0.24 g sodium dihydrogen phosphate and dissolve them in 800 mL secondary water, adjust the pH value to 7.4 with 0.5 M hydrochloric acid solution, transfer to a 1 L volumetric flask, and dilute to obtain a PBS buffer solution with a molar concentration of 10 mM (pH = 7.4).

[0125] 2. Mix 50% ethanol and the above PBS buffer according to V 乙醇 :V PBS =1:1 mixed solution was used as solvent, and the fluorescent probe prepared in Example 2 was configured into a series of 10 μM 50% ethanol-PBS buffer dispersion solutions.

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

[0127] 4. Selective testing:

[0128] Take 18 portions of 2 mL of 10 μM 50% ethanol-PBS buffer dispersion solution of the fluorescent probe prepared in Example 2, and add K + 、Na + , Ca2+ Mg 2+ 、Zn 2+ , Cu 2+ , Fe 3+ 、NO 3 - 、NO 2 - , GSH, Cys, H 2 S.ONOO - , OH, H 2 O 2 , HClO and NO in aqueous solution, the final concentrations of the interfering substances are: K + 、Na + , Ca 2+ Mg 2+ 、Zn 2+ , Cu 2 + , Fe 3+ 、NO 3 - 、NO 2 - and GSH were 1 mM, Cys, H 2 S.ONOO - , OH, H 2 O 2 , HClO and NO were all 100 μM. After being placed at room temperature for 5 minutes, the fluorescence spectra of each group of solutions were measured, and the excitation wavelength was 725 nm. The logarithm of the fluorescence intensity ratio of each group of solutions at 816 nm and 762 nm is as follows Figure 8 As shown in the figure, it can be analyzed that the ratio fluorescence of the probe does not change significantly when several common metal cations, acid anions and active species are added, indicating that the fluorescent probe of the present invention has excellent specific recognition performance in response to NO.

[0129] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe that responds to both adenosine triphosphate and nitric oxide, having a structure shown in the following formula I:

2. A method for preparing a near-infrared fluorescent probe that responds to both adenosine triphosphate and nitric oxide, comprising the following steps: (1) reacting rhodamine B with diethylenetriamine to obtain an intermediate compound 1; (2) reacting the intermediate compound 1 with IR-780 dye to obtain a near-infrared fluorescent probe represented by 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), preferably 1:(2-5); and / or, The reaction temperature is 60-80° C.; and / or the reaction time is 8-12 hours.

4. The preparation method according to claim 2 or 3, characterized in that: In step (1), the reaction is carried out in a first organic solvent, and the first organic solvent comprises a C1-C4 low-carbon alcohol; Preferably, the first organic solvent comprises ethanol.

5. The preparation method according to any one of claims 2 to 4, 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 to 80° C.; and / or, In step (2), the reaction time is 6-24 hours, preferably 6-12 hours.

6. 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, and the second organic solvent comprises one or more nitrile and amide organic solvents; Preferably, the second organic solvent comprises acetonitrile and / or N,N-dimethylformamide.

7. The preparation method according to any one of claims 2 to 6, characterized in that: In step (2), the reaction is carried out in the presence of an organic weak base; Preferably, the organic weak base comprises aliphatic amine, preferably triethylamine and / or N,N-diisopropylethylamine; Preferably, the molar ratio of the IR-780 dye to the organic weak base is 1:(1-3).

8. Use of the near-infrared fluorescent probe according to claim 1 or the near-infrared fluorescent probe obtained by the preparation method according to any one of claims 2 to 7 in detecting adenosine triphosphate and / or nitric oxide.

9. The use according to claim 8, characterized in that: The applications include: (S1) contacting the sample to be tested with the fluorescent probe to obtain a mixture; (S2) measuring the fluorescence intensity of the mixture; Preferably, when detecting adenosine triphosphate in the sample to be tested, step (S2) comprises: measuring the fluorescence spectrum of the mixture at 570nm-650nm; Preferably, when detecting nitric oxide in the sample to be tested, step (S2) includes: measuring the fluorescence spectrum of the mixture at 740nm-875nm.

10. The use according to claim 9, characterized in that: In step (S1), the contacting includes: mixing the sample to be tested with the dispersion of the fluorescent probe to obtain a mixture; Preferably, 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.

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