A pH ratio fluorescent probe based on FRET mechanism and its application

Abstract

The present invention discloses a pH ratio change fluorescent probe based on the FRET mechanism. The probe is composed of three parts: an energy donor coumarin derivative fluorophore, a linker piperazine, and an energy acceptor (E)-3-(2-hydroxyethyl)-1,1-dimethyl-2-(4-methylphenyl)-1H-benz[e]indole-3-ium bromide. The probe is a spiropyran compound, and its chemical structure is shown in Formula I. The fluorescent probe prepared by the present invention is based on the FRET mechanism. As the pH value increases, the fluorescence intensity at 608nm gradually decreases, and the fluorescence intensity at 485nm gradually increases. Fluorescence intensity ratio I 485 / I 608 It shows a good linear relationship within the pH range of 7.2 to 8.0, and is expected to play a role in detecting changes in solution systems and intracellular pH values, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic small molecule fluorescent probes, and in particular relates to a pH ratio fluorescent probe based on the FRET mechanism and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] pH is a key parameter in a wide range of applications, including biomedical research, environmental water quality control, and material aging monitoring. It also plays a crucial role in various cellular behaviors, such as cell proliferation, migration, apoptosis, autophagy, ion transport, endocytosis, and cell polarization. Studies have shown that abnormal physiological pH is associated with a range of diseases, including cardiovascular disease, cancer, Parkinson's disease, and Alzheimer's disease. Therefore, real-time pH measurement in biological tissues is crucial for studying cellular physiological and pathological processes.

[0004] In recent decades, researchers have developed and utilized a variety of analytical methods for measuring pH, including nuclear magnetic resonance spectroscopy, microelectrodes, and high-performance liquid chromatography-mass spectrometry. However, these methods have significant limitations, such as low sensitivity, poor specificity, and irreversible damage to living cells. Consequently, fluorescent probe-based methods have seen significant development in recent years due to their high sensitivity, strong anti-interference capabilities, and non-invasive nature.

[0005] Most of the pH fluorescent probes reported so far are single-emission probes, and there have been no reports on the use of ratiometric fluorescent probes based on fluorescence resonance energy transfer (FRET) to measure the pH value in biological tissues in real time. This type of ratiometric fluorescent probe eliminates the influence of factors such as probe concentration, background fluorescence, and instrument sensitivity through the built-in two fluorescence emission peak corrections, and has broader development prospects, which urgently needs to be explored by technical personnel. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the present invention provides a pH ratio fluorescent probe based on the FRET mechanism, which is used to detect pH changes in water and cells.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention provides a pH ratio fluorescent probe based on the FRET mechanism, having the structure shown in Formula I:

[0009]

[0010] The chemical name of the fluorescent probe is: (E)-2-(4-(4-(7-(diethylamino)-2-oxo-2H-chroman-3-carbonyl)piperazin-1-yl)phenylvinyl)-3-(2-hydroxyethyl)-1,1-dimethyl-1H-benzo[e]indol-3-ium bromide (abbreviated as DPM);

[0011] Preferably, the fluorescent probe is composed of three parts: an energy donor coumarin derivative fluorophore, a connecting group piperazine, and an energy acceptor (E)-3-(2-hydroxyethyl)-1,1-dimethyl-2-(4-methylphenyl)-1H-benz[e]indol-3-ium bromide.

[0012] The second aspect of the present invention provides a method for preparing a pH ratio fluorescent probe based on the FRET mechanism, comprising the following steps:

[0013] S1, mixing 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine in an organic solvent for reaction to obtain compound 1;

[0014] S2, heating 2-bromoethane-1-ol and 1,1,2-trimethyl-1H-benzo[e]indole to reflux to obtain compound 2;

[0015] S3. Dissolve piperazine, compound 1, and compound 2 in an organic solvent and heat them for reaction to obtain the fluorescent probe DPM.

[0016] Preferably, in step S1, the molar ratio of 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 3-6:3-6:4-8:1-3.

[0017] Further preferably, the molar ratio of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 5:5:6:2.

[0018] Preferably, in step S1, the organic solvent is dichloromethane.

[0019] Preferably, in step S1, the reaction temperature is room temperature and the reaction time is 15 to 20 hours.

[0020] More preferably, the mixing reaction time is 18.5 h.

[0021] Preferably, in step S2, the molar ratio of 2-bromoethane-1-ol to 1,1,2-trimethyl-1H-benzo[e]indole is 4-8:3-7.

[0022] More preferably, the molar ratio of the 2-bromoethane-1-ol to the 1,1,2-trimethyl-1H-benzo[e]indole is 6:5.

[0023] Preferably, in step S2, the temperature of the heating reflux reaction is 60-100° C., and the time is 6-10 h.

[0024] More preferably, the temperature of the heating reflux reaction is 80° C. and the time is 8 h.

[0025] Preferably, in step S3, the molar ratio of piperazine, compound 1 and compound 2 is 0.02-0.06:0.8-1.2:0.8-1.2.

[0026] Further preferably, the molar ratio of piperazine, compound 1 and compound 2 is 0.04:1:1.

[0027] Preferably, in step S3, the organic solvent is ethanol.

[0028] Preferably, in step S3, the heating reaction temperature is 60-100° C. and the time is 6-10 h.

[0029] More preferably, the heating reaction temperature is 80° C. and the time is 8 hours.

[0030] The third aspect of the present invention provides an application of the pH ratio fluorescent probe based on the FRET mechanism described in the first aspect in pH-responsive fluorescence detection.

[0031] Preferably, the probe is used as a pH sensor in biological analysis, cell imaging, organelle targeting, and drug delivery and release.

[0032] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0033] (1) The ratiometric fluorescent probe prepared by the present invention, based on the FRET mechanism, exhibits variable fluorescence as the pH value of the system changes, with a linear relationship between the fluorescence and pH within a certain range. This fluorescent probe can not only quantitatively detect the pH value of a solution system, but can also be used for pH detection in living cells.

[0034] (2) The ratiometric fluorescent probe based on the FRET mechanism prepared by the present invention is expected to play a role in detecting the pH value of water samples and biological samples and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The response mechanism of the fluorescent probe prepared in Example 1 of the present invention to pH;

[0036] Figure 2 The fluorescent probe prepared in Example 1 of the present invention is sensitive to various analytes (such as HS - 、HSO3 - 、NO3 - 、SCN - 、CO3 2- 、F - Br - , I - , ClO - 、SO4 2- Mg 2+ 、Fe 3+ , Ca 2+ 、Al 3+ 、Cu 2+ 、Zn 2+ , Hcy, Cys, H2O2, GSH) response selectivity and anti-interference ability;

[0037] Figure 3 Titration spectra of the fluorescent probe prepared in Example 1 of the present invention at different pH values ​​(a) and a linear relationship diagram in the pH range of 7.2 to 8.0 (b);

[0038] Figure 4 Confocal fluorescence imaging of Hela cells at different pH values ​​using the fluorescent probe (2 μM) prepared in Example 1 of the present invention, blue channel (440-525 nm), red channel (575-700 nm);

[0039] Wherein: (a) is the cell imaging under the conditions shown; (b) is the fluorescence intensity ratio (I 485 / I 608 ) and pH value linear correlation diagram;

[0040] Figure 5 Schematic diagram of Hela cell imaging of the fluorescent probe targeted to mitochondria prepared in Example 1 of the present invention (co-localization imaging diagram);

[0041] Wherein: (a) is the probe imaging diagram of the present invention, λ ex =405nm, blue channel: 440-545nm; (b) is the commercial dye Mito-Tracker targeting mitochondria TM Deep Red imaging, λ ex =644 nm, red channel: 664-700 nm; (c) is the overlay of (a) and (b). DETAILED DESCRIPTION

[0042] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0043] The first typical embodiment of the present invention provides a pH ratio fluorescent probe based on the FRET mechanism, having the structure shown in Formula I:

[0044]

[0045] The chemical name of the fluorescent probe is: (E)-2-(4-(4-(7-(diethylamino)-2-oxo-2H-chroman-3-carbonyl)piperazin-1-yl)phenylvinyl)-3-(2-hydroxyethyl)-1,1-dimethyl-1H-benz[e]indol-3-ium bromide (abbreviated as DPM).

[0046] In one or more examples of this embodiment, the fluorescent probe is composed of three parts: an energy donor coumarin derivative fluorophore, a linker piperazine, and an energy acceptor (E)-3-(2-hydroxyethyl)-1,1-dimethyl-2-(4-methylphenyl)-1H-benz[e]indol-3-ium bromide.

[0047] A second exemplary embodiment of the present invention provides a method for preparing a pH ratio fluorescent probe based on the FRET mechanism, comprising the following steps:

[0048] S1, mixing 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine in an organic solvent for reaction to obtain compound 1;

[0049] S2, heating 2-bromoethane-1-ol and 1,1,2-trimethyl-1H-benzo[e]indole to reflux to obtain compound 2;

[0050] S3. Dissolve piperazine, compound 1, and compound 2 in an organic solvent and heat them for reaction to obtain the fluorescent probe DPM.

[0051] In one or more examples of this embodiment, in step S1, the molar ratio of 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 3-6:3-6:4-8:1-3.

[0052] In one or more examples of this embodiment, the molar ratio of 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 5:5:6:2.

[0053] In one or more examples of this embodiment, in step S1, the organic solvent is dichloromethane.

[0054] In one or more examples of this embodiment, in step S1, the reaction temperature is room temperature and the reaction time is 15 to 20 hours.

[0055] In one or more examples of this embodiment, the mixing reaction time is 18.5 hours.

[0056] In one or more examples of this embodiment, in step S2, the molar ratio of 2-bromoethane-1-ol to 1,1,2-trimethyl-1H-benzo[e]indole is 4-8:3-7.

[0057] In one or more examples of this embodiment, the molar ratio of 2-bromoethane-1-ol to 1,1,2-trimethyl-1H-benzo[e]indole is 6:5.

[0058] In one or more examples of this embodiment, in step S2, the temperature of the heating reflux reaction is 60-100° C., and the time is 6-10 h.

[0059] In one or more examples of this embodiment, the temperature of the heating reflux reaction is 80° C. and the time is 8 hours.

[0060] In one or more examples of this embodiment, in step S3, the molar ratio of piperazine, compound 1 and compound 2 is 0.02-0.06:0.8-1.2:0.8-1.2.

[0061] In one or more examples of this embodiment, the molar ratio of piperazine, compound 1, and compound 2 is 0.04:1:1.

[0062] In one or more examples of this embodiment, in step S3, the organic solvent is ethanol.

[0063] In one or more examples of this embodiment, in step S3, the heating reaction temperature is 60-100° C. and the time is 6-10 hours.

[0064] In one or more examples of this embodiment, the temperature of the heating reaction is 80° C. and the time is 8 hours.

[0065] A third typical embodiment of the present invention provides an application of the above-mentioned pH ratio fluorescent probe based on the FRET mechanism in pH-responsive fluorescence detection.

[0066] In one or more examples of this embodiment, the probe is used as a pH sensor for applications in biological analysis, cell imaging, organelle targeting, and drug delivery and release.

[0067] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are intended to explain the present invention rather than to limit it.

[0068] Example 1: This example provides a pH ratio fluorescent probe based on the FRET mechanism and a preparation method.

[0069]

[0070] The specific preparation method comprises the following steps:

[0071] S1. 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid (1.31 g, 5 mmol), 4-(piperazin-1-yl)benzaldehyde (950 mg, 5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.16 g, 6 mmol) and 4-dimethylaminopyridine (611 mg, 2 mmol) were stirred in dichloromethane (120 mL) at room temperature for 18.5 h to obtain compound 1, 4-(4-(7-(diethylamino)-2-oxo-2H-benzopyran-3-carbonyl)piperazin-1-yl)benzaldehyde (Compound 1);

[0072] S2. 2-Bromoethane-1-ol (1.50 g, 12 mmol) and 1,1,2-trimethyl-1H-benz[e]indole (2.09 g, 10 mmol) were heated under reflux at 80° C. for 8 h in acetonitrile (80 mL) to obtain compound 2;

[0073] S3. Piperazine (3.6 mg, 0.04 mmol), compound 1 (433.2 mg, 1 mmol) and compound 2 (333.1 mg, 1 mmol) were dissolved in ethanol (4 mL) and heated under reflux at 80°C for 6 h to obtain the fluorescent probe DPM with a yield of 56.1%. The melting point was 228-230°C. IR (KBr, cm -1 ):3407.95,2868.28,1713.26,1620.46,1521.94,1167.87. 1HNMR (400MHz, DMSO-d6) δ8.44(d,J=16.0Hz,1H),8.39(d,J=7.8Hz,1H),8.22(d,J=9.2Hz,1H),8.17(d,J=6.8 Hz,1H),8.10(d,J=9.2Hz,2H),8.05(s,1H),8.01(d,J=9.0Hz,1H),7.78(t,J=7.0Hz,1H),7.67(t,J=8.1Hz,1H ),7.52(d,J=9.0Hz,1H),7.46(d,J=16.1Hz,1H),7.12(d,J=9.4Hz,2H),6.77(dd,J=8.9,2.4Hz,1H),6.58(d, J=2.6Hz,1H),5.20(s,1H),4.80(s,2H),3.93(s,2H),3.79–3.47(m,12H),2.02(s,6H),1.14(t,J=7.0Hz,6H). 13 C NMR (101MHz, DMSO-d6) δ182.88,164.82,158.99,157.19,154.33,153.05,151. 83,144.77,139.51,137.40,134.02,133.11,130.99,130.71,130.46,128.68, 127.30,127.00,124.47,123.29,116.05,114.20,113.83,109.95,107.63,96. 77,59.34,56.49,53.50,47.04,46.28,44.68,26.70,19.04,12.79.HR-ESI-MS m / z=669.3433for[M+H] + (calcd forC 42 H 45 N4O4, 669.3436). The specific response mechanism of the fluorescent probe DPM to pH is as follows Figure 1 shown.

[0074] Example 2: This example provides a test of the selectivity and anti-interference ability of the fluorescent probe prepared in Example 1 in response to various analytes at different pH values.

[0075] First, test solutions (PBS buffer / ethanol, v / v=9:1) with different pH values ​​(6.0, 7.6, 9.0) were prepared. Then, 1 mM HS - 、HSO3 -、NO3 - 、SCN - 、CO3 2- 、F - Br - , I - , ClO - 、SO4 2- Mg 2+ 、Fe 3+ , Ca 2+ 、Al 3+ 、Cu 2+ 、Zn 2+ , Hcy (homocysteine), cysteine ​​(Cys), H2O2, glutathione (GSH). Fluorescence detection was performed after 60 seconds of reaction.

[0076] The results are as follows Figure 2 As shown, under the same pH conditions, no I 485 / I 608 The significant change in pH indicates that the fluorescent probe DPM has high stability in pH determination, that is, the fluorescent probe prepared by the present invention has strong anti-interference ability and has the potential to detect pH values ​​in complex physiological environments.

[0077] Example 3: This example provides a fluorescence performance test of the fluorescent probe prepared in Example 1 at different pH values.

[0078] 2.5 μM of the fluorescent probe DPM prepared in Example 1 of the present invention was added to a 10 mL volumetric flask of a PBS buffer / ethanol (v / v=9:1) solution system with different pH values ​​(6.0, 7.2, 7.4, 7.6, 7.8, 8.0), and fluorescence testing was performed after 20 minutes.

[0079] The results are as follows Figure 3 As shown in (a), as the pH value increases, the fluorescence intensity at 608 nm gradually decreases, and the fluorescence intensity at 485 nm gradually increases; and as shown in Figure 3 As shown in (b), the fluorescence intensity ratio I 485 / I 608 In the pH range of 7.2 to 8.0, a good linear relationship is shown, and the linear equation is y = 3.12006x + 21.84956, R 2 It is 0.9922.

[0080] Example 4: Intracellular fluorescence imaging test

[0081] Experimental procedure: (1) Set the laser confocal microscope to an excitation wavelength of 405 nm, a blue channel collection wavelength of 440-525 nm, and a red channel collection wavelength of 575-700 nm.

[0082] (2) Hela cells were transferred to small glass bottles and incubated for 24 hours before grouping for experiments.

[0083] (3) Hela cells were incubated with 5 μM probe for 30 min, and then incubated in buffers with different pH values ​​(6, 7, 8, 9, 10) for 30 min. The fluorescence emission data were collected under excitation at 405 nm.

[0084] The results are as follows Figure 4 As shown, bright field imaging was first performed to observe the general outline of the cells. Then, blue and red light were used to excite the cells and observe the fluorescence imaging after adding buffers with different pH values. As the pH value increased, the blue fluorescence gradually increased, while the red fluorescence intensity decreased significantly.

[0085] Example 5: Fluorescence imaging test of intracellular targeting

[0086] Experimental procedure: Hela cells were first incubated with 5 μM DPM for 30 min, and then commercial dye Mito-Tracker was added. TM The cells were incubated with deep Red for 30 min, and imaging was performed using a laser confocal microscope.

[0087] The results are as follows Figure 5 As shown, at this time, blue light (λ ex =405nm) to excite the blue light, which is the fluorescence emitted by the DPM probe in response to pH. ex =644nm) for excitation, red light can be observed, which is the red light emitted by the commercial dye mitochondrial red. It can be processed with confocal microscope software to obtain a colocalization coefficient of DPM in mitochondria of 0.71, indicating that the fluorescent probe DPM prepared by the present invention can target mitochondria.

Claims

1. A pH ratio fluorescent probe based on the FRET mechanism, characterized in that: The fluorescent probe has a structure shown in Formula I: Formula I.

2. The pH ratio fluorescent probe based on the FRET mechanism as claimed in claim 1, wherein The fluorescent probe is composed of an energy donor coumarin derivative fluorophore, a connecting group piperazine and an energy acceptor ( E )-3-(2-hydroxyethyl)-1,1-dimethyl-2-(4-methylphenyl)-1H-benz[e]indol-3-ium bromide is composed of three parts.

3. A method for preparing a pH ratio fluorescent probe based on the FRET mechanism, characterized in that: The following steps are involved: S1, mixing 7-(diethylamino)-2-oxo-2H-benzopyran-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine in an organic solvent for reaction to obtain compound 1; S2, heating 2-bromoethane-1-ol and 1,1,2-trimethyl-1H-benzo[e]indole to reflux to obtain compound 2; S3. Dissolve piperazine, compound 1, and compound 2 in an organic solvent and heat them for reaction to obtain the fluorescent probe DPM.

4. The preparation method according to claim 3, wherein In step S1, the molar ratio of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 3-6:3-6:4-8:1-3.

5. The preparation method according to claim 4, wherein The molar ratio of 7-(diethylamino)-2-oxo-2H-chromene-3-carboxylic acid, 4-(piperazin-1-yl)benzaldehyde, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 5:5:6:

2.

6. The preparation method according to claim 3, wherein In step S1, the organic solvent is dichloromethane; the temperature of the mixing reaction is room temperature, and the time is 15 to 20 hours.

7. The preparation method according to claim 6, wherein In step S1, the mixing reaction time is 18.5 hours.

8. The preparation method according to claim 3, wherein In step S2, the molar ratio of 2-bromoethane-1-ol to 1,1,2-trimethyl-1H-benzo[e]indole is 4-8:3-7.

9. The preparation method according to claim 3, wherein In step S2, the molar ratio of 2-bromoethane-1-ol and 1,1,2-trimethyl-1H-benzo[e]indole is 6:5; The heating reflux reaction temperature is 60-100° C. and the time is 6-10 h.

10. The preparation method according to claim 3, wherein In step S2, the heating reflux reaction temperature is 80° C. and the time is 8 h.

11. The preparation method according to claim 3, wherein In step S3, the molar ratio of piperazine, compound 1 and compound 2 is 0.02-0.06:0.8-1.2:0.8-1.

2.

12. The preparation method according to claim 11, characterized in that In step S3, the molar ratio of piperazine, compound 1 and compound 2 is 0.04:1:

1.

13. The preparation method according to claim 3, wherein In step S3, the organic solvent is ethanol; the heating reaction temperature is 60-100° C., and the time is 6-10 h.

14. The preparation method according to claim 13, wherein In step S3, the heating reaction temperature is 80° C. and the time is 8 h.

15. Use of the pH ratio fluorescent probe based on the FRET mechanism according to any one of claims 1 to 2 in the preparation of a pH sensor.

16. The use according to claim 15, characterized in that The probe is used as a pH sensor in cell imaging.

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