pH fluorescent probe containing carbazole-merocyanine structure and its preparation and application

Abstract

The present invention discloses a pH fluorescent probe containing a carbazole-merocyanine structure and a preparation and application thereof, and relates to the technical field of pH fluorescent probes. The present invention provides a novel pH fluorescent probe, which exhibits good cell permeability, non-toxicity and excellent anti-photobleaching performance, and can change fluorescence intensity in response to local environmental changes. The probe can be used not only to detect solution pH, but also to simultaneously monitor mitochondria and lysosomes to reflect the state of mitochondrial autophagy, and respond to environmental changes according to the state of organelles in mitochondrial autophagy (the fluorescence intensity in mitochondria decreases, and the fluorescence intensity in lysosomes increases).

Description

Technical field:

[0001] The invention relates to the technical field of pH fluorescent probes, and in particular to a pH fluorescent probe containing a carbazole-merocyanine structure and preparation and application thereof. Background technology:

[0002] As we all know, pH is one of the most important parameters reflecting the acid-base balance state in a system. Controlling pH is a key factor in many chemical and biological processes and has important application value in environmental monitoring, chemistry and biology.

[0003] Intracellular pH plays an important role in regulating and maintaining a variety of biological processes such as cell growth, apoptosis, autophagy, and homeostasis. Abnormal changes in intracellular pH, as a cell health indicator, are often associated with various functional disorders and diseases such as cancer, stroke, and Alzheimer's disease. Therefore, measuring intracellular pH with high sensitivity and selectivity has become an extremely important scientific research in cell function, physiology, and pathology.

[0004] Detection techniques such as microelectrodes, nuclear magnetic resonance, magnetic resonance spectroscopy, molecular spectroscopy, and chemical exchange saturation transfer have been widely used to monitor biological pH. However, the development of low-cost, simple to operate, real-time, highly sensitive, and selective monitoring systems for intracellular pH levels remains a strong demand. Fluorescent probes combined with their imaging are considered important detection systems because of their high spatiotemporal resolution and are a useful tool. It remains a complex issue in the design and construction of useful fluorescent probes for solution and intracellular pH monitoring. Summary of the invention:

[0005] The technical problem to be solved by the present invention is to provide a fluorescent probe and a preparation method and application thereof. The fluorescent probe molecule has the advantages of simple structure, easy synthesis, high sensitivity, good optical stability and low cytotoxicity. Under pH induction, it itself changes from salt type to electrical neutrality. Molecules with different charge states have different fluorescence emission spectra. Different fluorescence can be emitted in the solution to distinguish acid and alkaline solutions. In the cell, mitochondria and lysosomes can be targeted separately according to different pH values ​​to observe the intracellular pH changes in real time through confocal imaging.

[0006] The technical problem to be solved by the present invention is achieved by adopting the following technical solutions:

[0007] One of the purposes of the present invention is to provide a pH fluorescent probe, referred to as CZ-OH, the structural formula of the pH fluorescent probe is as follows:

[0008]

[0009] The second object of the present invention is to provide a method for preparing the pH fluorescent probe, firstly, 1,1,2-trimethyl-1H-benzo[e]indole is reacted with 2-iodoethanol to synthesize an intermediate, and then the intermediate is reacted with N-ethylcarbazole-3-carboxaldehyde to synthesize the pH fluorescent probe.

[0010] The reaction equation is as follows:

[0011]

[0012] The third object of the present invention is to provide the application of the pH fluorescent probe in visual real-time detection of solution pH.

[0013] The fourth object of the present invention is to provide the application of the pH fluorescent probe in visualizing and real-time monitoring of pH changes in cell lysosomes and mitochondria.

[0014] The pH fluorescent probe skeleton of the present invention contains two intramolecular charge transfer (ICT) emission peaks, of which carbazole (emission ~435nm) and merocyanine (emission ~655nm) are used to stain mitochondria and lysosomes. In PBS buffer at pH = 7.4, the fluorescent probe can emit blue fluorescence of carbazole itself. Under acidic conditions, the conjugated system in the fluorescent probe skeleton can easily bind to H + The reaction causes the molecular fluorescence to red shift, emitting red fluorescence.

[0015] When the probe is in an acidic environment, the probe undergoes a proton-triggered ring-opening process; as the alkalinity increases, the C=N double bond undergoes nucleophilic addition to the hydroxyl group, and the CZ-OH ring is closed. The reaction mechanism is shown below:

[0016]

[0017] Therefore, the present invention proposes a new double labeling strategy, which can distinguish acid and base solutions through fluorescence changes in acid and base solutions, and the fluorescent probe changes the spectral behavior of the environment in the mitochondrial and lysosomal organelles, and locates mitochondria and lysosomes by emitting fluorescence of different colors. When the fluorescent probe is used to label mitochondria, the emission peak wavelength is 435nm under 405nm wavelength excitation, presenting blue imaging; when the fluorescent probe is used to label lysosomes, the emission peak wavelength is 655nm under 528nm wavelength excitation, presenting red imaging.

[0018] The beneficial effects of the present invention are as follows: the present invention provides a novel pH fluorescent probe, which exhibits good cell permeability, non-toxicity and excellent anti-photobleaching performance, and can change fluorescence intensity in response to local environmental changes. It can be used not only to detect solution pH, but also to simultaneously monitor mitochondria and lysosomes to reflect the state of mitochondrial autophagy, and respond to environmental changes according to the state of organelles in mitochondrial autophagy (the fluorescence intensity in mitochondria decreases, and the fluorescence intensity in lysosomes increases). Description of the drawings:

[0019] Figure 1 The fluorescent probe CZ-OH of the present invention 1 H NMR spectrum;

[0020] Figure 2 The fluorescent probe CZ-OH of the present invention 13 C NMR spectrum;

[0021] Figure 3 is the HRMS spectrum of the fluorescent probe CZ-OH of the present invention;

[0022] Figure 4 The ultraviolet absorption spectra of the fluorescent probe CZ-OH of the present invention in acidic, neutral and alkaline media;

[0023] Figure 5 The fluorescence titration spectra of the fluorescent probe CZ-OH of the present invention in solutions with different pH values ​​under 527nm excitation;

[0024] Figure 6 The fluorescence titration spectra of the fluorescent probe CZ-OH of the present invention in solutions with different pH values ​​under 370nm excitation;

[0025] Figure 7 This is a fluorescence intensity diagram of the fluorescent probe CZ-OH of the present invention in solutions with different pH values;

[0026] Figure 8 The fluorescence intensity of the fluorescent probe CZ-OH of the present invention when different analytes are present in different buffer solutions; the black filling is the selectivity under 370nm excitation, and the gray filling is the selectivity under 527nm excitation;

[0027] Fig. 9 This is a graph showing the survival rate of HeLa cells under the action of different concentrations (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, and 10 μM) of the fluorescent probe CZ-OH of the present invention;

[0028] Fig.10 This is a confocal fluorescence imaging image of HeLa cells mitochondria co-stained with the fluorescent probe CZ-OH (2 μM) of the present invention and a commercial mitochondrial dye (1 μM);

[0029] Fig.11 This is a confocal fluorescence imaging image of HeLa cells lysosomes co-stained with the fluorescent probe CZ-OH (2 μM) of the present invention and a commercial lysosomal dye (200 nM);

[0030] Fig.12 This is a confocal fluorescence imaging diagram of the pH change of the fluorescent probe CZ-OH of the present invention in the intracellular lysosomal channel;

[0031] Fig.13 This is a confocal fluorescence imaging diagram of the pH change of the fluorescent probe CZ-OH of the present invention in the mitochondrial channel in the cell. Specific implementation method:

[0032] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0033] The present invention provides a pH fluorescent probe containing a carbazole-merocyanine structure, referred to as CZ-OH, and the structural formula of the pH fluorescent probe is as follows:

[0034]

[0035] The invention provides a method for preparing the pH fluorescent probe, which comprises firstly reacting 1,1,2-trimethyl-1H-benzo[e]indole with 2-iodoethanol to synthesize an intermediate, and then reacting the intermediate with N-ethylcarbazole-3-carboxaldehyde to synthesize the pH fluorescent probe.

[0036] The reaction equation is as follows:

[0037]

[0038] Preferably, the molar ratio of 1,1,2-trimethyl-1H-benzo[e]indole to 2-iodoethanol is 1:(1.1-1.2). An appropriate excess of 2-iodoethanol can allow 1,1,2-trimethyl-1H-benzo[e]indole to participate in the reaction as much as possible, thereby increasing the conversion rate of 1,1,2-trimethyl-1H-benzo[e]indole and further increasing the yield of the intermediate.

[0039] Preferably, the 1,1,2-trimethyl-1H-benzo[e]indole reacts with 2-iodoethanol at room temperature. Of course, the reaction can also be carried out under heating conditions.

[0040] Preferably, the molar ratio of the intermediate to N-ethylcarbazole-3-carboxaldehyde is 1:(1.1-1.2). An appropriate excess of N-ethylcarbazole-3-carboxaldehyde can allow the intermediate to participate in the reaction as much as possible, thereby increasing the conversion rate of the intermediate and further increasing the yield of CZ-OH.

[0041] Preferably, the intermediate reacts with N-ethylcarbazole-3-carboxaldehyde under reflux, and piperidine is added as a catalyst. Of course, the reaction temperature can be lower than the boiling point of the solvent used, and other catalysts can also be used.

[0042] The present invention also provides application of the pH fluorescent probe in visual real-time detection of solution pH.

[0043] The present invention also provides the application of the pH fluorescent probe in visually monitoring pH changes in cell lysosomes and mitochondria in real time.

[0044] The technical solution of the present invention is described in detail below through specific embodiments:

[0045] Example 1: Synthesis of CZ-OH

[0046] Step 1: Dissolve 1,1,2-trimethyl-1H-benzo[e]indole (1 g, 4.78 mmol) in 30 mL of acetone, then add 2-iodoethanol (0.447 mL, 5.73 mmol), stir at room temperature for 8 h, filter, and obtain intermediate 1.

[0047] Step 2: Dissolve the intermediate 1 (0.5 g, 2.08 mmol) obtained in step 1 in 20 mL of acetonitrile, then add N-ethylcarbazole-3-carboxaldehyde (0.5582 g, 2.5 mmol), and then drop two drops of piperidine. Reflux and stir to react for 8 h, and column chromatography to obtain CZ-OH.

[0048] 1H NMR(400MHz,Chloroform-d)δ8.20(s,1H),8.11(d,J=7.7Hz,1H),8.04(d,J=8.5Hz,1H),7.8 2(d,J=8.2Hz,1H),7.74(d,J=8.6Hz,1H),7.65(d,J=9.6Hz,1H),7.43(t,J=25.1,7.7Hz,4H), 7.30(t,J=7.5Hz,1H),7.18–7.09(m,2H),6.39(d,J=15.8Hz,1H),4.42–4.33(m,2H),3.89(d, J=5.2Hz,2H),3.68–3.55(m,2H),1.84(s,3H),1.55(s,1H),1.46–1.41(m,6H),1.25(s,1H). Such as Figure 1 shown.

[0049] 13 C NMR (101 MHz, Chloroform-d) δ 140.47, 133.51, 130.88, 130.47, 129.47, 129.10, 126.32, 125.95, 124.70, 123.36, 123.06, 122.79, 122.72, 122.30, 120.61, 119.14, 119.10, 114.12, 110.70, 108.72, 108.66, 63.48, 50.22, 49.73, 37.75, 26.58, 21.69, 13.90. Figure 2 shown.

[0050] In positive ion mode, the peak of CZ-OH [M+H] + found, m / z = 458.272 and the actual value m / z = 458.273 also match. Figure 3 shown.

[0051] Example 2: Spectral testing of CZ-OH in solvents with different pH values

[0052] In order to explore the UV and fluorescence changes of CZ-OH in solvents at different pH values, CZ-OH was dissolved in DMSO to prepare a 2 mM mother solution, and the UV and fluorescence spectra of CZ-OH (10 μM) in aqueous solutions at different pH values ​​were tested.

[0053] In acidic solution, the absorbance of CZ-OH increases at 500nm; in alkaline solution, the absorbance increases at 300nm, such as Figure 4 As shown. Under 527nm excitation, the red fluorescence decreases with the increase of pH, as shown Figure 5As shown. Under 370nm wavelength excitation, the blue fluorescence increases with the increase of pH, as shown Figure 6 shown. Figure 7 The dots in the figure are the fluorescence titration spectra under 527nm excitation, and the square dots are the fluorescence titration spectra under 370nm excitation. The above results show that the fluorescent probe CZ-OH of the present invention can effectively detect the pH of the solution and can be used as a pH detector.

[0054] In addition, the present invention further investigated the response of CZ-OH to other biologically related species, and CZ-OH (2 μM) was added to the following ion solutions (100 μM): Ca 2+ , Ser, K + ;,Cys,DNA,BSA,Lys,Zn 2+ Mg 2+ , GSH, ClO - 、H2O、H2S、RNA、O2、O 2- or Na2SO3, detect the fluorescence intensity, the result is as follows Figure 8 The results show that the fluorescent probe CZ-OH of the present invention has no response to the above biologically relevant species, indicating that it is not affected by other species.

[0055] Example 3: Cytotoxicity test

[0056] The cytotoxicity of CZ-OH was tested by MTT (5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide) method. Various concentrations (0 μM, 2 μM, 4 μM, 6 μM, 8 μM, 10 μM) of CZ-OH were added to living HeLa cells and tested after incubation for 24 hours. The results are shown in Figure 2. Fig. 9 The results show that the fluorescent probe CZ-OH of the present invention has low biological toxicity and can be used for biological imaging applications.

[0057] Example 4: Labeling of mitochondria

[0058] In order to study the cell mitochondrial localization performance of CZ-OH, the present invention uses the commercial mitochondrial dye MTDR and CZ-OH to perform co-localization studies in HeLa cells. The results are as follows Fig.10 As shown. The results show that the fluorescence images of the blue channel of CZ-OH and MTDR (λem=660-670nm, λex=644nm) overlap well. This proves that the fluorescent probe CZ-OH of the present invention can be used to label mitochondria.

[0059] Example 5: Lysosome labeling

[0060] In the case of mitochondrial autophagy, the number of lysosomes will increase to maintain homeostasis in the cell. The present invention uses PMA to treat cells, uses CZ-OH to label red particles, and uses commercial lysosomal dye LTDR and CZ-OH to co-stain cells to detect red particles. The results are as follows Fig.11 As shown. The results show that the red channel of CZ-OH and the fluorescence image of LTDR (λem=585-595nm, λex=577nm) overlap well. This proves that the fluorescent probe CZ-OH of the present invention can be used to label lysosomes.

[0061] In summary, the fluorescent probe CZ-OH of the present invention can stain mitochondria and lysosomes with blue and red respectively.

[0062] Example 6: pH detection of CZ-OH on cell mitochondria

[0063] In order to further explore the pH detection ability of CZ-OH on cell mitochondria and lysosomes, the present invention co-incubated CZ-OH with HeLa cells whose intracellular pH was homogenized by nigericin and high potassium ion buffer solution. Fig.12 and Fig.13 As shown. The results show that after CZ-OH was co-incubated with HeLa cells for 20 minutes, the red fluorescence of channel I (λem=600-650nm, λex=488nm) increased as the intracellular pH decreased; the blue fluorescence of channel II (λem=600-650nm, λex=405nm) decreased as the intracellular pH decreased. This proves that the fluorescent probe CZ-OH of the present invention is suitable for intracellular pH detection.

[0064] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A pH fluorescent probe, characterized in that: The structural formula of the pH fluorescent probe is as follows:

2. The method for preparing the pH fluorescent probe according to claim 1, characterized in that: First, 1,1,2-trimethyl-1H-benzo[e]indole reacts with 2-iodoethanol to synthesize an intermediate, and then the intermediate reacts with N-ethylcarbazole-3-carboxaldehyde to synthesize the pH fluorescent probe; The reaction equation is as follows:

3. The preparation method according to claim 2, characterized in that: The molar ratio of the 1,1,2-trimethyl-1H-benzo[e]indole to 2-iodoethanol is 1:(1.1-1.2).

4. The preparation method according to claim 2, characterized in that: The 1,1,2-trimethyl-1H-benzo[e]indole is reacted with 2-iodoethanol at room temperature.

5. The preparation method according to claim 2, characterized in that: The molar ratio of the intermediate to N-ethylcarbazole-3-carboxaldehyde is 1:(1.1-1.2).

6. The preparation method according to claim 2, characterized in that: The intermediate reacts with N-ethylcarbazole-3-carboxaldehyde under reflux, and piperidine is added as a catalyst.

7. Use of the pH fluorescent probe according to claim 1 in preparing a reagent for visualizing and real-time detecting solution pH.

8. Use of the pH fluorescent probe according to claim 1 in preparing a reagent for visualizing and real-time monitoring of pH changes in cell lysosomes and mitochondria.

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