Pyridine-based water-soluble dual-response fluorescent probe and preparation method and application thereof

Abstract

The present invention discloses a pyridine-based water-soluble dual-response fluorescent probe and a preparation method and application thereof, and relates to the technical field of fluorescent probes. The technical scheme is as follows: the pyridine-based water-soluble dual-response fluorescent probe has the following structure:. The present invention uses acetyl as the recognition site of carboxylesterase and terminal benzyl pinacol ester as the reaction site of hypochlorite to prepare an on-off and ratio fluorescent probe that can simultaneously respond to hypochlorite and carboxylesterase; in addition, the pyridinium salt provides an effective mitochondrial targeting site, which enables the probe to detect mitochondrial hypochlorite and carboxylesterase in living cells.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a pyridine-based water-soluble dual-response fluorescent probe and a preparation method and application thereof. Background Art

[0002] Hypochlorite is mainly produced by the reaction of hydrogen peroxide and chloride ions in mitochondria. As one of the most important reactive oxygen species, hypochlorite is involved in a variety of physiological processes, and its excessive presence is associated with many diseases. Carboxylesterases (CEs), as a serine hydrolase, also play an important role in the metabolism of various ester and amide molecules. However, carboxylesterase dysfunction may lead to atherosclerosis, cholesterol-induced liver damage, and type II diabetes. Studies have shown that carboxylesterases are mainly present in the endoplasmic reticulum and mitochondria. Mitochondria not only play an important role in cellular physiological processes, but also have a close relationship between changes in their structure and function and diseases. Therefore, the effective detection of hypochlorite and carboxylesterase in living cells, especially in subcellular organelles such as mitochondria, is of great significance for understanding their physiological and pathological effects.

[0003] Recently, fluorescent probes have attracted extensive attention due to their advantages such as high sensitivity, instantaneous response and easy operation. In this regard, many one-to-one probes for single fluorescence detection of hypochlorite and carboxylesterase have been developed using various fluorophores (such as coumarin, fluorescein, rhodamine, cyanine, boron-dipyrromethene and 1,8-naphthalimide). However, fluorescent probes that can simultaneously identify hypochlorite and carboxylesterase have not been reported. In comparison, this one-to-two fluorescent probe has more application prospects and lower cost than the one-to-one type of fluorescent probe. Although some hypochlorite probes targeting mitochondria have been developed, probes for detecting mitochondrial carboxylesterase are still rare. Poor water solubility is also a problem for most of the current chlorate / carboxylesterase probes. Therefore, there is still a great demand for designing water-soluble mitochondrial-targeted probes that can simultaneously detect hypochlorite and carboxylesterase. Summary of the invention

[0004] The technical problem to be solved by the present invention is: to overcome the shortcomings of the prior art, to provide a pyridine-based water-soluble dual-response fluorescent probe and a preparation method and application thereof, to use an acetyl group as a recognition site for carboxylesterase and a terminal benzyl pinacol ester as a reaction site for hypochlorite, to prepare an on-off and ratio fluorescent probe capable of simultaneously responding to hypochlorite and carboxylesterase; in addition, the pyridinium salt provides an effective mitochondrial targeting site, which enables the probe to detect mitochondrial hypochlorite and carboxylesterase in living cells.

[0005] The technical solution of the present invention is:

[0006] In a first aspect, the present invention provides a pyridine-based water-soluble dual-response fluorescent probe having the following structure:

[0007] .

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned pyridine-based water-soluble dual-response fluorescent probe, comprising the following steps:

[0009] S1: adding 4-methylpyridine to an organic solution of p-hydroxybenzaldehyde to obtain a mixture;

[0010] S2 After the mixture is refluxed and stirred for reaction, the solvent is evaporated to obtain a crude product; the crude product is dissolved in CH2Cl2, washed and dried; the solvent is removed in vacuum, and recrystallized to obtain compound A;

[0011] S3: dissolving compound A in an organic solvent and reacting with 4-bromomethylphenylboronic acid pinacol ester at room temperature;

[0012] After the S4 reaction was completed, the precipitate was filtered and dried in vacuum, and the product was recrystallized to obtain a pyridine-based water-soluble dual-responsive fluorescent probe.

[0013] Preferably, in step S1, the solvent of the organic solution is acetic anhydride.

[0014] Preferably, in step S1, the molar ratio of 4-methylpyridine to p-hydroxybenzaldehyde is 1:(0.6-1).

[0015] Preferably, in step S2, the reflux stirring reaction time is 20-25h.

[0016] Preferably, in step S2, washing is performed with a saturated aqueous solution of NaHCO3 and then drying is performed with anhydrous Na2SO4.

[0017] Preferably, in step S2, compound A is obtained by recrystallization from a mixture of ethanol and water in a volume ratio of 7:3.

[0018] Preferably, in step S3, the organic solvent is acetonitrile, and the molar ratio of compound A to 4-bromomethylphenylboronic acid pinacol ester is 1:(1-1.2).

[0019] Preferably, in step S3, the reaction time is 3-5 h.

[0020] In a third aspect, the present invention provides an application of the above-mentioned pyridine-based water-soluble dual-responsive fluorescent probe, wherein the pyridine-based water-soluble dual-responsive fluorescent probe is used as a hypochlorite and carboxylesterase fluorescent probe for mitochondrial fluorescence imaging in living cells.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention prepares a pyridine-based water-soluble dual-responsive fluorescent probe through a condensation reaction. The raw materials are readily available, and the synthesis and post-treatment methods are simple. The purification yield of the prepared pyridine-based water-soluble dual-responsive fluorescent probe can reach more than 68%. The pyridine-based water-soluble dual-responsive fluorescent probe prepared by the present invention exhibits high fluorescence recognition performance for hypochlorite and carboxylesterase among a variety of common anions and cations, amino acids and biological enzyme species. The PBS buffer solution of the fluorescent probe is titrated with different concentrations of hypochlorite and carboxylesterase. 460 There was a good linear correlation in the detection range of 0-110 μM, and the detection limit was 65 nM; 510 / F 460 The detection limit of carboxylesterase was 1.1×10 -6 U / mL. Therefore, the pyridine-based water-soluble dual-response fluorescent probe prepared by the present invention can be used for the quantitative detection of hypochlorite and carboxylesterase.

[0023] 2. The pyridinyl-based water-soluble dual-response fluorescent probe prepared by the present invention shows strong blue emission in pure PBS buffer solution due to weak intermolecular charge transfer (ICT) effect. In the presence of carboxylesterase, both acetate and 4-bromomethylphenylboronic acid pinacol ester are hydrolyzed, and the ICT effect is enhanced, resulting in strong green emission. However, for hypochlorite, its addition leads to the elimination of 4-bromomethylphenylboronic acid pinacol ester, but greatly quenches the fluorescence of the probe. At this point, the probe can be used as an on-off and ratio fluorescence probe for hypochlorite and carboxylesterase, respectively. In addition, the pyridinium salt provides an effective mitochondrial targeting site, which enables the synthesized probe to detect mitochondrial hypochlorite and carboxylesterase in living cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 of the present invention is 1 H NMR spectrum.

[0025] Figure 2 The pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 of the present invention is 13 C NMR spectrum.

[0026] Figure 3 This is the mass spectrometry of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 of the present invention.

[0027] Figure 4It is a fluorescence spectrum and competition bar graph of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 of the present invention titrating hypochlorite and carboxylesterase at different concentrations; wherein, Figure 4 (a) is the fluorescence response of the pyridine-based water-soluble dual-response fluorescent probe (10 μM, 10 mM PBS, pH=7.4) prepared in Example 1 to carboxylesterase (0-0.035 U / mL); Figure 4 (b) is a competition bar graph of the pyridine-based water-soluble dual-response fluorescent probe (10 μM) prepared in Example 1 in the presence of different analytes and the addition of carboxylesterase (0.015 U / mL); Figure 4 (c) is the fluorescence response of the pyridine-based water-soluble dual-response fluorescent probe (10 μM, 10 mM PBS, pH=7.4) prepared in Example 1 to hypochlorite (0-150 μM); Figure 4 (d) is a competition bar graph of the pyridine-based water-soluble dual-response fluorescent probe (10 μM) prepared in Example 1 in the presence of different analytes and the addition of hypochlorite (150 μM); the excitation wavelength is 360 nm.

[0028] Figure 5 The pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 of the present invention was added with 0.02 U / mL of CEs and 150 μM of ClO in a PBS buffer solution (10 mM, pH = 7.4). - The fluorescence response time is 360 nm.

[0029] Figure 6 This is a co-stained fluorescence imaging image of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 and the commercial mitochondrial localization dye Mito-TrackerGreen in C6 cells; wherein Figure 6 (a) is a bright field image; Figure 6 (b) is the green channel image; Figure 6 (c) is the image of the red channel; Figure 6 (d) is the image after the green channel and the red channel are superimposed; Figure 6 (e) is the image after the bright field, green channel and red channel are superimposed; Figure 6 (f) is a scatter plot of fluorescence intensity of green channel and red channel; Figure 6 (g) is the intensity distribution diagram of the linear region of the red channel and the green channel.

[0030] Figure 7 is a fluorescence imaging diagram of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 with hypochlorite and carboxylesterase in C6 cells; 10 μM of the above probe was incubated with C6 cells at 37° C. for 30 min, wherein Figure 7 (a) is a bright field image of C6 cells; Figure 7 (b) is the image of the blue channel; Figure 7 (c) is the green channel image; Figure 7 (d) is the image after the blue channel and the green channel are superimposed; Figure 7 (e) is the image after the bright field, blue channel and green channel are superimposed; C6 cells were pre-incubated with BNPP100μM at 37℃ for 30min, and then incubated with 10μM of the above probe at 37℃ for 30min. Figure 7 (f) is a bright field image of C6 cells; Figure 7 (g) is the image of the blue channel; Figure 7 (h) is the green channel image; Figure 7 (i) is the image after the blue channel and the green channel are superimposed; Figure 7 (j) is the image after the bright field, blue channel and green channel are superimposed; C6 cells were incubated with 10μM of the above probe at 37℃ for 30min, and then incubated with 50μM ClO- for 30min, and then incubated at 37℃ for another 30min. Figure 7 (k) is a bright field image of C6 cells; Figure 7 (l) is the image of the blue channel; Figure 7 (m) is the green channel image; Figure 7 (n) is the image after the blue channel and the green channel are superimposed; Figure 7 (o) is an image obtained by superimposing the bright field, blue channel, and green channel. C6 cells were incubated with 10 µM of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 at 37°C for 30 min, and then incubated with 2 µg / mL of LPS at 37°C for 30 min. Figure 7 (p) is a bright field image of C6 cells; Figure 7 (q) is the image of the blue channel; Figure 7 (r) is the green channel image; Figure 7 (s) is the image after the blue channel and the green channel are superimposed; Figure 7 (t) is the image after the bright field, blue channel and green channel are superimposed.

[0031] Figure 8 is a fluorescence imaging diagram of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 with hypochlorite and carboxylesterase in zebrafish; 10 μM of the above probe was incubated with zebrafish for 30 minutes, wherein Figure 8 (a) is a bright field image; Figure 8 (b) is the image of the blue channel; Figure 8 (c) is the green channel image; Figure 8 (d) is the image after the blue channel and the green channel are superimposed; Figure 8 (e) is the image after the bright field, blue channel and green channel are superimposed; Figure 8(f) is a bright field image of zebrafish; Figure 8 (g) is the image of the blue channel; Figure 8 (h) is the green channel image; Figure 8 (i) is the image after the blue channel and the green channel are superimposed; Figure 8 (j) is the image after the bright field, blue channel and green channel are superimposed; zebrafish were pre-incubated with 100 µM BNPP at 37°C for 30 min, and then incubated with 10 µM of the above probe at 37°C for 30 min. Figure 8 (k) is a bright field image of a zebrafish; Figure 8 (l) is the image of the blue channel; Figure 8 (m) is the green channel image; Figure 8 (n) is the image after the blue channel and the green channel are superimposed; Figure 8 (o) is the image after the bright field, blue channel and green channel are superimposed; zebrafish were incubated with 10 μM of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 at 37°C for 30 min, and then incubated with 50 μM ClO - Incubate for 30 minutes. Figure 8 (p) is a bright field image of zebrafish; Figure 8 (q) is the image of the blue channel; Figure 8 (r) is the green channel image; Figure 8 (s) is the image after the blue channel and the green channel are superimposed; Figure 8 (t) is an image obtained by superimposing the bright field, blue channel, and green channel. After the zebrafish was incubated with 10 µM of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 at 37°C for 30 min, it was then incubated with 2 µg / mL of LPS at 37°C for 30 min. Figure 8 (u) is a bright field image of zebrafish; Figure 8 (v) is the image of the blue channel; Figure 8 (w) is the green channel image; Figure 8 (x) is the image after the blue channel and the green channel are superimposed; Figure 8 (y) is the image after the bright field, blue channel and green channel are superimposed. DETAILED DESCRIPTION

[0032] In order to enable persons skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0033] Example 1

[0034] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of this embodiment comprises the following steps:

[0035] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 5 g (41 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0036] After the S2 mixture was stirred under reflux for 24 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 32%);

[0037] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.24 g (4.2 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 3 h;

[0038] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum, and the product purification yield was 75%, obtaining a pyridine-based water-soluble dual-responsive fluorescent probe.

[0039] The synthesis steps of the pyridine-based water-soluble dual-response fluorescent probe of this embodiment are as follows:

[0040] .

[0041] The prepared pyridine-based water-soluble dual-response fluorescent probe was analyzed by nuclear magnetic resonance, and the results are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 9.02 (dd, J =39.8, 6.4 Hz, 2H), 8.22 (dd, J =45.1, 5.4 Hz, 2H), 8.05 (d, J =16.3 Hz, 2H), 7.83 (dd, J =13.8, 8.3 Hz, 2H),7.78–7.65 (m, 1H), 7.59–7.42 (m, 2H), 7.40–7.23 (m, 1H), 7.17–6.91 (m, 2H),5.78 (d, J =21.9 Hz, 1H), 5.05 (dd, J =23.5, 5.9 Hz, 2H), 2.35–2.19 (m, 3H), 1.18(d, J =86.6 Hz, 12H). The specific H NMR spectrum is as follows Figure 1 shown.

[0042] 13 C NMR (101 MHz, DMSO- d 6) δ: 169.51, 153.80, 152.51, 144.96, 144.61,140.80, 138.06, 135.59, 133.25, 129.89, 129.46, 128.49, 124.73, 123.82,123.13, 122.10, 84.37, 74.00, 62.67, 21.36. The specific NMR carbon spectrum is as follows: Figure 2 shown.

[0043] Mass spectrum ESI-MS: m / z=456.2363 forC 28 H 31 BBrNO4[M-Br] + (calc. 456.23). The specific mass spectrum is as follows Figure 3 shown.

[0044] Example 2

[0045] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of this embodiment comprises the following steps:

[0046] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 6 g (50 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0047] After the S2 mixture was stirred under reflux for 20 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 28%);

[0048] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.36 g (4.62 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 4 h;

[0049] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum, and the product purification yield was 70%, obtaining a pyridine-based water-soluble dual-responsive fluorescent probe.

[0050] Example 3

[0051] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of this embodiment comprises the following steps:

[0052] S1: Add 6 mL (62 mmol) of 4-methylpyridine to 7.6 g (62 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0053] After the S2 mixture was stirred under reflux for 25 h, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 25%);

[0054] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.49 g (5.04 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 5 h;

[0055] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum, and the product purification yield was 68%, obtaining a pyridine-based water-soluble dual-responsive fluorescent probe.

[0056] Comparative Example 1

[0057] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of Comparative Example 1 comprises the following steps:

[0058] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 3.8 g (31 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0059] After the S2 mixture was stirred under reflux for 24 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 20%);

[0060] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.24 g (4.2 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 3 h;

[0061] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum. The product was purified with a yield of 63%, and a pyridine-based water-soluble dual-responsive fluorescent probe was obtained.

[0062] Comparative Example 2

[0063] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of Comparative Example 2 comprises the following steps:

[0064] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 8.3 g (68 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0065] After the S2 mixture was stirred under reflux for 24 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 21%);

[0066] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.24 g (4.2 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 3 h;

[0067] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum. The product was purified with a yield of 60%, and a pyridine-based water-soluble dual-responsive fluorescent probe was obtained.

[0068] Comparative Example 3

[0069] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of Comparative Example 3 comprises the following steps:

[0070] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 5 g (41 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0071] After the S2 mixture was stirred under reflux for 24 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 32%);

[0072] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.11 g (3.8 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 3 h;

[0073] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum. The product was purified with a yield of 65%, and a pyridine-based water-soluble dual-responsive fluorescent probe was obtained.

[0074] Comparative Example 4

[0075] The preparation method of the pyridine-based water-soluble dual-response fluorescent probe of Comparative Example 4 comprises the following steps:

[0076] S1 Add 6 mL (62 mmol) of 4-methylpyridine to 5 g (41 mmol) of acetic anhydride solution (20 mL) of p-hydroxybenzaldehyde to obtain a mixture;

[0077] After the S2 mixture was stirred under reflux for 24 hours, the solvent was evaporated to obtain a crude product; the crude product was dissolved in CH2Cl2, washed twice with a saturated aqueous solution of NaHCO3, and then dried over anhydrous Na2SO4; finally, the solvent was removed in vacuo, and recrystallized from a mixture of ethanol and water in a volume ratio of 7:3 to obtain compound A (yield: 32%);

[0078] S3: 1 g (4.2 mmol) of compound A was dissolved in acetonitrile and reacted with 1.61 g (5.46 mmol) of 4-bromomethylphenylboronic acid pinacol ester at room temperature for 3 h;

[0079] After the S4 reaction was completed, the precipitate was filtered and dried in a vacuum. The product was purified with a yield of 60%, and a pyridine-based water-soluble dual-responsive fluorescent probe was obtained.

[0080] It can be seen from Comparative Examples 1-4 that when preparing a pyridine-based water-soluble dual-response fluorescent probe, the amount of raw material added has a great influence on the yield of the reaction product. Only when the amount of raw material added is appropriate can a high yield of the reaction product be guaranteed.

[0081] The optical properties of hypochlorite and carboxylesterase were measured for the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1:

[0082] The fluorescence response of the pyridine-based water-soluble dual-response fluorescent probe (10 μM, 10 mM PBS, pH = 7.4) to CEs (0-0.035 U / mL) prepared in Example 1 is as follows: Figure 4 (a) shows the F 510 / F 460 and fluorescence intensity ratio of CEs concentration (0-0.018U / mL). Figure 4 (b) shows the fluorescence response of the pyridine-based water-soluble dual-responsive fluorescent probe upon the addition of different analytes, including CEs, amino acids, enzymes, cations, and anions; and the competition experiment of the pyridine-based water-soluble dual-responsive fluorescent probe (10 μM) with the addition of CEs (0.015 U / mL) in the presence of different analytes. Figure 4 (c) Shows the pyridine-based water-soluble dual-responsive fluorescent probe (10 μM, 10 mM PBS, pH = 7.4) for ClO - (0-150 μM) fluorescence response, the figure shows F 460 and ClO - Fluorescence intensity ratio of the concentrations (0-110 μM). Figure 4(d) shows the fluorescence response of the pyridine-based water-soluble dual-responsive fluorescent probe upon addition of different analytes, including ClO - , amino acids, reactive oxygen species, cations and anions; and the probe (10 μM) was added with ClO in the presence of different analytes - (150 μM) competition experiment, the excitation wavelength is 360 nm. Figure 4 It can be seen that the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 is sensitive to CEs and ClO in these biologically relevant analytes. - It has remarkable specificity, with a detection limit of 65nM for hypochlorite and 1.1×10 -6 U / mL. Therefore, the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 can be used for the quantitative detection of hypochlorite and carboxylesterase.

[0083] Through Figure 5 From the fluorescence response time shown, it can be seen that after the pyridyl-based water-soluble dual-response fluorescent probe prepared in Example 1 was added with hypochlorite and carboxylesterase, its fluorescence intensity at 460nm changed significantly, reaching stability at 30s and 20min, respectively. It is worth noting that after the addition of hypochlorite, the fluorescence intensity of the probe at 460nm rapidly weakened within 30s, which is better than most known probes; at the same time, the fluorescence intensity remained constant for a long period of time. These results show that the pyridyl-based water-soluble dual-response fluorescent probe prepared in Example 1 can be used as a useful tool for the rapid detection of hypochlorite and carboxylesterase.

[0084] The pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 was used to detect hypochlorite and carboxylesterase in cells. The operation steps are as follows:

[0085] In C6 cells, the co-staining fluorescence imaging of the pyridine-based water-soluble dual-response fluorescent probe and the commercial mitochondrial localization dye Mito-TrackerGreen was performed. Figure 6 As shown by Figure 6 It can be seen that the green fluorescence signal of the pyridine-based water-soluble dual-responsive fluorescent probe can overlap well with the red fluorescence signal of the mitochondrial red fluorescent dye, and the calculated Pearson's correlation coefficient is 0.81, which indicates that the pyridine-based water-soluble dual-responsive fluorescent probe prepared in Example 1 can better target mitochondria.

[0086] In C6 cells, the fluorescence imaging of the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 with hypochlorite and carboxylesterase is shown in FIG. Figure 7 As shown. Figure 7As can be seen from (b) and (c), when the pyridine-based water-soluble dual-response fluorescent probe was added to C6 cells, the blue and green channels showed strong fluorescence. However, after the addition of hypochlorite, the fluorescence of the blue channel was significantly weakened (e.g. Figure 7 (l)); after adding carboxylesterase inhibitor, the green channel fluorescence also weakened significantly (as shown in Figure 7 (h)). Combined with the excellent mitochondrial targeting ability of the pyridine-based water-soluble dual-response fluorescent probe in Example 1, the endogenous hypochlorite stimulator lipopolysaccharide was used to stimulate cells to produce endogenous hypochlorite, and the blue channel fluorescence was also significantly weakened (as shown in Figure 7 (q)). Therefore, it is believed that the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 can efficiently monitor the changes of carboxylesterase in cell mitochondria through ratiometric fluorescence signals, as well as detect the concentration changes of endogenous and exogenous hypochlorite.

[0087] The pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 was used to detect hypochlorite and carboxylesterase in zebrafish, and the operating steps were as follows:

[0088] In zebrafish, a pyridyl-based water-soluble dual-responsive fluorescent probe was used for fluorescence imaging of hypochlorite and carboxylesterase. Figure 8 As shown. Figure 8 As can be seen from (b) and (c), when the pyridine-based water-soluble dual-response fluorescent probe is added to zebrafish, strong fluorescence appears in the green channel and weak fluorescence appears in the blue channel. After the carboxylesterase inhibitor is added, the fluorescence of the green channel is significantly weakened, while the fluorescence of the blue channel is significantly enhanced (e.g. Figure 8 (l)). The obvious color change in zebrafish imaging indicates that the pyridyl-based water-soluble dual-response fluorescent probe prepared in Example 1 has tissue permeability. Comparison of the significant changes in the dual-channel fluorescence color before and after carboxylesterase incubation indicates that the pyridyl-based water-soluble dual-response fluorescent probe prepared in Example 1 can be used for ratiometric detection of carboxylesterase in zebrafish. After exogenous hypochlorite was added to the zebrafish to which the pyridyl-based water-soluble dual-response fluorescent probe was added, the blue channel fluorescence was significantly weakened (as shown in Figure 2). Figure 8 (q)); Under the same conditions, the endogenous hypochlorite stimulant was added to stimulate the production of endogenous hypochlorite in zebrafish imaging (as shown in Figure 8 (v)), the blue channel fluorescence is consistent with the channel color change when exogenous hypochlorite is added. This shows that the pyridine-based water-soluble dual-response fluorescent probe prepared in Example 1 can be used for endogenous and exogenous ClO in zebrafish. - Imaging experiments.

Claims

1. A water-soluble dual-response fluorescent probe based on pyridine, characterized in that: It has the following structure: 。 2. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 1, characterized in that: The following steps are involved: S1: adding 4-methylpyridine to an organic solution of p-hydroxybenzaldehyde to obtain a mixture; S2: After the mixture is refluxed and stirred for reaction, the solvent is evaporated to obtain a crude product; the crude product is dissolved in CH2Cl2, washed and dried; the solvent is removed in a vacuum, and recrystallized to obtain compound A. The structural formula of compound A is as follows: ； S3: dissolving compound A in an organic solvent and reacting with 4-bromomethylphenylboronic acid pinacol ester at room temperature; S4: After the reaction is completed, the precipitate is filtered and dried in vacuum, and the product is recrystallized to obtain a pyridine-based water-soluble dual-responsive fluorescent probe.

3. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S1, the solvent of the organic solution is acetic anhydride.

4. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S1, the molar ratio of 4-methylpyridine to p-hydroxybenzaldehyde is 1:(0.6-1).

5. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S2, the reflux stirring reaction time is 20-25h.

6. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S2, the product is washed with a saturated aqueous solution of NaHCO3 and then dried over anhydrous Na2SO4.

7. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S2, compound A is obtained by recrystallization from a mixture of ethanol and water in a volume ratio of 7:

3.

8. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S3, the organic solvent is acetonitrile, and the molar ratio of compound A to 4-bromomethylphenylboronic acid pinacol ester is 1:(1-1.2).

9. The method for preparing a pyridine-based water-soluble dual-response fluorescent probe according to claim 2, characterized in that: In step S3, the reaction time is 3-5h.

Images