Preparation method and application of a mitochondria-targeted biothiol near-infrared ratio fluorescent probe

Abstract

The present application relates to the field of optical imaging, in particular to a preparation method and application of a biological thiol near-infrared ratio fluorescent probe targeting mitochondria. The present application synthesizes a red solid fluorescent probe, (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide, through a series of chemical reactions based on 4-(diethylamino)salicylaldehyde and salicylaldehyde. The fluorescent probe not only has a simple synthesis route, strong operability, easily available raw materials and low cost, but also can detect biological thiols in living cells in a ratio, has high sensitivity, good selectivity and large stokes shift, and can target mitochondria for imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical imaging, in particular to a preparation method and application of a biological thiol near-infrared ratio fluorescent probe targeting mitochondria. BACKGROUND

[0002] Small molecule biological thiols, such as cysteine (Cys), glutathione (GSH) and homocysteine (Hcy), play a key role in maintaining normal physiological functions. However, abnormal levels of these biological thiols in the body can cause adverse reactions and serve as biomarkers for many diseases. For example, Cys concentration below normal levels can cause edema, liver damage, skin damage, lethargy, growth retardation and weakness, etc. Elevated Cys or Hcy concentration is associated with diseases such as Parkinson's disease and Alzheimer's disease. Abnormal GSH levels can lead to cancer, AIDS and neurodegenerative diseases, etc. Therefore, it is of great significance to develop effective biological thiol detection methods.

[0003] According to relevant literature reports, chromene derivatives have good biological thiol detection sites. However, most of the reported fluorescent probes exhibit single-wavelength "on" or "off" response, which is easily disturbed by probe distribution, instrument efficiency and environmental conditions, etc. Ratio fluorescent probes can eliminate the influence of the above factors and provide more accurate analysis by using the intensity ratio of two fluorescent emission peaks for self-calibration. Mitochondria, as the center of metabolic activity and a potential target for cancer treatment, have attracted increasing attention, and the development of mitochondrial-targeting fluorescent probes has become a research hotspot, but there are few reports on such fluorescent probes. Near-infrared fluorescent probes can effectively improve tissue penetration, reduce photodamage and background interference, and have significant advantages in biological fluorescence imaging. Large Stokes shift can effectively reduce the overlap between the absorption spectrum and the fluorescence spectrum, thereby largely eliminating self-absorption and minimizing autofluorescence interference, which can effectively improve sensitivity. However, there is no biological thiol fluorescent probe with the advantages of ratio detection, mitochondrial targeting, near-infrared emission and large Stokes shift. Therefore, it is of great significance to design and synthesize a new type of biological thiol fluorescent probe with chromene derivatives as recognition groups. SUMMARY

[0004] In view of the above problems, the present application provides a new near-infrared ratio fluorescent probe for recognizing biological thiols and targeting mitochondria. The fluorescent probe has a short response time, can realize ratio detection of biological thiols, has extremely high selectivity and sensitivity, and has a large Stokes shift. In addition, it can also be targeted to mitochondria, thereby realizing imaging analysis at a specific location.

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

[0006] A mitochondria-targeting biothiol near-infrared ratio fluorescent probe is (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide, and a structural formula is as follows:

[0007]

[0008] A preparation method of a mitochondria-targeting biothiol near-infrared ratio fluorescent probe comprises the following steps:

[0009] Under argon protection, (E)-7-diethylamino-3-(4-pyridyl)vinyl coumarin (compound 2) and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one (compound 4) are dissolved in acetonitrile, heated to reflux and stirred to react until the raw material is completely reacted, the reaction liquid is cooled to room temperature, solid is precipitated with ethyl acetate, filtered and washed with ethyl acetate, and the solid crude product is collected to obtain (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide (compound 1) through silica gel column chromatography.

[0010]

[0011] Further, the molar ratio of the (E)-7-diethylamino-3-(4-pyridyl)vinyl coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one is 1:1.2-2.0.

[0012] Still further, the stirring reaction temperature is 90-100°C, and the stirring reaction time is 3-14h.

[0013] Still further, the reaction until the raw material is completely reacted is monitored by TLC.

[0014] The route realizes green and efficient synthesis of the mitochondria-targeting biothiol near-infrared ratio fluorescent probe (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide.

[0015] The application of a mitochondria-targeting biothiol near-infrared ratio fluorescent probe is applied to ratio detection of biothiols, and to intracellular biothiol and mitochondria-targeting imaging.

[0016] Compared with the prior art, the application has the following advantages:

[0017] 1. The preparation method of the fluorescent probe provided by the application is simple, easy to operate, and has low cost.

[0018] 2. The fluorescent probe provided by the application can detect biothiols in a ratio, has extremely high sensitivity and selectivity, and has a large Stokes shift.

[0019] 3. The fluorescent probe provided by the application has good biocompatibility and can be applied to intracellular biothiol imaging.

[0020] 4. The application utilizes the characteristics of the negative membrane potential of mitochondria, designs a positively charged fluorescent probe, and realizes targeted mitochondria imaging through electrostatic attraction.

[0021] The fluorescent probe provided by the application realizes detection of biothiols through the change of the ratio (F 541nm / F 650nm ) of the fluorescent intensity at two wavelengths, 542 nm and 650 nm, and overcomes the characteristics of low accuracy of single-wavelength detection in the prior art. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the probe of the application.

[0023] Figure 2 is the nuclear magnetic resonance carbon spectrum of the probe of the application.

[0024] Figure 3 is the ultraviolet and fluorescence spectrum of the fluorescent probe of the application before and after reaction with biothiols.

[0025] Figure 4 is the ultraviolet spectrum of the fluorescent probe of the application for detecting 30 μM Cys with time change.

[0026] Figure 5 is the fluorescence spectrum of the fluorescent probe of the application for detecting 30 μM Cys with time change.

[0027] Figure 6 is the fluorescence ratio (F 541nm / F 650nm ) change graph of the fluorescent probe of the application for different Cys concentration titration.

[0028] Figure 7is the fluorescence ratio (F 541nm / F 650nm ) change chart of the fluorescent probe of the present application after adding biothiols and different amino acids.

[0029] Figure 8 is the imaging of the fluorescent probe of the present application to the biothiols in living cells.

[0030] Figure 9 is the imaging of the fluorescent probe of the present application targeted to mitochondria. DETAILED DESCRIPTION

[0031] Example 1: Specific synthesis process of the fluorescent probe of the present application

[0032] (1) The specific synthesis steps of compound 2 are as follows:

[0033]

[0034] Compound 9 (19.33 g, 100 mmol), diethyl malonate (19.22 g, 120 mmol) and piperidine (1 mL) were dissolved in anhydrous ethanol (100 mL), heated to 102°C and stirred for 14 h. After the reaction was completed, the solvent was evaporated, and glacial acetic acid (80 mL) and concentrated hydrochloric acid (80 mL) were added. The reaction was stirred at 116°C for 14 h. After the reaction was completed, the reaction liquid was poured into ice water (300 mL), and the pH was adjusted to about 5.0 with 40% sodium hydroxide solution. A large amount of solid was precipitated, and stirring was carried out for 30 min. The crude product was obtained by filtration under reduced pressure, and the solid product compound 8 (yield 75%) was obtained by recrystallization with toluene, which was 7-diethylaminocoumarin.

[0035]

[0036] Under argon protection, anhydrous N,N-dimethylformamide (14 mL) was cooled to 0°C in an ice bath, and phosphorus oxychloride (14 mL) was slowly added to it under ice bath. Then 7-diethylaminocoumarin (compound 8) (10.86 g, 50 mmol) was dissolved in N,N-dimethylformamide (20 mL) and slowly added to the above reaction liquid. After the addition was completed, the reaction was stirred at 60°C for 14 h. After the reaction was completed, the reaction liquid was poured into ice water (300 mL), and the pH was adjusted to precipitate a large amount of solid with 20% sodium hydroxide solution. The crude product was obtained by filtration under reduced pressure, and the solid product compound 3 (yield 50%) was obtained by recrystallization with anhydrous ethanol, which was 7-diethylaminocoumarin-3-carboxaldehyde.

[0037]

[0038] Compound 2 (yield 20%) was obtained as a solid product, i.e. (E)-7-diethylamino-3-(4-pyridyl)vinylcoumarin, by column chromatography (ethyl acetate / dichloromethane = 1:1) of the crude product obtained by dissolving 7-diethylaminocoumarin-3-carbaldehyde (compound 3) (2.46 g, 10 mmol) and 4-methylpyridine (1.40 g, 15 mmol) in anhydrous N,N-dimethylformamide (20 mL) and adding p-toluenesulfonic acid monohydrate (3.81 g, 20 mmol) and heating to 140 °C to reflux for 8 h, after which the reaction was poured into ice water (150 mL) and stirred for 30 min, after which a solid precipitated, which was filtered under reduced pressure to obtain the crude product, which was separated by column chromatography (ethyl acetate / dichloromethane = 1:1) to obtain the solid product, compound 2 (yield 20%).

[0039] (2) The specific synthesis steps of compound 4 are as follows:

[0040]

[0041] Compound 6 (yield 40%) was obtained as a solid product, i.e. 2-hydroxy-5- hydroxymethylbenzaldehyde, by column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) of the crude product obtained by dissolving salicylaldehyde (compound 7) (5.0 mL, 50 mmol) in concentrated hydrochloric acid (50 mL) under argon protection, then adding a 37% mass fraction aqueous formaldehyde solution (4 mL), stirring the mixture at room temperature for 24 h, filtering after the reaction was completed, washing the solid with ultrapure water, collecting the solid, dissolving it in dimethyl sulfoxide (DMSO, 30 mL) and ultrapure water (15 mL), then adding copper sulfate pentahydrate (12.48 g, 50 mmol), heating the mixture to 110 °C and stirring for 3 h, cooling to room temperature after the reaction was completed, extracting with dichloromethane, washing the combined organic phases with brine, drying over anhydrous sodium sulfate, and concentrating the filtrate to obtain the crude product, which was separated by column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) to obtain the solid product, compound 6 (yield 40%).

[0042]

[0043] Compound 5 (yield 26%) was obtained as a solid product, i.e. 7-(hydroxymethyl)-3,3a- dihydrocyclopenta[b]chromen-1(2H)-one, by column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) of the crude product obtained by dissolving 2-hydroxy-5- hydroxymethylbenzaldehyde (compound 6) (1.52 g, 10 mmol), 2-cyclopenten-1-one (1.23 g, 15 mmol) and imidazole (1.02 g, 15 mmol) in tetrahydrofuran (THF, 5 mL) and ultrapure water (5 mL) under argon protection, stirring the mixture at room temperature for 72 h, extracting with dichloromethane after the reaction was completed, washing the combined organic phases with ultrapure water and brine, drying over anhydrous sodium sulfate, and concentrating the filtrate to obtain the crude product, which was separated by column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) to obtain the solid product, compound 5 (yield 26%).

[0044]

[0045] Under argon protection, 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one (compound 5) (432.4 mg, 2 mmol) and triphenylphosphine (1.05 g, 4 mmol) were dissolved in dichloromethane (DCM, 20 mL), and then N-bromosuccinimide (NBS, 711.9 mg, 4 mmol) was added. The mixture was stirred at room temperature for 2 h, and then washed with water and brine successively, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:5~1:4) to obtain a solid product, compound 4 (yield 33%), 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one.

[0046] Example 2: The specific synthesis steps of the fluorescent probe are as follows:

[0047]

[0048] Under argon protection, (E)-7-diethylamino-3-(4-pyridyl)vinylcoumarin (compound 2) (128.2 mg, 0.4 mmol) and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one (compound 4) (139.6 mg, 0.5 mmol) were dissolved in acetonitrile (5 mL), heated to 90°C and stirred for 3 h, and then cooled to room temperature. The solid was precipitated with ethyl acetate, filtered and washed with ethyl acetate, and the solid crude product was collected and separated by silica gel column chromatography (dichloromethane / methanol = 20:1) to obtain a solid product, compound 1 (yield 88%), (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide. 1HNMR (400 MHz, d6-DMSO, ppm) δ 8.96 (d, J = 6.8 Hz, 2H), 8.24 (s, 1H), 8.19 (d, J = 6.8 Hz, 2H), 7.87 (d, J = 15.6 Hz, 1H), 7.70 (d, J = 16.0 Hz, 1H), 7.60 (d, J = 1.6 Hz, 1H), 7.56 (d, J = 8.8 Hz, 1H), 7.51 (dd, J = 1.6 Hz, J = 8.4 Hz, 1H), 7.21 (d, J = 1.6 Hz, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.81 (dd, J = 2.0 Hz, J = 8.8 Hz, 1H), 6.61 (d, J = 1.6 Hz, 1H), 5.65 (s, 2H), 5.34-5.31 (m, 1H), 3.51 (q, J = 6.8 Hz, 4H), 2.65-2.58 (m, 1H), 2.46-2.34 (m, 2H), 2.08-1.97 (m, 1H), 1.16 (t, J = 6.8 Hz, 6H). 13 CNMR (100 MHz, d6-DMSO, ppm) δ 200.98, 159.60, 156.38, 155.29, 153.74, 152.05, 143.88, 137.37, 132.96, 132.81, 130.95, 130.84, 128.35, 125.36, 123.63, 122.53, 122.25, 116.93, 113.59, 110.09, 96.24, 75.57, 44.40, 36.66, 27.62, 12.39.

[0049] Example 3: UV and fluorescence spectra of the fluorescent probe of the present application before and after reaction with biological thiols.

[0050] After 30 μM of Cys, GSH or Hcy was added into the solution of the fluorescent probe of the present application (10 μM, PBS:DMSO = 1:4) for 20 min, the UV and fluorescence spectra were determined. The results are shown in Figure 3 .

[0051] As shown in (a) of Figure 3 , the maximum UV absorption peaks of the solution of the fluorescent probe of the present application before and after addition of Cys, GSH or Hcy are located at 508 nm and 448 nm, respectively. From (b) of Figure 3 , it can be found that the change of fluorescence emission spectrum (500 nm excitation, slit: 5 nm / 10 nm) is blue-shifted from 650 nm to 541 nm. It indicates that the fluorescent probe of the present application has reacted with biological thiols (Cys, GSH and Hcy) and can be used for tracing biological thiols.

[0052] Example 4: UV and fluorescence spectra of the fluorescent probe of the present application for detecting Cys over time.

[0053] The UV and fluorescence spectra over time were measured after adding 30 μM Cys to the fluorescent probe solution (10 μM, PBS:DMSO = 1:4) of the present application. The results are shown in Figure 4 and Figure 5 .

[0054] As shown in (a) of Figure 4 , after adding 30 μM Cys to the fluorescent probe solution of the present application, the absorbance at 508 nm rapidly decreased, the absorbance at 448 nm gradually increased, and the color of the solution changed from red to yellow; from (b) of Figure 4 , it can be found that within 20 min, the absorbance at 508 nm reached a minimum, while the absorbance at 448 nm reached a maximum. Meanwhile, the fluorescence spectrum also changed obviously, as shown in (a) of Figure 5 , after adding 30 μM Cys to the fluorescent probe solution of the present application, the fluorescence intensity at 650 nm gradually decreased, and the fluorescence intensity at 541 nm rapidly increased. Under a 365 nm UV lamp, the fluorescence color of the solution changed from red to green; from (b) of Figure 5 , it can be found that within 20 min, the fluorescence ratio (F 541nm / F 650nm ) changed obviously and reached a maximum. It is indicated that the fluorescent probe of the present application can realize the detection of biological thiols through the fluorescence ratio (F 541nm / F 650nm ), and has a faster response speed and a larger Stokes shift (142 nm).

[0055] Example 5: Fluorescence ratio (F 541nm / F 650nm ) change graph of the fluorescent probe of the present application for different Cys concentration titration.

[0056] The fluorescence ratio (F 541nm / F 650nm ) over time was measured after adding different concentrations of Cys to the fluorescent probe solution (10 μM, PBS:DMSO = 1:4) of the present application. The results are shown in Figure 6 .

[0057] As shown in Figure 6 , the fluorescence ratio (F 541nm / F 650nm ) change of the fluorescent probe solution of the present application after adding different concentrations of Cys (500 nm excitation, slit: 5 nm / 10 nm). From (a) of Figure 6 , it can be found that with the increase of Cys concentration, the fluorescence ratio (F 541nm / F650nm ) increased more and faster, and reached the maximum when the Cys concentration was 30 μM; Figure 6 In (b), it can be found that the fluorescence ratio (F 541nm / F 650nm ) has good linearity with Cys in the low concentration range, indicating that the fluorescent probe has high sensitivity.

[0058] Example 6: Fluorescence ratio (F) of the fluorescent probe of the present invention after adding biothiol and different amino acids 541nm / F 650nm )Change graph.

[0059] 30 μM of cysteine ​​(Cys), glutathione (GSH), homocysteine ​​(Hcy), alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), histidine (His), leucine (Leu), lysine (Lys), proline (Pro) and serine (Ser) were added to the fluorescent probe solution of the present invention (10 μM, PBS:DMSO=1:4) and the fluorescence spectrum was measured after 6 minutes. The results are shown in Figure 7 .

[0060] like Figure 7 As shown, only biothiols (Cys, GSH and Hcy) cause the fluorescence ratio (F 541nm / F 650nm ) significantly increased, while other amino acids remained almost unchanged. This indicates that the fluorescent probe of the present invention has good selectivity for biothiols (Cys, GSH, and Hcy) and can specifically recognize biothiols to a certain extent, providing a good foundation for biological imaging.

[0061] Example 7: Imaging of biothiols in living cells using the fluorescent probe of the present invention.

[0062] Take out two confocal dishes containing HeLa cells, directly add 5uM of the fluorescent probe of the present invention to one of the confocal dishes and incubate for 30 minutes before imaging; the other confocal dish is first incubated with 1mM thiol scavenger N-ethylmaleimide (NEM) for 30 minutes, washed with phosphate buffered saline (PBS), and then added with 5uM of the fluorescent probe of the present invention and incubated for 30 minutes before imaging. The excitation wavelength is 458nm, the green window fluorescence collection wavelength is 500-570nm, and the red window fluorescence collection wavelength is 620-690nm. The results are shown in Figure 8 .

[0063] from Figure 8It was found that in cells treated only with the fluorescent probe of the present invention, the green window fluorescence was stronger than the red window fluorescence, and the fluorescence intensity ratio (green window / red window) was larger. In contrast, in cells treated first with NEM and then with the fluorescent probe of the present invention, the green window fluorescence was weaker than the red window fluorescence, and the fluorescence intensity ratio (green window / red window) was smaller. This demonstrates that the fluorescent probe can ratiometrically detect changes in intracellular biothiol concentrations.

[0064] Example 8: Imaging of the fluorescent probe of the present invention targeted to mitochondria.

[0065] Take a confocal dish containing HeLa cells, replace the culture medium with 1 mL of fresh culture medium, incubate with 1 mM thiol scavenger N-ethylmaleimide (NEM) for 30 minutes, wash with phosphate buffered saline (PBS), and then add 5 uM of the fluorescent probe of the present invention and 500 nM of the commercial mitochondrial targeting fluorescent probe Mito-TrackerGreen and incubate for 30 minutes before imaging. The excitation wavelength is 488 nm, the green window fluorescence collection wavelength of the fluorescent probe of the present invention is 620-690 nm, and the red window fluorescence collection wavelength of Mito-TrackerGreen is 495-530 nm. The results are shown in Figure 9 .

[0066] from Figure 9 It can be found that the fluorescent probe of the present invention has good overlap with the commercial mitochondria-targeted fluorescent probe, indicating that the fluorescent probe of the present invention can target mitochondria.

[0067] The above embodiments are preferred implementations of the present invention, but the implementation of the present invention is not limited to the above embodiments. For ordinary technicians in this field, several modifications and improvements can be made without departing from the principles of the present invention, which are all included in the scope of protection of the present invention.

[0068] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A preparation method of a mitochondria-targeted biothiol near-infrared ratio fluorescent probe and its application, characterized in that, The targeting mitochondria biothiol near-infrared ratio fluorescent probe is (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide, and the structural formula is as follows: 。 2. A method for preparing a mitochondria-targeted biothiol near-infrared ratio fluorescent probe, characterized in that, The method comprises the following steps: (E)-7-diethylamino-3-(4-pyridyl)vinyl coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one are dissolved in acetonitrile under the protection of argon, and the reaction is stirred and heated to reflux until the raw material is consumed, then the reaction liquid is cooled to room temperature, solid is precipitated from ethyl acetate, and the solid is filtered and washed with ethyl acetate, and the solid crude product is collected, and (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide is obtained by silica gel column chromatography.

3. The method for preparing a mitochondria-targeted biothiol near-infrared ratio fluorescent probe according to claim 2, characterized in that, The molar ratio of (E)-7-diethylamino-3-(4-pyridyl)vinyl coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one is 1:1.2-2.

0.

4. The method according to claim 2, wherein, The stirring reaction temperature is 90-100 DEG C, and the stirring reaction time is 3-14 h.

5. The method according to claim 2, wherein, The reaction is monitored by TLC until the raw material is consumed. 6.The application of the mitochondria-targeted biothiol near-infrared ratio fluorescent probe according to claim 1, characterized in that, The near-infrared ratio fluorescent probe is applied to prepare a reagent for rapidly detecting biothiols, and is applied to prepare a reagent for imaging biothiols in cells and targeting mitochondria.

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