Fluorescent probe for cell imaging and its synthesis method and application

Abstract

The present invention discloses a fluorescent probe for cell imaging, its synthesis method and application. The fluorescent probe is a compound with the structure shown in formula (I); in the formula, the groups R1, R2, and R3 are each independently hydrogen, a substituted or unsubstituted alkyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heteroaryl group; the group X is O, S, or NH; the group Y is O, S, or N; the group Z is O, S, or N; the group Ar is a substituted or unsubstituted aryl group or heteroaryl group. The fluorescent probe of the present invention can be used for targeted mitochondrial fluorescence imaging and also has potential applications in imaging other organelles. #imgabs0#

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent labeling, and in particular to a fluorescent probe for cell imaging and a synthesis method and application thereof. Background Art

[0002] As a subcellular organelle, mitochondria play a vital role in many cellular processes, such as metabolism and apoptosis. As the energy factory of cells, mitochondria mainly provide ATP required for life through oxidative phosphorylation. In addition, the intrinsic pathway of apoptosis is mainly triggered by the release of activated caspase proteins by cytochrome C, which is the result of changes in the permeability of the mitochondrial outer membrane. Because many cellular activities are closely related to mitochondria, many disease pathologies related to mitochondrial metabolic disorders have been discovered in recent decades, and mitochondria have also become key therapeutic targets. For example, inhibiting mitochondrial respiration may limit the energy supply to cancer cells. Therefore, the localization and distribution of mitochondria in cells are of great significance.

[0003] Fluorescent probes are a type of fluorescent molecule that has characteristic fluorescence in the ultraviolet-visible-near infrared region, and whose fluorescence properties (excitation and emission wavelengths, intensity, lifetime, polarization, etc.) can change sensitively with the properties of the environment, such as polarity, refractive index, viscosity, etc. Fluorescent probes are usually composed of three parts: a fluorescent group, a recognition group, and a linking group. The fluorescent group and the recognition group are assembled into a probe through a linking group, and the change in fluorescence signal is achieved by the specific reaction of the recognition group with the substance being measured.

[0004] In recent years, fluorescent probes have played a vital role in scientific research and industrial applications. Since fluorescent probes can detect extremely low concentrations of substances while maintaining high selectivity and specificity, they can be used to study the internal mechanisms of cell molecules and develop new molecular probes and bioimaging technologies. They can also provide accurate detection and analysis in drug analysis, environmental analysis, and food safety testing. In short, as an efficient analytical tool, fluorescent probes not only play an irreplaceable role in scientific research, but also provide strong technical support in the industrial and medical fields.

[0005] Most of the commercially available mitochondrial targeting probes are lipophilic and cationic, and their accumulation in the mitochondrial matrix and the resulting toxicity are one of the main drawbacks. The self-quenching phenomenon and spectral overlap caused by the narrow Stokes shift also limit the practical application of fluorescent probes (for example, the Stokes shifts of rhodamine 123 and JC-1 are both less than 30 nm).

[0006] In recent years, fluorescent probes for detecting various analytes have been designed one after another, and a wide variety of fluorophores have been used. Among them, 2-(2′-hydroxyphenyl)benzothiazole has attracted widespread attention due to its strong photostability, large Stokes shift and high quantum yield. However, the shortcomings of existing 2-phenylbenzothiazoles are that their emission and absorption wavelengths are short. The absorption and emission wavelengths of unsubstituted 2-phenylbenzothiazoles are lower than 365nm. In cell imaging, short-wavelength emission probes cause great damage to cells. In addition, most 2-phenylbenzothiazoles use the excited state intramolecular proton transfer (ESIPT) process to display fluorescence. The disadvantage is that the ESIPT process in protic solvents is easily affected by hydrogen bonds between solute-solvent molecules. Its proton transfer ability in aqueous solution is relatively weak, which is not conducive to detection in living cells.

[0007] Therefore, it is of great significance to develop a class of small molecule fluorescent probes that can be easily synthesized and targeted to mitochondria. Summary of the invention

[0008] The present invention provides a fluorescent probe for cell imaging, a synthesis method and application thereof. The fluorescent probe of the present invention can target mitochondrial fluorescence imaging and has potential application in imaging of other cell organelles.

[0009] The technical solution of the present invention is as follows:

[0010] A fluorescent probe for cell imaging, wherein the fluorescent probe is a compound having a structure shown in formula (I):

[0011] ;

[0012] In the formula, R1, R2, and R3 groups are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

[0013] The X group is O, S or NH;

[0014] The Y group is O, S or N;

[0015] The Z group is O, S or N;

[0016] The Ar group is a substituted or unsubstituted aryl or heteroaryl group.

[0017] Preferably, R1 and R2 groups are each independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;

[0018] R3 is .

[0019] Preferably, the X group is O;

[0020] The Y group is N;

[0021] The Z group is O, S or N.

[0022] Preferably, the Ar group is selected from:

[0023] .

[0024] Preferably, the fluorescent probe is a compound having a structure represented by formula (I-1), formula (I-2) or formula (I-3):

[0025] .

[0026] The invention also provides application of the fluorescent probe in fluorescent microscopic imaging of mitochondria in living cells.

[0027] The fluorescent probe is a compound with a structure shown in formula (I-1), with an excitation wavelength of 453 nm and an emission wavelength of 524 nm.

[0028] The fluorescent probe is a compound with a structure shown in formula (I-2), with an excitation wavelength of 466 nm and an emission wavelength of 616 nm.

[0029] The fluorescent probe is a compound with a structure shown in formula (I-3), with an excitation wavelength of 440 nm and an emission wavelength of 495 nm.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The method of the invention can significantly red-shift the absorption and emission wavelengths of the original 2-phenylbenzoxazole.

[0032] The method of the invention has the advantages of simple and readily available raw materials, mild reaction conditions, wide applicability of substrates, etc., and meets the requirements for developing green and environmentally friendly chemistry.

[0033] The small molecule fluorescent probe of the present invention has a larger Stokes shift than traditional fluorescent dyes (such as fluorescein, rhodamine, and anthocyanin). The emission wavelength of the excitation can be significantly changed by selecting different aromatic cyclomethylamines, which is of great significance to small molecule fluorescent dyes. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the H NMR spectrum of the fluorescent probe Dye-1 prepared in Example 1 of the present invention;

[0035] Figure 2 is the C NMR spectrum of the fluorescent probe Dye-1 prepared in Example 1 of the present invention;

[0036] Figure 3 is the H NMR spectrum of the fluorescent probe Dye-2 prepared in Example 2 of the present invention;

[0037] Figure 4 is the C NMR spectrum of the fluorescent probe Dye-2 prepared in Example 2 of the present invention;

[0038] Figure 5 is the H NMR spectrum of the fluorescent probe Dye-3 prepared in Example 3 of the present invention;

[0039] Figure 6 is the C NMR spectrum of the fluorescent probe Dye-3 prepared in Example 3 of the present invention;

[0040] Figure 7 Graph showing the maximum absorption wavelength and maximum emission wavelength of the fluorescent probe Dye-1 prepared in Example 1 of the present invention;

[0041] Figure 8 Graph showing the maximum absorption wavelength and maximum emission wavelength of the fluorescent probe Dye-2 prepared in Example 2 of the present invention;

[0042] Fig. 9 Graph showing the maximum absorption wavelength and maximum emission wavelength of the fluorescent probe Dye-3 prepared in Example 3 of the present invention;

[0043] Fig.10 This is the confocal image of the fluorescent probe Dye-1 prepared in Example 1 of the present invention on MDA-MB-231 cells. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be pointed out that the embodiments described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0045] Example 1

[0046]

[0047] In a 20 mL reaction bottle equipped with a magnetic stirrer, first add S1 (0.2 mmol) and sodium periodate (1.0 mmol), and dissolve in 3 mL of water and 1 mL of acetonitrile. After full dissolution, add S2-1 (1.0 mmol). If there is viscous liquid adhering to the wall, add 2 mL of acetonitrile. Stir the reaction at room temperature for 3 h. After the reaction is completed, stop stirring, extract with ethyl acetate, and perform crude separation by column chromatography (petroleum ether: ethyl acetate = 3:1). Then, separate the pale yellow solid Dye-1 (6 mg, 5%) using a preparative plate (dichloromethane: ethyl acetate = 10:1). The H NMR and C NMR spectra of Dye-1 are shown as follows: Figure 1 and Figure 2 shown.

[0048] 1 H NMR (400 MHz, Chloroform- d ) δ 8.24 – 8.17 (m, 2H), 7.78 – 7.71 (m,2H), 7.65 (d, J = 8.3 Hz, 2H), 7.55 (d, J = 8.1 Hz, 2H), 7.33 (s, 1H), 6.59 (s,1H), 6.11 (s, 1H), 5.47 (d, J = 6.9 Hz, 1H), 4.56 (d, J = 5.1 Hz, 2H), 4.50 –4.44 (m, 1H), 3.70 (s, 3H), 3.21 (d, J = 14.6 Hz, 1H), 3.04 (dd, J = 14.7, 9.2Hz, 1H), 1.40 (s, 9H).

[0049] Example 2

[0050]

[0051] In a 20 mL reaction bottle equipped with a magnetic stirrer, first add S1 (0.2 mmol) and sodium periodate (0.8 mmol), and dissolve in 3 mL of water and 1 mL of acetonitrile. After full dissolution, add S2-2 (0.8 mmol). If there is viscous liquid adhering to the wall, add 2 mL of acetonitrile. Stir the reaction at room temperature for 3 h. After the reaction is completed, stop stirring, extract with ethyl acetate, and perform crude separation by column chromatography (petroleum ether: ethyl acetate = 3:1). Then, separate the pale yellow solid Dye-2 (15.4 mg, 11%) using a preparative plate (dichloromethane: ethyl acetate = 10:1). The H NMR and C NMR spectra of Dye-2 are shown as follows: Figure 3 and Figure 4 shown.

[0052] 1 H NMR (400 MHz, Chloroform- d ) δ 8.27 – 8.20 (m, 2H), 7.93 – 7.87 (m,2H), 7.86 – 7.77 (m, 6H), 7.64 – 7.44 (m, 8H), 7.35 (s, 1H), 6.70 (s, 1H), 5.94 (s, 1H), 5.47 (d,J = 6.9 Hz, 1H), 4.60 (d, J = 4.9 Hz, 2H), 4.54 (td, J =7.4, 4.2 Hz, 1H), 3.70 (s, 3H), 3.24 (d, J = 14.6 Hz, 1H), 3.08 (dd, J = 14.7,8.7 Hz, 1H), 1.41 (s, 9H).

[0053] Example 3

[0054]

[0055] In a 20 mL reaction bottle equipped with a magnetic stirrer, first add S1 (0.2 mmol) and sodium periodate (1.0 mmol), and dissolve in 3 mL of water and 1 mL of acetonitrile. After full dissolution, add S2-3 (1.0 mmol). If there is viscous liquid adhering to the wall, add 2 mL of acetonitrile. Stir the reaction at room temperature for 3 h. After the reaction is completed, stop stirring, extract with ethyl acetate, and perform crude separation by column chromatography (petroleum ether: ethyl acetate = 4:1). Then, separate the pale yellow solid Dye-3 (21.5 mg, 20%) using a preparative plate (dichloromethane: ethyl acetate = 15:1). The H NMR spectrum and C NMR spectrum of Dye-3 are shown as follows: Figure 5 and Figure 6 shown.

[0056] 1 H NMR (400 MHz, Chloroform- d ) δ 8.11 – 8.04 (m, 2H), 7.59 – 7.55 (m,2H), 7.50 – 7.46 (m, 2H), 7.39 (d, J = 8.2 Hz, 2H), 7.29 (s, 1H), 6.64 (s, 1H), 5.70 (s, 1H), 5.44 (d, J = 7.0 Hz, 1H), 4.54 – 4.43 (m, 3H), 3.68 (s, 3H), 3.21 (s, 2H), 3.06 (s, 2H), 1.40 (s, 9H).

[0057] Performance Testing

[0058] The excitation wavelength of Dye-1 detected in acetonitrile was 453 nm and the emission wavelength was 524 nm (e.g. Figure 7 The excitation wavelength of Dye-2 detected in acetonitrile is 466 nm, and the emission wavelength is 616 nm (as shown in Figure 8 The excitation wavelength of Dye-3 detected in acetonitrile is 440 nm, and the emission wavelength is 495 nm (as shown in Fig. 9 As shown). In addition to these three examples, different aromatic methylamines can be used to obtain fluorescent probes with different wavelengths. The emission wavelength is used as the horizontal axis and the fluorescence intensity is used as the vertical axis to obtain a wavelength-fluorescence intensity correlation spectrum.

[0059] Fluorescent probe Dye-1 and commercial organelle probes were used to perform co-localization tests on MDA-MB-231 cells. The steps included:

[0060] (1) Preparation: A dimethyl sulfoxide solution with a concentration of 200 μM for the fluorescent probe Dye-1 (referred to as storage culture medium); a dimethyl sulfoxide solution with a concentration of 200 μM for the mitochondrial deep red fluorescent probe (Mito-Tracker Deep Red FM).

[0061] (2) Cell culture: The revived MDA-MB-231 cells were cultured in a medium containing 10% bovine embryonic serum, 1% double antibody, and 89% DMEM at 37°C and 5% CO2 for 24 h. The MDA-MB-231 cells cultured in the above medium for 24 h were placed in a confocal dish and cultured in a medium containing 10% bovine embryonic serum, 1% double antibody, and 89% DMEM at 37°C and 5% CO2 for another 24 h. The inoculation size was 3×10 5 cell / mL, for future use.

[0062] (3) Take 2 μL of Mito-Tracker Deep Red FM at a concentration of 200 μM and add it to 1 mL of culture medium to prepare a 400 nM probe working solution. Incubate the above cells at 37°C, 5% CO2 for 20 min. Discard the culture medium and wash with PBS 2 to 3 times. Take 2 μL of Dye-1 storage culture medium and add it to 1 mL of culture medium to prepare a 400 μM probe working solution. Incubate at 37°C, 5% CO2 for another 1 h and discard the culture medium. Wash with PBS 2 to 3 times. Add 1 mL of culture medium and perform fluorescence imaging under a confocal microscope. The commercial mitochondrial probe is the red channel, with an excitation wavelength of 640 nm and a collection wavelength of 665 nm. The Dye-1 probe is the green channel, with an excitation wavelength of 405 nm and a collection wavelength of 524 nm.

[0063] Fluorescent probe imaging of mitochondria in MDA-MB-231 cells Fig.10 As shown: (a) is a picture of Dye-1 being able to image mitochondria in MDA-MB-231 cells; (b) is a picture of commercial deep red mitochondrial dye being able to image mitochondria in MDA-MB-231 cells; (c) is a superposition of the first two signal channels. This shows that Dye-1 can accurately locate mitochondria in cells, and linear mitochondria in MDA-MB-231 cells are clearly visible.

[0064] The embodiments described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A fluorescent probe for cell imaging, characterized in that: The fluorescent probe is a compound having a structure shown in formula (I-1), formula (I-2) or formula (I-3): 。 2. Use of a compound of formula (I-1), formula (I-2) or formula (I-3) as claimed in claim 1 in the preparation of a fluorescent probe for mitochondrial fluorescence microscopy imaging in living cells.

3. The use according to claim 2, characterized in that: The compound of the structure shown in formula (I-1) has an excitation wavelength of 453 nm and an emission wavelength of 524 nm; 。 4. The use according to claim 2, characterized in that: The compound of the structure shown in formula (I-2) has an excitation wavelength of 466 nm and an emission wavelength of 616 nm; 。 5. The use according to claim 2, characterized in that: The compound of the structure shown in formula (I-3) has an excitation wavelength of 440 nm and an emission wavelength of 495 nm; 。

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