Chromoazole near-infrared viscosity sensitive probe, preparation method thereof and application of probe in living cell imaging

Abstract

The invention relates to the technical field of biochemical sensing and fluorescence imaging, in particular to a chromazole near-infrared viscosity-sensitive probe, a preparation method thereof and application of the probe in living cell imaging. The preparation method of the probe comprises the following steps: dissolving tryptazole tetrafluoroborate and benzaldehyde or a derivative thereof in acetonitrile, stirring at room temperature to react, evaporating to remove a solvent after the reaction is finished, and separating and purifying through column chromatography to obtain the tryptazole near-infrared viscosity sensitive probe. The application of the probe in living cell nucleus imaging is to mark chromosomes in a cell mitosis process, so that chromosome migration behaviors are visualized. The probe is high in utilization rate, mild in preparation reaction condition, simple and convenient to operate, green and efficient. The cell nucleus of the living cell can be quickly dyed at low concentration, the cytoplasm, the nuclear membrane and the nucleolus of the living cell can be distinguished through the fluorescence brightness, and the mitosis process of the cell can be traced. A feasible strategy is provided for developing a near-infrared probe with a rapid cell nucleus staining function.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry sensing and fluorescence imaging, in particular to a kind of azole near-infrared viscosity sensitive probe and its preparation method and the application of probe in live cell imaging. BACKGROUND

[0002] Although there are many commercialized live cell nucleus staining probes at present, their emission wavelengths are all in the visible light band (less than 700 nm), which will cause non-negligible phototoxicity and biological background signal. Some probes also have non-specific staining, interfere with normal cell function and other defects. Therefore, it is a meaningful work to design and prepare a new type of near-infrared fluorescent probe with emission wavelength greater than 700 nm, which has the ability of rapid staining of live cell nucleus and does not interfere with the normal function of cells by chemical synthesis.

[0003] Aniline and dimethylaminobenzene, which have freely rotatable aromatic ring fragments, will cause the probe molecule to relax in the excited state, i.e. fluorescence is turned off, when the molecule is in a low viscosity environment or the molecular structure is not restricted by free rotation. When the molecule is in a high viscosity environment or the free rotation is restricted, the outer electrons of the probe can normally fall from the excited state to the ground state, and the fluorescence is turned on.

[0004] Azole molecules contain α, β-unsaturated aromatic ring structure. This structure fragment may bind to the small groove near the base in the DNA double helix structure, which is different from the base binding mode of embedding, and does not affect the biological activity of DNA.

[0005] In the present application, azole tetrafluoroborate is used as the skeleton, and rotatable aromatic ring structure is introduced under mild conditions. The prepared azole near-infrared viscosity sensitive probe can distinguish the cytoplasm, nuclear membrane, chromosome and nucleolus of various cells by fluorescence brightness, and observe the physiological process of live cell nucleus such as mitosis by laser confocal microscope. Therefore, the azole viscosity sensitive probe is very promising as a potential cell nucleus staining probe, and its prospect is worth paying attention to. SUMMARY

[0006] In order to solve the problems of existing live cell nucleus staining probes, which are all visible emission wavelength and have non-specific staining, the present application provides a kind of azole near-infrared viscosity sensitive probe and its preparation method and the application of probe in live cell imaging.

[0007] The specific technical solutions are as follows: Firstly, the present application provides an azole near-infrared viscosity sensitive probe, which has the following general structure: , Wherein, R = one of H, NH2 or NO2. The probe is a new substance. When R is NH2, the probe has a wavelength of 710 nm, and the existing cell nucleus probe does not exceed 700 nm. The fluorescence intensity of the probe of the present application is significantly enhanced in a high viscosity environment, and the viscosity sensitivity is high.

[0008] Secondly, the application further provides a preparation method of the probe, comprising the following steps: The azole tetrafluoroborate and benzaldehyde or its derivative are dissolved in acetonitrile solvent, and the reaction is carried out under stirring at room temperature. After the reaction is completed, the solvent is evaporated, and then column chromatography is used for separation and purification to obtain the azole near-infrared viscosity sensitive probe.

[0009] The synthesis of the probe is represented by the following formula: .

[0010] Further, the molar ratio of the azole tetrafluoroborate and benzaldehyde or its derivative is 1:1 to 1:10, and the ratio of the amount of azole tetrafluoroborate to acetonitrile is 1 mmol:5 to 50 mL.

[0011] Further, the eluent used in the separation and purification is a mixed solvent of dichloromethane and methanol.

[0012] Further, in the column chromatography process, the volume ratio of dichloromethane to methanol is gradient adjusted within 20:1 to 1:1.

[0013] Further, the benzaldehyde or its derivative is a compound in which the para-substituent of the benzene ring is one of hydrogen atoms, amino groups or nitro groups.

[0014] Further, the stirring reaction is carried out for 2 hours, and the separation and purification is carried out by a silica gel column.

[0015] Thirdly, the application further provides the application of the probe in imaging of the nucleus of living cells. The probe is used for labeling the nuclear membrane, chromosomes and nucleolus structure in the nucleus of living cells.

[0016] Compared with the prior art, the application has the beneficial effects that a new near-infrared fluorescent probe with azole tetrafluoroborate as the skeleton is developed. The probe can have an emission wavelength of 710 nm, and the fluorescence intensity thereof is highly sensitive to the microenvironment viscosity, and can present a significantly enhanced fluorescence signal under high viscosity conditions.

[0017] In terms of preparation, the synthesis process of the probe has the characteristics of high atom utilization rate, mild reaction conditions, simple operation and green efficiency. The whole process does not need to be carried out under harsh anhydrous and anaerobic conditions, and does not involve metal reagents, and is environmentally friendly.

[0018] In the biological application level, the core advantage of the probe is its excellent nuclear staining ability. It can quickly stain live cells at low concentration, and clearly distinguish subcellular structures such as cytoplasm, nuclear membrane and nucleolus by virtue of its fluorescence brightness. In addition, the probe can also track the whole process of cell mitosis, providing a powerful visualization tool for studying the life activities of the nucleus, and providing a feasible strategy for developing near-infrared probes with fast nuclear staining function. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 A nuclear magnetic resonance hydrogen spectrum diagram of the probe YY-1 of the present application is shown in the figure; Figure 2 A high-resolution mass spectrum diagram of the probe YY-1 of the present application is shown in the figure; Figure 3 A nuclear magnetic resonance hydrogen spectrum diagram of the probe YY-2 of the present application is shown in the figure; Figure 4 A high-resolution mass spectrum diagram of the probe YY-2 of the present application is shown in the figure; Figure 5 A nuclear magnetic resonance hydrogen spectrum diagram of the probe YY-3 of the present application is shown in the figure; Figure 6 A high-resolution mass spectrum diagram of the probe YY-3 of the present application is shown in the figure; Figure 7 The excitation and emission wavelength diagrams of YY-1, YY-2 and YY-3 of the present application in different solvents are shown in the figure; Figure 8 The viscosity sensitivity linear regression equation calculation results of the probe YY-2 of the present application are shown in the figure; Figure 9 The docking results of the probe YY-2 of the present application and the commercial nuclear staining probe Hoechst 33342 with DNA are shown in the figure; Figure 10 The cell imaging results of the probe YY-2 of the present application staining the nucleus of live cells are shown in the figure. DETAILED DESCRIPTION

[0020] The technical solutions of the present application will be described in detail below through examples, but the protection scope of the present application is not limited to the examples. EXAMPLE

[0021] Add 4 mL of acetonitrile to a round-bottom flask, then dissolve 0.2 mmol, 70 mg of tryptophan salt and 0.2 mmol, 30 mg of p-nitrobenzaldehyde in the acetonitrile. React at room temperature with stirring for 2 h. After reaction, evaporate the solvent, and then use a silica gel column chromatography process with an eluent gradient of V(dichloromethane):V(methanol) = 20:1-1:1 to separate and purify the target compound, nitrophenyltryptophan tetrafluoroborate (compound YY-1) ((E)-7-(dimethylamino)-4-(4-nitrostyryl)-2-phenylchromenylium tetrafluoroborate), with the following general structural formula: , The nitrophenylchrome tetrazole tetrafluoroborate (compound YY-1) obtained in this example is a dark reddish-brown solid with a yield of 31%. Its proton nuclear magnetic resonance (NMR) spectral data are as follows: 1 H NMR (600 MHz, DMSO- d 6) delta 8.74 (d, J = 9.7 Hz, 1H), 8.57(d, J = 16.0 Hz, 1H), 8.46 (s, 1H), 8.41 (dd, J = 23.3, 8.3 Hz, 5H), 8.27 (d, J =8.3 Hz, 2H), 7.77 (d, J = 7.3 Hz, 1H), 7.73 (t, J = 7.5 Hz, 2H), 7.52 (dd, J = 9.6, 2.6 Hz, 1H), 7.38 (d, J = 2.5 Hz, 1H), 3.37 (s, 7H), its specific 1H NMR spectrum is as follows: Figure 1 As shown. High-resolution mass spectrometry data are: HRMS m / z: calcd for C 25 H 21 N2O3 + 397.1547, found 397.1556 [M] + Its specific high-resolution mass spectrometry is as follows: Figure 2 As shown. Example

[0022] The same steps and process parameters as in Example 1 were followed, with 0.2 mmol of tryptophan salt and 1 mmol of p-nitrobenzaldehyde reacted. After 2 h of reaction, the yield was 26%. Example

[0023] The same steps and process parameters as in Example 1 were followed, with 0.2 mmol of tryptophan salt and 2 mmol of p-nitrobenzaldehyde reacted. After 2 h of reaction, the yield was 23%. Example

[0024] By substituting p-nitrobenzaldehyde with p-aminobenzaldehyde, and following the same steps as in Example 1, the target compound, aminophenylchromezolium tetrafluoroborate (compound YY-2) ((E)-4-(4-aminostyryl)-7-(dimethylamino)-2-phenylchromenylium tetrafluoroborate), has the following general structural formula: , The aminophenylchrome tetrafluoroborate (compound YY-2) obtained in this example is a dark greenish-brown solid with a yield of 44%. Its proton nuclear magnetic resonance (NMR) spectral data are as follows: 1 H NMR (600 MHz, DMSO- d 6) delta 8.59 (d, J = 9.7 Hz, 1H), 8.55 (d, J = 15.1 Hz, 1H), 8.36 (dt, J = 6.8, 1.6 Hz, 2H), 8.29 (s, 1H), 7.87 (d, J =8.3 Hz, 2H), 7.83 (d, J = 15.1 Hz, 1H), 7.73 – 7.65 (m, 3H), 7.28 (dd, J = 9.5, 2.6 Hz, 1H), 7.14 (d, J = 2.6 Hz, 1H), 6.91 (s, 2H), 6.75 – 6.70 (m, 2H), 3.28 (s, 6H), its specific 1H NMR spectrum is as follows: Figure 3 As shown. High-resolution mass spectrometry data are: HRMS m / z: calcd for C 25 H 23 N2O + 367.1805, found 367.1810 [M] + Its specific high-resolution mass spectrometry is as follows:Figure 4 As shown. Example

[0025] 0.2 mmol of tryptophan salt and 1 mmol of p-aminobenzaldehyde were added, with other steps and process parameters the same as in Example 1. The reaction was carried out for 2 h, with a yield of 35%. Example

[0026] 0.2 mmol of tryptophan salt and 2 mmol of p-aminobenzaldehyde were added, with other steps and process parameters the same as in Example 1. The reaction was carried out for 2 h, with a yield of 30%. Example

[0027] Benzaldehyde was substituted for p-nitrobenzaldehyde, and the other steps were the same as in Example 1, to obtain the target compound, phenylchromenylium tetrafluoroborate (compound YY-3) ((E)-7-(dimethylamino)-2-phenyl-4-styrylchromenyliumtetrafluoroborate), whose general structural formula is as follows: , The phenylchromel tetrafluoroborate (compound YY-3) obtained in this example is a dark reddish-brown solid with a yield of 56.9%. Its proton nuclear magnetic resonance (NMR) spectral data are as follows: 1 H NMR (600 MHz, DMSO- d 6) delta 8.75 (d, J = 9.7 Hz, 1H), 8.57 (d, J = 15.8 Hz, 1H), 8.49 (s, 1H), 8.47 – 8.44 (m, 2H), 8.25 (d, J = 15.9Hz, 1H), 8.09 – 8.06 (m, 2H), 7.79 – 7.70 (m, 3H), 7.62 – 7.55 (m, 3H), 7.49(dd, J = 9.6, 2.6 Hz, 1H), 7.35 (d, J = 2.5 Hz, 1H), 3.37 (s, 6H), its specific 1H NMR spectrum is as follows: Figure 5 As shown. High-resolution mass spectrometry data are: HRMS m / z: calcd for C 25 H 22 NO + 352.1696, found 352.1699 [M] + Its specific high-resolution mass spectrometry is as follows: Figure 6 As shown. Examples

[0028] 0.2 mmol of color salt and 1 mmol of benzaldehyde, other steps and process parameters are the same as example 1. Reaction 2 h, yield is 50%. Examples

[0029] 0.2 mmol of color salt and 2 mmol of benzaldehyde, other steps and process parameters are the same as example 1. Reaction 2 h, yield is 41%.

[0030] It can be seen that example 1 has the best yield in examples 1-3, example 4 has the best yield in examples 4-6, and example 7 has the best yield in examples 7-9.

[0031] The excitation emission wavelength of the product of example 1, 4, 7 is tested.

[0032] (1) Three kinds of probe test solutions are prepared in glycerol, dichloromethane, methanol and PBS buffer solution respectively, the concentration is 0.5 μM, and the ultraviolet-visible absorption spectrum test is carried out.

[0033] (2) Three kinds of probe test solutions are prepared in glycerol, oleic acid, ethanol and PBS buffer solution respectively, the concentration is 0.5 μM, and the fluorescence spectrum test is carried out.

[0034] The test results are shown in Figure 7 The results show that the emission wavelength of the probe YY-2 is in the near infrared region 700-900 nm, and the existing cell nucleus probe does not exceed 700, the wavelength of the probe is 710 nm, and the fluorescence intensity is affected by the viscosity.

[0035] The viscosity sensitivity of the probe YY-2 is tested. The PBS buffer solution containing 0%, 20%, 40%, 60%, 80%, 90% and 100% glycerol is prepared, and the YY-2 test solution with a concentration of 0.5 μM is prepared with these solutions as mother liquor, and the fluorescence spectrum test is carried out, and the linear regression equation related to the viscosity sensitivity is calculated according to the test results.

[0036] The test and calculation results are shown in Figure 8 The results show that the fluorescence intensity of the probe YY-2 increases linearly with the increase of the viscosity, that is, when the molecular skeleton rotation is limited, the fluorescence intensity of the probe will be enhanced.

[0037] Molecular simulation is carried out by using AutoDockTools-1.5.6, and the docking results of the probe YY-2 and the commercial dye nucleus probe Hoechst33342 with DNA are as follows Figure 9As shown, probe YY-2 binds to the minor groove of the DNA double helix structure close to the base, while the commercial nuclear staining probe binds to the minor groove of the DNA double helix structure away from the base. The inhibition constant indicates that YY-2 has lower toxicity to DNA.

[0038] After 20 min of administration of probe YY-2 (concentration 0.2 μM, λex = 640 nm, λem = 700 nm), the nuclear membrane, nucleolus and chromosomes of KYSE-150 cells were photographed using a laser confocal microscope, and the cell mitosis process was tracked. The test cell imaging results are shown in FIG. 6. Figure 10 As shown, A1-L1 blue is the commercial cell nuclear dye Hoechst 33342 probe channel, and A2-L2 pink is the probe YY-2 channel. The red arrow indicates the nuclear membrane, the orange arrow indicates the nucleolus, the green arrow indicates the gradually condensed chromosomes, the yellow arrow indicates the migration direction of the chromosomes, and the white arrow indicates the gap formed in the middle of the paired chromosomes during cell division.

[0039] In the art, the prior art generally uses probes with a concentration of 1 μM or more, and the administration time of 30 min to 1 h is considered relatively fast. In the present application, the concentration reaches 0.2 μM, which is significantly lower than the concentration used in the prior art, and the administration time of 20 min can achieve staining effect. The present application can quickly stain the nuclear membrane, chromosomes and nucleolus of living cells at a low concentration.

[0040] The above-mentioned technologies not specifically mentioned refer to the prior art.

[0041] As described above, although the present application has been shown and described with reference to specific preferred embodiments, it is to be understood that such is by way of illustration and not of limitation. Various changes and modifications can be made therein without departing from the spirit and scope of the application as defined in the appended claims.

Claims

1. A tryptophan near-infrared viscosity-sensitive probe, characterized in that, It has the following general formula structure: ， R = one of H, NH2 or NO2.

2. The method for preparing the probe according to claim 1, characterized in that, Includes the following steps: The tryptophan tetrafluoroborate and benzaldehyde or its derivatives were dissolved in acetonitrile and reacted with stirring at room temperature. After the reaction was completed, the solvent was evaporated and the mixture was then separated and purified by column chromatography to obtain a tryptophan near-infrared viscosity-sensitive probe.

3. The preparation method according to claim 2, characterized in that: The molar ratio of tryzofluridine tetrafluoroborate to benzaldehyde or its derivative is 1:1 to 1:10, and the volume ratio of tryzofluridine tetrafluoroborate to acetonitrile is 1 mmol: 5 to 50 mL.

4. The preparation method according to claim 2, characterized in that: The eluent used for separation and purification is a mixed solvent of dichloromethane and methanol.

5. The preparation method according to claim 4, characterized in that: During the column chromatography process, the volume ratio of dichloromethane to methanol is adjusted in a gradient from 20:1 to 1:

1.

6. The preparation method according to claim 2, characterized in that, The benzaldehyde derivative is a compound in which the para-substituent of the benzene ring is either amino or nitro.

7. The preparation method according to claim 2, characterized in that: The stirring reaction was carried out for 2 hours, and the separation and purification were performed using a silica gel column.

8. The application of the probe according to claim 1 in live cell nuclear imaging, characterized in that: The probe is used to label the nuclear membrane, chromosomes, and nucleolus structures within the nucleus of living cells.

Images