CN111333677A - 488nm excited mitochondria fluorescent probe and preparation and biological application thereof - Google Patents

Abstract

The invention provides 488nm excited mitochondrial probes and preparation and biological application thereof, and the mitochondrial probes solve the problems of low brightness and poor stability of the mitochondrial probes in the wave band to a certain extent. The 488nm excited mitochondrial probe takes naphthalimide as a parent body, and different rigid structures are introduced to 4, 5-positions of the naphthalimide, so that energy loss caused by torsion is inhibited, the brightness and the stability of the mitochondrial probe are improved, the fluorescence quantum yield in different solvents is more than 0.80 (phi is 0.81 in water), and the molar extinction coefficient is more than 40000M^‑1cm^‑1. Meanwhile, the sensitivity of the probe to micro environments such as pH, temperature, polarity and the like is reduced, and the fluorescent signal in a complex physiological environment is ensuredThe method has the advantages of accuracy, capability of realizing rapid and accurate dyeing of living cell mitochondria and wide application prospect in the field of fluorescence microscopic imaging.

Description

A class of mitochondrial fluorescent probes excited at 488 nm and their preparation and biological applications

technical field

The invention belongs to the field of mitochondrial fluorescent probes, in particular to a class of mitochondrial probes excited at 488 nm and their preparation and biological applications.

Background technique

Mitochondria are an important hub of cell life activities, and are constantly dividing, merging and redistributing with the metabolic state of cells. Therefore, the study of mitochondrial dynamics is of great significance for understanding the life state of cells. Among many research tools, fluorescence imaging technology is widely used in the monitoring of such life processes due to its in-situ non-destructive, fast and sensitive characteristics, and the parameters such as brightness and stability of fluorescent probes as mediators of fluorescence imaging and life activities directly affect the quality of fluorescence imaging.

The most widely used mitochondrial probe excited at 488 nm is Mito Tracker Green. The dye is based on cyanine dye, which is easily attacked by singlet oxygen and quenched by fluorescence, with low brightness and poor stability. With the advent of the era of super-resolution fluorescence imaging, the brightness and stability of fluorescent dyes need to jump to new heights to achieve real-time and accurate imaging under high-power lasers. Therefore, such mitochondrial probes excited at 488 nm are still extremely scarce, and it is particularly urgent to develop high-stability mitochondrial probes excited at this wavelength.

SUMMARY OF THE INVENTION

One of the objectives of the present invention is to provide a class of mitochondrial probes with high stability excited at 488 nm, which can rapidly and accurately label the mitochondria of living cells.

Another object of the present invention is to provide a method for preparing a class of highly stable mitochondrial probes excited at 488 nm, which has the advantages of simple steps, easy separation, and low cost of raw materials.

The present invention provides a class of highly stable mitochondrial probes excited at 488 nm. Through the strong restriction of intramolecular torsion of naphthalimide, the molecules can achieve fluorescence stability and greatly improve the brightness, and the quantum yield of the probe molecules in water can be improved. up to 0.80.

A class of mitochondrial probes excited at 488nm, this class of mitochondrial fluorescent probes has the following structure:

其中，若R₁为H，则R₂不为H；R₃为C1-4烷基；n为0-2整数。R ₁ , R ₂ are H respectively,

Wherein, if R ₁ is H, then R ₂ is not H; R ₃ is C1-4 alkyl; n is an integer of 0-2.

The preparation method of a class of mitochondrial probes excited at 488nm, the synthetic route of this series of fluorescent probes, is as follows:

R ₁ , R ₂ are H respectively,

Wherein, if R ₁ is H, then R ₂ is not H; R ₃ is C1-4 alkyl; n is an integer of 0-2, and R ₄ is

The specific synthesis steps are as follows:

(1) Synthesis of intermediate N-hydroxyalkyl-4,5-disubstituted-1,8-naphthalene anhydride:

Dissolve 4,5-disubstituted-1,8-naphthalimide and amino alcohol in ethanol, heat up to 50-90°C, stir for 1-10h, distill off the solvent under reduced pressure, and separate the residue through a silica gel column to obtain Off-white solid N-hydroxyalkyl-4,5-disubstituted-1,8-naphthalene anhydride.

(2) Synthesis of intermediate N-bromoalkyl-4,5-disubstituted-1,8-naphthalene anhydride:

N-Hydroxyalkyl-4,5-disubstituted-1,8-naphthalene anhydride was dissolved in ethyl acetate, phosphorus tribromide was added dropwise to it, the temperature was slowly raised to 60-80°C and stirred for 4-12h. The solvent was removed under pressure, and N-bromoalkyl-4,5-disubstituted-1,8-naphthalene anhydride was obtained by silica gel column chromatography.

(3) Synthesis of intermediate N-triphenylphosphinoalkyl-4,5-disubstituted-1,8-naphthalene anhydride:

Dissolve N-bromoalkyl-4,5-disubstituted-1,8-naphthalene anhydride and triphenylphosphine in acetonitrile, raise the temperature to 120-140°C, remove the solvent under reduced pressure after the reaction for 18-30h, and chromatograph on silica gel Column separation gave N-triphenylphosphinoalkyl-4,5-disubstituted-1,8-naphthalene anhydride.

(4) Synthesis of mitochondrial probes:

Dissolve N-triphenylphosphinoalkyl-4,5-disubstituted-1,8-naphthalene anhydride in ethylene glycol methyl ether, add dropwise aliphatic amine to it, raise the temperature to 100-150°C and stir, react for 10- After 15 h, the solvent was removed under reduced pressure, and the mitochondrial probe was obtained by silica gel chromatography.

In step (1): the mass ratio of 4,5-disubstituted-1,8-naphthalimide to amino alcohol is 1.25-5:1; the mass ratio of 4,5-disubstituted-1,8-naphthalimide The mass to ethanol volume ratio is 10-20:1 mg/mL.

In step (2): the mass ratio of N-hydroxyalkyl-4,5-disubstituted-1,8-naphthalene anhydride to phosphorus tribromide is 1:1.7-5; N-hydroxyalkyl-4,5- The mass ratio of disubstituted-1,8-naphthalene anhydride to ethyl acetate was 20-30:1 mg/mL.

In step (3): the mass ratio of N-bromoalkyl-4,5-disubstituted-1,8-naphthalene anhydride to triphenylphosphine: 1:2.7-8; N-bromoalkyl-4,5 - The mass ratio of disubstituted-1,8-naphthalene anhydride to acetonitrile is 15-30:1 mg/mL.

In step (4): the mass ratio of N-triphenylphosphinoalkyl-4,5-disubstituted-1,8-naphthalene anhydride to aliphatic amine is: 1.6-2.4:1; N-triphenylphosphino The mass ratio of alkyl-4,5-disubstituted-1,8-naphthalene anhydride to ethylene glycol methyl ether is 5.3-24:1.

Application of a class of mitochondrial fluorescent probes excited at 488 nm in fluorescence imaging and fluorescence sensing.

The present invention has the following features:

The mitochondrial probe involved in the invention has the advantages of low cost of synthetic raw materials, simple method, easy separation and the like.

The spectral properties of the mitochondrial probe involved in the invention change little with the environment, and the maximum absorption wavelength in various solvents is around 488 nm, which is suitable for excitation and imaging at this wavelength. The fluorescence intensity basically does not change with the changes of ambient temperature, acidity and alkalinity.

The mitochondrial probe involved in the invention has high brightness, and the quantum yield of some compounds in water can reach 0.80.

Compared with commercial dyes, the mitochondrial probe involved in the present invention has extremely high stability. Taking Mito-DAC and Mito-DAze as examples, after being irradiated by a 500W tungsten lamp for 10 hours, the fluorescence intensity is maintained at 95% of the initial value, respectively. 96%.

The mitochondrial probe involved in the present invention has good membrane permeability. Taking Mito-DAze as an example, the fluorescence intensity in the cell reaches the maximum within five minutes after adding the dye.

The mitochondrial probe involved in the invention has accurate localization, and can achieve accurate localization of mitochondria in HeLa, MCF, C3A, RWPE and other cells, and can be used for super-resolution imaging of mitochondria due to the improvement of brightness and stability. The study of interactions with other organelles.

Description of drawings

FIG. 1 is the hydrogen nuclear magnetic spectrum of the fluorescent dye Mito-Aze prepared in Example 1.

FIG. 2 is the hydrogen nuclear magnetic spectrum of the fluorescent dye Mito-DAC prepared in Example 2. FIG.

FIG. 3 is the hydrogen nuclear magnetic spectrum of the fluorescent dye Mito-DAze prepared in Example 3. FIG.

Fig. 4 is the ultraviolet absorption and fluorescence emission spectrum of the fluorescent dye Mito-DAC prepared in Example 2 in ethanol, the abscissa is the wavelength, and the ordinate is the absorbance and the fluorescence intensity, respectively. The concentration of fluorescent dye was 10 μM.

Fig. 5 is a fluorescent image of HeLa cells of the fluorescent dye Mito-Aze prepared in Example 1, and the final concentration of the fluorescent probe is 1 μM.

Fig. 6 is a fluorescent image of C3A cells of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

Fig. 7 is a fluorescent image of HT29 cells of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

FIG. 8 is a fluorescent image of MCF cells of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

Fig. 9 is a fluorescence image of HeLa cells of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

Fig. 10 is a fluorescent image of CHO cells of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

Fig. 11 is a micro-imaging image of the HeLa cell structure of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

Fig. 12 is a micro-imaging image of the MCF cell structure of the fluorescent dye Mito-DAC prepared in Example 2, and the final concentration of the fluorescent probe is 1 μM.

FIG. 13 is a curve showing the change of intracellular fluorescence intensity with time during the staining of MCF cells by Mito-DAze prepared in Example 3. FIG. Figures (a), (b), (c), and (d) are confocal images at 0s, 60s, and 120s, respectively; Figure (e) is the change of fluorescence intensity with time during incubation, the abscissa is time, the ordinate is time is the relative value of fluorescence intensity.

FIG. 14 is a confocal fluorescence imaging image of MCF cells of Mito-Daze prepared in Example 3, and the final concentration of fluorescent probe is 1 μM.

FIG. 15 is a confocal fluorescence imaging image of Mito-Daze prepared in Example 3 of HT29 cells, and the final concentration of fluorescent probe is 1 μM.

Figure 16 is a confocal fluorescence image of Mito-Daze prepared in Example 3 of RWPE cells, and the final concentration of the fluorescent probe is 1 μM.

Figure 17 is a micro-imaging image of the RWPE cell structure of Mito-Daze prepared in Example 3, and the final concentration of the fluorescent probe is 1 μM.

Figure 18 is a micro-imaging image of the HT29 cell structure of Mito-Daze prepared in Example 3, and the final concentration of the fluorescent probe is 1 μM.

Detailed ways

Example 1

Synthesis of the mitochondrial fluorescent dye Mito-Aze.

Synthesis of intermediate N-(6-hydroxyhexyl)-4-bromo-1,8-naphthalene anhydride:

4-Bromo-1,8-naphthalene anhydride (0.50g, 1.81mmol) and 6-amino-1-hexanol (0.64g, 5.44mmol) were mixed in 10mL of ethanol, the temperature was raised to 80°C, and the reaction was completed for 8h and then reduced in pressure The solvent was removed, and the residue was separated on a silica gel column (dichloromethane/methanol=80/1, V/V) to obtain 0.58 g of an off-white solid with a yield of 86%.

Its high-resolution mass spectrometry data are as follows:

High resolution mass spectrometry C ₁₈ H ₁₈ BrNO ₃ ⁺ [M] ⁺ calcd: 376.0548; found: 376.0521.

After testing, the product structure is N-(6-hydroxyhexyl)-4-bromo-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-bromohexyl)-4-bromo-1,8-naphthalene anhydride

N-(6-hydroxyhexyl)-4-bromo-1,8-naphthalene anhydride (0.30 g, 0.80 mmol) was dissolved in 10 mL of ethyl acetate, and phosphorus tribromide (0.64 g, 2.4 mmol) was added dropwise thereto, The temperature was slowly raised to 70° C. and stirred for 6 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=100/1, V/V) to obtain 0.22 g of an off-white solid with a yield of 63%.

Its nuclear magnetic spectrum hydrogen spectrum data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (dd, J=7.3, 0.9 Hz, 1H), 8.57 (dd, J=8.5, 0.8 Hz, 1H), 8.41 (d, J=7.9 Hz, 1H) ,8.04(d,J=7.9Hz,1H),7.85(dd,J=8.4,7.4Hz,1H),4.23–4.03(m,2H),3.41(t,J=6.8Hz,2H),1.95– 1.82 (m, 2H), 1.81–1.66 (m, 2H), 1.58–1.34 (m, 4H).

Its nuclear magnetic spectrum and carbon spectrum data are as follows:

¹³ C NMR (101MHz, CDCl ₃ ) δ163.64, 163.62, 133.30, 132.07, 131.26, 131.12, 130.64, 130.29, 129.01, 128.10, 123.09, 122.22, 40.39, 33.83, 32.6, 2.6.72

After testing, the product structure is N-(6-bromohexyl)-4-bromo-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-triphenylphosphinohexyl)-4-bromo-1,8-naphthalene anhydride

N-(6-Bromohexyl)-4-bromo-1,8-naphthalene anhydride (0.30 g, 0.69 mmol) and triphenylphosphine (0.91 g, 3.45 mmol) were mixed in 5 mL of acetonitrile, and the temperature was raised to 140 in a sealed tube Stir at °C for 10 h, remove the solvent under reduced pressure after the reaction, and separate the residue through a silica gel column (dichloromethane/methanol=100/1, V/V) to obtain 0.39 g of a white solid with a yield of 82%.

Its nuclear magnetic spectrum hydrogen spectrum data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (d, J=7.3 Hz, 1H), 8.51 (d, J=8.5 Hz, 1H), 8.31 (d, J=7.9 Hz, 1H), 7.99 (d , J=7.9Hz, 1H), 7.82 (ddd, J=12.8, 10.1, 7.6Hz, 10H), 7.71 (td, J=7.6, 3.4Hz, 6H), 4.07 (t, J=7.3Hz, 2H) ,3.78–3.68(m,2H),1.70–1.60(m,4H),1.45–1.36(m,2H).

After detection, the product structure is N-(6-triphenylphosphinohexyl)-4-bromo-1,8-naphthalene anhydride.

Synthesis of mitochondrial probe Mito-Aze

N-(6-triphenylphosphinohexyl)-4-bromo-1,8-naphthalene anhydride (0.1 g, 0.14 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and azetidine ( 42 mg, 0.72 mmol), heated to 120 °C and stirred for 10 h, after the reaction was completed, the solvent was removed under reduced pressure, and the residue was separated by silica gel column (dichloromethane/methanol=50/1, V/V) to obtain 33 mg of orange solid, yield 40 %.

Its nuclear magnetic spectrum hydrogen spectrum is shown in Figure 1 below, and the specific data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.48 (d, J=7.2 Hz, 1H), 8.33 (d, J=8.1 Hz, 1H), 8.26 (d, J=8.4 Hz, 1H), 7.80 (dt , J=14.3, 7.3Hz, 9H), 7.71 (dd, J=7.1, 2.7Hz, 6H), 7.51 (t, J=7.8Hz, 1H), 6.38 (d, J=8.4Hz, 1H), 4.51 (t, J=7.4Hz, 4H), 4.06(t, J=7.0Hz, 2H), 3.69(s, 2H), 2.65–2.54(m, 2H), 1.80(s, 2H), 1.67(d, J=11.6Hz, 4H), 1.39(s, 2H).

Its nuclear magnetic spectrum and carbon spectrum data are as follows:

¹³ C NMR(101MHz,CDCl ₃ )δ164.72,164.04,152.58,135.05,133.72,133.62,133.28,131.09,130.60,130.48,130.25,123.74,118.71,117.85,116.16,106.23,55.44,29.70,27.47,26.35,22.29 , 17.10.

Its high-resolution mass spectrum data are as follows:

High Resolution Mass Spec. Calculated for C ₃₉ H ₃₈ N ₂ O ₂ P ⁺ [M] ⁺ : 1054.4134; found: 1054.4212.

After testing, the product structure is shown in the above formula Mito-Aze, the maximum absorption wavelength of the compound in water is 480nm, and the maximum emission wavelength is 555nm, which is suitable for 488nm live cell mitochondria imaging.

Example 2

Synthesis of fluorescent probe Mito-DAC.

Synthesis of Intermediate N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

4-Bromo-5-nitro-1,8-naphthalimide (1.30 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 6-amino-1-hexanol (363 mg, 3.11 mmol) was added dropwise thereto. After 1 h at 70°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether:dichloromethane=2:1, V/V) to obtain 620 mg of an off-white solid with a yield of 53%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.71(d,J=7.8Hz,1H),8.51(d,J=7.9Hz,1H),8.22(d,J=7.9Hz, 1H), 7.93(d, J=7.8Hz, 1H), 4.25–4.07(m, 2H), 3.65(t, J=6.5Hz, 2H), 1.75(dt, J=14.4, 7.0Hz, 2H), 1.59(dd,J=13.2,6.5Hz,2H),1.48–1.43(m,4H).

The carbon nuclear magnetic spectrum data are as follows: ¹³ C NMR (101MHz, CDCl ₃ )δ162.83,162.06,151.31,135.98,132.36,131.24,130.55,125.74,124.15,123.55,122.45,121.23,62.77,5.26,26.6.32. .

High-resolution mass spectrometry data are as follows: C ₁₈ H ₁₈ BrN ₂ O ₅ [M+H] ⁺ calcd: 421.0399, found: 421.0396.

After verification, the above structure is N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (500 mg, 1.19 mmol) was dissolved in dichloromethane, and phosphorus tribromide (1.61 g) was added dropwise thereto. , 5.95 mmol). After stirring at 70° C. for 6 h, the organic phase was washed with saturated sodium carbonate solution. The obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was separated through a silica gel column (dichloromethane:petroleum ether=1:1, V/V) to obtain 230 mg of a white solid with a yield of 40%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.71(d,J=7.8Hz,1H),8.52(d,J=7.9Hz,1H),8.22(d,J=7.9Hz, 1H), 7.93 (d, J=7.8Hz, 1H), 4.22–4.11 (m, 2H), 3.41 (t, J=6.8Hz, 2H), 1.94–1.83 (m, 2H), 1.75 (dt, J =15.0,7.6Hz,2H),1.58–1.49(m,2H),1.44(dd,J=14.8,5.8Hz,2H).

High-resolution mass spectrometry data are as follows: C ₁₈ H ₁₆ Br ₂ N ₂ O ₄ [M+H] ⁺ calcd: 481.9477, found: 481.9482.

After verification, the above structure is N-(6-bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

N-(6-Bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (200 mg, 0.41 mmol) and triphenylphosphine (1.08 g, 4.13 mmol) were dissolved in 10 mL of anhydrous acetonitrile , and placed in a sealed tube. After reacting at 140° C. for 24 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=400:1, V/V) to obtain 485 mg of a white solid with a yield of 60%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.66(d,J=7.3Hz,1H),8.47(d,J=8.0Hz,1H),8.20(d,J=7.3Hz, 1H), 8.01–7.40 (m, 16H), 4.11 (t, J=6.8Hz, 2H), 3.72 (s, 2H), 1.80–1.33 (m, 8H).

核磁碳谱数据如下： ¹³ C NMR(101MHz,CDCl ₃ )δ162.73,161.96,151.21,135.98,135.13,133.77,133.67,132.32,132.13,132.03,131.96,131.25,130.64,130.52,128.56,128.44,125.68,124.05 ,123.59,122.40,121.16,118.57,117.71,53.46,40.58,30.11,29.95,27.43,26.55.

High resolution mass spectrometry data are as follows: C ₃₆ H ₃₁ N ₂ O ₄ P ⁺ [M] ⁺ Calculated: 665.1205, found: 665.1208.

After verification, the above structure is N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Fluorescent Probe Mito-DAC

N-(6-Triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (100 mg, 0.13 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 1 , 2-diaminocyclohexanediamine (60 mg, 0.52 mmol). The reaction solution was slowly heated to 120 °C and reacted for 12 h. The ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain 40 mg of a yellow solid with a yield of 89%.

The hydrogen nuclear magnetic spectrum of the compound is shown in Figure 2, and the specific data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.04(d, J=8.5Hz, 2H), 7.83 (t, J=6.8Hz, 3H), 7.68 (dd,J＝13.9,6.4Hz,12H),6.83(d,J＝8.5Hz,2H),5.86(s,2H),4.02(t,J＝6.5Hz,2H),3.42–3.31(m, 2H), 3.18(d, J＝9.7Hz, 2H), 2.33(d, J＝12.5Hz, 2H), 1.80(d, J＝8.2Hz, 2H), 1.63(s, 4H), 1.48(d, J=9.7Hz, 2H).

The carbon nuclear magnetic spectrum data are as follows: ¹³ C NMR (101MHz, CDCl ₃ )δ164.31,153.34,135.46,134.31,133.53,133.43,130.75,130.63,118.30,117.44,111.04,109.26,107.18,52.7.29,38. ,25.53,23.65.

High-resolution mass spectrometry data are as follows: C ₄₂ H ₄₃ N ₁₀ O ₂₁ P ⁺ [M] ⁺ Calculated: 652.3087, found: 652.3128.

It has been verified that the structure of this compound is shown in Mito-DAC, which is suitable for the imaging of mitochondria in live cells under various physiological conditions, and its optical properties are not affected by the microenvironment. Tracking, the fluorescence emission wavelength is around 481nm.

Example 3

Synthesis of fluorescent probe Mito-DAze.

Synthesis of Intermediate N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

4-Bromo-5-nitro-1,8-naphthalimide (1.30 g, 3.11 mmol) was dissolved in 50 mL of ethanol, and 6-amino-1-hexanol (363 mg, 3.11 mmol) was added dropwise thereto. After 1 h at 70°C, the solvent was distilled off under reduced pressure, and the residue was separated through a silica gel column (petroleum ether:dichloromethane=2:1, V/V) to obtain 620 mg of an off-white solid with a yield of 53%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.71(d,J=7.8Hz,1H),8.51(d,J=7.9Hz,1H),8.22(d,J=7.9Hz, 1H), 7.93(d, J=7.8Hz, 1H), 4.25–4.07(m, 2H), 3.65(t, J=6.5Hz, 2H), 1.75(dt, J=14.4, 7.0Hz, 2H), 1.59(dd,J=13.2,6.5Hz,2H),1.48–1.43(m,4H).

The carbon nuclear magnetic spectrum data are as follows: ¹³ C NMR (101MHz, CDCl ₃ )δ162.83,162.06,151.31,135.98,132.36,131.24,130.55,125.74,124.15,123.55,122.45,121.23,62.77,5.26,26.6.32. .

High-resolution mass spectrometry data are as follows: C ₁₈ H ₁₈ BrN ₂ O ₅ [M+H] ⁺ calcd: 421.0399, found: 421.0396.

After verification, the above structure is N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

The compound N-(6-hydroxyhexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (500 mg, 1.19 mmol) was dissolved in dichloromethane, and phosphorus tribromide (1.61 mmol) was added dropwise thereto. g, 5.95 mmol), after stirring at 70° C. for 6 h, the organic phase was washed with saturated sodium carbonate solution. The obtained organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was separated through a silica gel column (dichloromethane:petroleum ether=1:1, V/V) to obtain 230 mg of a white solid with a yield of 40%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.71(d,J=7.8Hz,1H),8.52(d,J=7.9Hz,1H),8.22(d,J=7.9Hz, 1H), 7.93 (d, J=7.8Hz, 1H), 4.22–4.11 (m, 2H), 3.41 (t, J=6.8Hz, 2H), 1.94–1.83 (m, 2H), 1.75 (dt, J =15.0,7.6Hz,2H),1.58–1.49(m,2H),1.44(dd,J=14.8,5.8Hz,2H).

High-resolution mass spectrometry data are as follows: C ₁₈ H ₁₆ Br ₂ N ₂ O ₄ [M+H] ⁺ calcd: 481.9477, found: 481.9482.

After verification, the above structure is N-(6-bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Intermediate N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride

Compound N-(6-bromohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (200 mg, 0.41 mmol) and triphenylphosphine (1.08 g, 4.13 mmol) were dissolved in 10 mL of anhydrous acetonitrile and placed in a sealed tube. After reacting at 140° C. for 24 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=400:1, V/V) to obtain 485 mg of a white solid with a yield of 60%.

The hydrogen nuclear magnetic spectrum data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.66(d,J=7.3Hz,1H),8.47(d,J=8.0Hz,1H),8.20(d,J=7.3Hz, 1H), 8.01–7.40 (m, 16H), 4.11 (t, J=6.8Hz, 2H), 3.72 (s, 2H), 1.80–1.33 (m, 8H).

核磁碳谱数据如下： ¹³ C NMR(101MHz,CDCl ₃ )δ162.73,161.96,151.21,135.98,135.13,133.77,133.67,132.32,132.13,132.03,131.96,131.25,130.64,130.52,128.56,128.44,125.68,124.05 ,123.59,122.40,121.16,118.57,117.71,53.46,40.58,30.11,29.95,27.43,26.55.

High resolution mass spectrometry data are as follows: C ₃₆ H ₃₁ N ₂ O ₄ P ⁺ [M] ⁺ Calculated: 665.1205, found: 665.1208.

It has been verified that the above structure is shown by N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride.

Synthesis of Fluorescent Probe Mito-DAze

The compound N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalene anhydride (100 mg, 0.13 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and nitrogen was added to it Hetidine (30 mg, 0.52 mmol). The reaction solution was slowly heated to 120 °C and reacted for 12 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain 40 mg of a yellow solid with a yield of 89%.

The hydrogen nuclear magnetic spectrum of the compound is shown in Figure 3, and the specific data are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ8.31 (d, J=8.4Hz, 2H), 7.76 (dd, J=21.9, 9.4Hz, 15H) ,6.38(d,J=8.4Hz,2H),4.22–3.83(m,10H),3.50(s,2H),2.43(s,4H),1.66(s,4H),1.38(s,4H).

The carbon nuclear magnetic spectrum data are as follows: ¹³ C NMR (101MHz, CDCl ₃ )δ155.69,135.22,133.65,133.55,132.86,130.68,130.56,118.51,109.73,107.73,106.34,55.05,39.31,296.6,2.27.5 .

High-resolution mass spectrometry data are as follows: C ₄₂ H ₄₃ N ₃ O ₂ P ⁺ [M] ⁺ Calculated: 652.3088, found: 652.3109.

After testing, the structure of the above product is Mito-DAze, the compound can be quickly and accurately located in mitochondria in live cell imaging experiments, with high brightness and strong stability.

The probe was dissolved in DMSO solution, prepared into 2mM stock solution, and prepared into test solutions of different concentrations as required to detect changes in its fluorescence spectrum and intracellular mitochondrial fluorescence imaging.

Fluorescence emission test of the mitochondrial fluorescent probe Mito-DAze obtained in Example 3 in ethanol. Take 20 μL of the fluorescent dye stock solution, add it into 4 mL of ethanol, prepare a 10 μM fluorescent dye test solution, and test the fluorescence emission spectrum. The normalized fluorescence intensity is shown in Figure 4.

The fluorescence probe Mito-DAze has a maximum emission wavelength of 500 nm in ethanol, which is suitable for imaging with excitation light of 488 nm.

Example 4

Fluorescence image of the Mito-Aze obtained in Example 1 after staining the living cells. Dissolve 0.5 μL of Mito-Aze stock solution in 2 mL of cell culture medium, incubate at 37 °C for 10 min under 5% CO ₂ and perform fluorescence confocal imaging respectively.

Figure 5 shows the results of confocal fluorescence imaging after incubating the HeLa cells in the cell culture medium of the compound Mito-Aze with a final concentration of 1 μM obtained in Example 1 for 10 minutes, and the linear mitochondria in the HeLa cells are clearly visible.

Example 5

Fluorescence imaging diagram of the Mito-DAC obtained in Example 2 after staining the living cells. Dissolve 0.5 μL of Mito-DAC stock solution in 2 mL of cell culture medium, incubate at 37°C under 5% CO ₂ for 10 minutes, and perform fluorescence confocal imaging and structured illumination imaging (SIM) imaging, respectively.

C3A cells were incubated with Mito-DAC at a final concentration of 1 μM in cell culture medium for 10 minutes, and then confocal fluorescence imaging was performed. As shown in Figure 6, linear mitochondria in C3A cells were clearly visible.

After incubating HT29 cells with Mito-DAC final concentration of 1 μM in cell culture medium for 10 minutes, confocal fluorescence imaging was performed. As shown in Figure 7, most of the mitochondria in HT29 cells were oval.

MCF cells were incubated with Mito-DAC final concentration of 1 μM in cell culture medium for 10 minutes, and then confocal fluorescence imaging was performed. As shown in Figure 8, linear mitochondria in MCF cells were clearly visible.

Confocal fluorescence imaging was performed after incubating HeLa cells with a final concentration of Mito-DAC of 1 μM in cell culture medium for 10 minutes. As shown in Figure 9, linear mitochondria in HeLa cells were clearly visible.

CHO cells were incubated with Mito-DAC final concentration of 1 μM in cell culture medium for 10 minutes, and then confocal fluorescence imaging was performed. As shown in Figure 10, linear mitochondria in CHO cells were clearly visible.

After incubating HeLa cells with a final concentration of 1 μM of Mito-DAC for 10 minutes, the structured illumination showed obvious micro-imaging. As shown in Figure 11, the linear mitochondria in HeLa cells were clearly visible, and the mitochondrial cristae could also be distinguished.

After incubating MCF cells with Mito-DAC final concentration of 1 μM for 10 minutes, the structured light imaging was obvious. As shown in Figure 12, the linear mitochondria in HeLa cells were clearly visible, and the mitochondrial cristae could also be distinguished.

Example 6

The Mito-DAze obtained in Example 3 was tested for the staining speed of living cells. Dissolve 0.5 μL of the stock solution in 1 mL of cell culture medium and perform real-time imaging.

The change curve of intracellular fluorescence intensity with time during the staining of MCF cells by compound Mito-DAze is shown in Figure 13 . The abscissa is time, and the ordinate is the relative value of fluorescence intensity. It can be seen from the figure that within 5 minutes after the probe is added to the cell culture medium, the intracellular fluorescence intensity reaches the maximum value, indicating that the dye has good permeability and is suitable for rapid imaging experiments of live cells.

Example 7

Fluorescence image of the Mito-DAze obtained in Example 3 after staining the living cells. Dissolve 0.5 μL of Mito-DAze stock solution in 2 mL of cell culture medium, incubate at 37 °C under 5% CO ₂ for 10 minutes, and perform fluorescence confocal imaging and structured illumination microscopy (SIM) imaging, respectively.

MCF cells were incubated with Mito-DAze at a final concentration of 1 μM in cell culture medium for 10 minutes, and confocal fluorescence imaging was performed. As shown in Figure 14, linear mitochondria in MCF cells were clearly visible.

HT29 cells were incubated with Mito-DAze at a final concentration of 1 μM for 10 minutes and then confocal fluorescence imaging was performed. As shown in Figure 15, most mitochondria in HT29 cells were oval.

After incubating RWPE cells with Mito-DAze at a final concentration of 1 μM for 10 minutes, confocal fluorescence imaging was performed. As shown in Figure 16, linear mitochondria in RWPE cells were clearly visible.

After incubating RWPE cells with Mito-DAC final concentration of 1 μM in cell culture medium for 10 minutes, the structured light imaging was obvious. As shown in Figure 17, the linear mitochondria in RWPE cells were clearly visible, and the mitochondrial cristae could also be distinguished.

After incubating HT29 cells with Mito-DAC final concentration of 1 μM cell culture medium for 10 minutes, the structured light imaging was obvious. As shown in Figure 18, most mitochondria in HT29 cells were oval, and the fine structure mitochondrial cristae could be distinguished.

Claims (7)

1. A488 nm excited mitochondria fluorescent probe is characterized in that the structure of the fluorescent probe is as follows:

R₁，R₂are respectively an oxygen atom (H) and an oxygen atom (H),

wherein, if R₁Is H, then R₂Is not H; r₃Is C1-4 alkyl; n is an integer of 0 to 2.

2. The method for synthesizing 488nm excited mitochondrial fluorescent probe according to claim 1, which comprises the following steps:

(1) synthesizing an intermediate N-hydroxyalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride:

dissolving 4, 5-disubstituted-1, 8-naphthalimide and amino alcohol in ethanol, heating to 50-90 ℃, stirring for 1-10h, distilling under reduced pressure to remove the solvent, and separating the residue by a silica gel column to obtain off-white solid N-hydroxyalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride;

(2) synthesizing an intermediate N-bromoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride:

adding N-hydroxyalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride into ethyl acetate, dropwise adding phosphorus tribromide into the ethyl acetate, slowly heating to 60-80 ℃, stirring for 4-12h, removing the solvent under reduced pressure after the reaction is finished, and separating by a silica gel chromatographic column to obtain N-bromoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride;

(3) synthesizing an intermediate N-triphenylphosphinoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride:

dissolving N-bromoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride and triphenylphosphine in acetonitrile, heating to 140 ℃, removing the solvent under reduced pressure after the reaction is finished for 18-30h, and separating by a silica gel chromatographic column to obtain N-triphenylphosphinoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride;

(4) synthesis of mitochondrial probe:

dissolving N-triphenylphosphine alkyl-4, 5-disubstituted-1, 8-naphthalic anhydride in ethylene glycol monomethyl ether, dripping fatty amine into the ethylene glycol monomethyl ether, heating to 100 ℃ and stirring, reacting for 10-15h, then decompressing and removing the solvent, and separating by a silica gel chromatographic column to obtain the mitochondrial probe.

3. The method for synthesizing 488nm excited mitochondrial fluorescent probe as claimed in claim 2, wherein the method comprises the following steps: in the step (1), the mass ratio of the 4, 5-disubstituted-1, 8-naphthalimide to the amino alcohol is 1.25-5: 1;

the volume ratio of the mass of the 4, 5-disubstituted-1, 8-naphthalimide to the volume of the ethanol is 10-20:1 mg/mL.

4. The method for synthesizing 488nm excited mitochondrial fluorescent probe as claimed in claim 2, wherein the method comprises the following steps: in the step (2), the mass ratio of the N-hydroxyalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the phosphorus tribromide is 1: 1.7-5;

the volume ratio of the mass of the N-hydroxyalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the ethyl acetate is 20-30:1 mg/mL.

5. The method for synthesizing 488nm excited mitochondrial fluorescent probe as claimed in claim 2, wherein the method comprises the following steps: in the step (3), the mass ratio of the N-bromoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the triphenylphosphine is as follows: 1: 2.7-8;

the volume ratio of the mass of the N-bromoalkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the acetonitrile is 15-30:1 mg/mL.

6. The method for synthesizing 488nm excited mitochondrial fluorescent probe as claimed in claim 2, wherein the method comprises the following steps: in the step (4), the mass ratio of the N-triphenyl phosphinyl alkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the fatty amine is as follows: 1.6-2.4: 1;

the volume ratio of the mass of the N-triphenyl phosphinyl alkyl-4, 5-disubstituted-1, 8-naphthalic anhydride to the ethylene glycol monomethyl ether is 5.3-24: 1.

7. The 488nm excited mitochondrial fluorescent probe as described in claim 1 is applied to the fields of fluorescence imaging and fluorescence sensing.

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