Two-photon fluorescent dye as well as preparation method and application thereof

Abstract

A preparation method of the two-photon fluorescent dye comprises the following steps: reacting 6-hydroxy-2-naphthaldehyde and dihalogenated hydrocarbon under the action of an alkaline substance to obtain monohalogenated alkyl aryl ether, and reacting the monohalogenated alkyl aryl ether with a trimethylamine aqueous solution to prepare S1, 4-(4-tolyl) pyridine-2, 3, 4-trimethylamine; according to the present invention, the intermediate S1 and the intermediate S2 are subjected to a reaction under the strong basicity effect, hydrolysis is performed to prepare the target product, and the dye has advantages of good cell membrane permeability, good dispersion in cytoplasm, preferential targeting of organelles in the cytoplasm, water solubility, low toxicity, and good stability, and can be used for the preparation of the target product. The two-photon absorption interface is high, and the material can be applied to two-photon fluorescence confocal microscopic imaging.

Description

Technical Field

[0001] The invention relates to a two-photon fluorescent dye and a preparation method and application thereof. Background Art

[0002] The excitation wavelength of two-photon excited fluorescence (TPEF) is generally longer than the emission wavelength, moving to the near-infrared region, avoiding the influence of ultraviolet lasers, with low photodamage, which can greatly reduce damage to living systems. In addition, TPEF will not be affected by background interference such as Rayleigh and Raman scattering with wavelengths far away from the near-infrared region (J Photochem Photobiol C Photochem Rev. 2022, 52: 100529). Two-photon excited fluorescence probes (TPEFP) can also overcome some limitations of single-photon excitation on imaging (such as out-of-plane photobleaching and scattering), have strong penetration in scattering media (>500μm), low tissue autofluorescence self-absorption, and high resolution. The excitation source of two-photon microscopy (TPM) uses two photons with longer wavelengths and lower energy. Combined with appropriate TPEFP, it can achieve long-term imaging inside intact tissues without generating tissue artifacts. TPEF imaging technology can perform functional imaging of biological tissues at the subcellular level, realize the detection of living tissues and living cells, and can be used to directly observe metabolism in living cells, cancer diagnosis, etc. It is very popular in biology and medicine (Phys. Chem. Chem. Phys., 2024, 26: 6008-6021.; Frontiers in Medicine. 2024, 10: 1-7.).

[0003] Cells are the basic building blocks of all biological structures and functions. Various life phenomena of biological organisms are mainly expressed and completed by cells. Diseases are the result of lesions in cells in local tissue structures of the human body. Fluorescence microscopy has become an important visual tool for studying cells, and fluorescent dyes have become the most important attachment tools in microscopic imaging research. However, most fluorescent dyes used for fluorescence imaging have poor water solubility and low two-photon absorption cross-section values ​​(δ), which limits their use in TPM and hinders progress in this field (Chem-An Asian J. 2011, 6: 58–69.). Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a two-photon fluorescent dye, which has a larger two-photon absorption cross section, stronger induced luminescence and better water solubility, so that it has better cell membrane permeability and can be used for biological imaging.

[0005] The second technical problem to be solved by the present invention is to provide a method for preparing the above two-photon fluorescent dye, which has simple steps, readily available raw materials and relatively loose reaction conditions.

[0006] The third technical problem to be solved by the present invention is to provide the application of the above two-photon fluorescent dye in the preparation of cell imaging reagents.

[0007] In order to solve the above-mentioned first technical problem, the present invention provides a two-photon fluorescent dye, the general structural formula of which is as follows:

[0008]

[0009] Wherein X is a halogen; R-, R', R", R'" are hydrocarbon groups; and M is a metal ion.

[0010] The benzene ring and naphthalene ring are added to the pyridine dicarboxylic acid unit. The naphthalene ring has a hydrocarbon oxy group as an electron-pushing group, and the pyridine ring is an electron-pulling group. The naphthalene ring is bridged with the benzene ring through a π bond, that is, it has a push-pull electron structure (donor-π-acceptor, D-π-A), the conjugated system is enlarged, the rigid planar structure is increased, the charge transfer within the molecule is enhanced, the absorption of light radiation is enhanced, the two-photon absorption cross section is increased, and the fluorescence intensity is increased. Its structure contains two hydrophilic carboxyl groups and one strongly hydrophilic quaternary ammonium group, so it has good water solubility. Its large two-photon absorption cross section, strong induced luminescence and good water solubility make it have good cell membrane permeability and can be used for biological imaging.

[0011] The two-photon fluorescent dye described in the present invention has good single-photon fluorescence properties, and its aqueous solution has no linear absorption above about 450nm, the maximum excitation wavelength and the emission wavelength are about 330nm and 460nm respectively, and the Stokes shift is relatively large, reaching about 130nm; it has good two-photon fluorescence properties at about 510nm, and has a large absorption cross-section in the range of 680nm to 820nm. When the excitation wavelength is 780nm, the two-photon absorption cross-section is the largest, reaching about 85GM, and has a wide two-photon application range.

[0012] The two-photon fluorescent dye described in the present invention has the characteristics of high fluorescence intensity, good water solubility, low fluorescence background, etc.; it is a cytoplasm and its organelle fluorescent dye, and can realize single-photon and two-photon imaging of cells and tissues.

[0013] In order to solve the above second technical problem, the present invention provides a method for preparing the above two-photon fluorescent dye, comprising the following steps:

[0014] (1) Preparation of intermediate S1:

[0015] The monohalogenated hydrocarbon aromatic ether obtained by reacting 6-hydroxy-2-naphthaldehyde with a dihalogenated hydrocarbon under the action of an alkaline substance is then reacted with an aqueous solution of trimethylamine to prepare S1:

[0016]

[0017] Wherein, X is a halogen; R-, R', R", R'" are hydrocarbon groups;

[0018] The molar ratio of 6-hydroxy-2-naphthaldehyde to dihalogenated hydrocarbon is 1:1.5-3.5, the reaction temperature is 20-115°C, the solvent is selected from one of methanol, ethanol, propanol, butanol, acetonitrile, tetrahydrofuran or a mixture thereof, the alkaline substance is carbonate or hydroxide, and the reaction time is 6-30h; when reacting with trimethylamine, the temperature is 25-120°C, the reaction time is 12-48h, the concentration of trimethylamine is 25%-40%, and the molar volume ratio of 6-hydroxy-2-naphthaldehyde to trimethylamine mol:L is 1:0.25-1.

[0019] (2) Preparation of intermediate S2:

[0020] The brominated product obtained by the reaction of diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate with N-bromosuccinimide is then reacted with triphenylphosphine to prepare S2:

[0021]

[0022] The molar ratio of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester to N-bromosuccinimide is 1:1-1:3, benzoyl peroxide or azobisisobutyronitrile is used as an initiator, wherein the initiator accounts for 10%-30% of the amount of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester, carbon tetrachloride is used as a solvent, and the reaction is carried out at reflux temperature for 12h-80h; when reacting with triphenylphosphine, the molar ratio of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester to triphenylphosphine is 1:0.8-3.0, toluene is used as a solvent, and the reaction is carried out at reflux for 3-10h.

[0023] (3) Preparation of target product L two-photon fluorescent dye substituted pyridine dicarboxylate:

[0024] After the intermediate S1 reacts with S2 under the action of strong alkali, it is hydrolyzed to prepare the target product L:

[0025]

[0026] Wherein X is a halogen; R-, R', R", R'" are hydrocarbon groups; M is a metal ion;

[0027] The molar ratio of S1 to S2 is 1:0.7 to 1:1.3, the strong base is selected from alcohol salts or hydroxides, the reaction temperature is -20 to 40°C, the solvent is selected from one of methanol, ethanol, propanol, acetonitrile or a mixture thereof, and the reaction time is 4 to 36 hours; the alkaline substance selected for hydrolysis is carbonate or hydroxide, and the reaction time is 10 to 60 hours.

[0028] Preferably, the alkaline substance in step (1) is selected from one of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide or a mixture thereof.

[0029] Preferably, in step (3), the strong base is selected from sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, potassium tert-butoxide, or a mixture thereof;

[0030] Preferably, the alkaline substance selected for hydrolysis in step (3) is one of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide, etc., or a mixture thereof.

[0031] The preparation method of the two-photon fluorescent dye of the present invention has reasonable method design, simple steps, readily available raw materials and relatively loose reaction conditions.

[0032] In order to solve the third technical problem mentioned above, the present invention provides the use of the above two-photon fluorescent dye in the preparation of cell imaging reagents.

[0033] In fluorescent confocal microscopy imaging, after mouse breast cancer cells (4T1) are stained with the two-photon fluorescent dye provided by the present invention, it can be observed that the two-photon fluorescent dye of the present invention is located in the cytoplasm and organelles of the 4T1 cells, single-photon fluorescence exhibits blue emission, while two-photon fluorescence exhibits strong and bright green light emission, and the resolution of two-photon fluorescent cell microscopy imaging is significantly increased compared with single-photon.

[0034] The two-photon fluorescent dye of the present invention is a water-soluble two-photon induced fluorescent material with very low toxicity. -1 Within this range, there is basically no obvious toxicity to 4T1 cells, and the cell survival rate is above 89%. It can be used to detect targets in cells and organisms within this range. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The invention discloses a method for preparing the two-photon fluorescent dye-substituted pyridine dicarboxylate.

[0036] Figure 2 a. Figure 2 b and Figure 2c are the UV-visible absorption spectrum, fluorescence excitation spectrum and fluorescence emission spectrum of the target product L aqueous solution.

[0037] Figure 3 a and Figure 3 b is the two-photon induced fluorescence spectrum of the target product L in water and the output fluorescence energy I out (au) and input laser energy I in (mW) logarithmic fitting curve.

[0038] Figure 4 is the two-photon absorption cross section value (GM) of the target product L in water.

[0039] Figure 5 This is the single-photon and two-photon fluorescence confocal microscopy imaging of the target product L on 4T1 cells: Figure 5 a is a single-photon fluorescence confocal micrograph of 4T1 cells stained with the target product L; Figure 5 b is a two-photon fluorescence confocal micrograph of 4T1 cells stained with the target product L.

[0040] Figure 6 It is between 0.02-25 μmol·L -1 Cell viability graph of 4T1 cells in aqueous solution of target product L within the concentration range. DETAILED DESCRIPTION

[0041] For the preparation of two-photon fluorescent dyes, see Figure 1 , including the following steps:

[0042] (1) Preparation of intermediate S1:

[0043] In a nitrogen atmosphere, 6-hydroxy-2-naphthaldehyde (7.11 g, 41.29 mmol), 1,6-dibromohexane (19 mL), and anhydrous potassium carbonate (9.69 g, 70.11 mmol) were added to acetonitrile (100 mL), and the mixture was stirred and refluxed for 24 h, cooled, filtered, and the residue was washed with acetonitrile 3 times. The filtrate was decompressed to remove the solvent, and the crude product was eluted by column chromatography with petroleum ether, ethyl acetate, and petroleum ether (1:9, v / v) to obtain 6.71 g of a light yellow solid. The light yellow solid (0.63 g, 1.88 mmol), acetonitrile (45 mL), and 30% trimethylamine aqueous solution (18 mL) were added to 5 90 mL polytetrafluoroethylene autoclaves, and the mixture was placed at 90 ° C for 30 h. After cooling, the reaction mixture was combined and the solvent was removed under reduced pressure. Acetonitrile was used as a solvent for recrystallization to obtain a nearly white solid S1 (2.01 g, 5.10 mmol) with a yield of 54%. mp195-197℃. IR(KBr),ν / cm -1:3027,3003,2945,2867,1682,1618,1471,1375,1269,1160,1006,893,820,776. 1 HNMR(600MHz,D2O,d ppm):9.58(s,1H,-CHO),6.68-7.80(6H,Naph–H),3.62(t,2H,–OCH2-),2.99-3.16(11H,–CH2N + (CH3)3),1.21-1.59(8H,-CH2CH2CH2CH2-. 13 C NMR(600MHz,D2O,d ppm):195.50,159.07,138.13,135.60,131.45,131.34,127.73,127.42,119.46,107.32,68 .28,66.61,57.49,52.84,28.05,25.31,24.83,22.26,16.86.HRMS(ESI,m / z):(1)Calcd.for C 20 H 28 NO2 + [M-Br] + 314.2215; found314.2108; (2)Calcd.for C 20 H 28 BrKNO2 + [M+K] + 432.0935;found432.0928;.(3)Calcd.forC 20 H 28 BrNNaO2 + [M+Na] + 416.1196; found 416.1191.

[0044] (2) Preparation of intermediate S2:

[0045] In anhydrous carbon tetrachloride (800 mL), diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate (3.89 g, 12.41 mmol), N-bromosuccinimide (NBS, 2.31 g, 12.98 mmol), dibenzoyl peroxide (BPO, 0.71 g, 2.93 mmol) were added to anhydrous carbon tetrachloride (800 mL) under nitrogen atmosphere. After stirring and reflux reaction for 18 h, NBS (0.77 g, 4.33 mmol) was added three times and stirred and refluxed for 24 h. Cool, filter, remove the solvent from the filtrate under reduced pressure, wash the residue (about 200 mL) with distilled water (150 mL) 5 times, and dry over anhydrous magnesium sulfate. Filter, remove the solvent from the filtrate under reduced pressure, and dry under vacuum at 50°C for 8 h. Eluted with ethyl acetate and petroleum ether (1:11, v / v) to obtain 4.25 g of a yellow solid. Add the yellow solid (3.93 g, 10.02 mmol) and triphenylphosphine (6.59 g, 25.13 mmol) obtained above to toluene (50 mL) under stirring in a nitrogen atmosphere, gradually increase the temperature and reflux for 7 h. After cooling, filter, wash the filter residue with toluene 3 times, and dry the crude product at 60 ° C in vacuum for more than 6 h, and then recrystallize with acetonitrile and ethyl acetate to obtain a yellow solid S2 (3.70 g, 5.65 mmol), with a yield of 56%. mp233-235℃.FT-IR(KBr),ν / cm-1:3054,3040,3000,2982,2961,2873,2836,2770, 1745,1722,1597,1437,1343,1245,1165,1110,1024,850. 1HNMR(400MHz,CD3OD,d ppm):8.42(s,2H,Py–H),7.27-7.94(19H,Ph–H),5.18(2H,Ph–CH2-),4.42-4.47(m,4H,-CH2-Me),1.43(t,6H,-CH3).13CNMR(400MHz,CD3OD,d ppm):164.35,149.15,149.82,136.40,135.21,134.12,132.07,130.12,127. 47,124.89,118.07,117.21,61.97,29.42,13.22.HRMS(ESI,m / z):Calcd.for C 36 H 33 NO4P+[M-Br]+574.2142; found574.2120.

[0046] (3) Preparation of target product L

[0047] Under stirring, in a nitrogen atmosphere at -10°C, to a 30 mL anhydrous methanol solution of S1 (0.61 g, 1.55 mmol) and S2 (1.05 g, 1.60 mmol), a 45 mL anhydrous methanol solution of potassium tert-butoxide (0.71 g, 6.33 mmol) was slowly added dropwise. After the addition, the reaction was continued at -5°C for about 8 hours, and then at room temperature for another 24 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the obtained yellow solid was added to tetrahydrofuran (70 mL) and stirred for 8 hours, filtered, and the filter residue was washed three times with tetrahydrofuran. Repeat the above operation three times. The filter residue was vacuum dried at 30°C for 8 hours. 1.01 g of a yellow solid was obtained. The yellow solid was added to an aqueous solution (12 mL) of KOH (0.27 g, 4.81 mmol) at room temperature, and the reaction was stirred at room temperature for 48 hours. After the reaction, acetonitrile (200 mL) was added and mixed, and then the mixture was allowed to stand for 6 hours, filtered, and the residue was washed with acetonitrile 3 times and vacuum dried at 30°C for 8 hours, then recrystallized with distilled water and isopropanol, and vacuum dried at 30°C for 48 hours. The target product L (0.63 g, 0.89 mmol) was obtained as a light yellow solid with a yield of 61%. FT-IR (KBr), ν / cm -1 :3026,2933,2851,1647,1622,1578,1480,1394,1339,1266,1211,1177,1061,1007,965,831,807,723,702. 1 H NMR(600MHz,CD3OD,d ppm):8.43(s,1H,Py–H),8.35(s,1H,Py–H),7.05-7.86(10H,Naph-H and Ph-H),6.83(d,1H,=C=CH-Ph),6.71(d,1H,-HC=C=),4.08(t,2H,-OCH2-),3.10-3.11(11H,-CH2N + (CH3)3),1.44-1.85(8H,-CH2CH2CH2CH2-). 13 C NMR(D2O,d ppm):171.43,160.06,154.76,129.54,129.06,126.95,126.90,126.50,126.45,126.22,121.87,121.7 9,66.38,56.11,52.33,52.20,52.10,52.00,28.68,25.71,25.41,22.49.HRMS(ESI,m / z):(1)Calcd.for C 34 H 35 N2O5 - [M-Br-2K] -551.2551;found 551.2556;(2)Calcd.for C 34 H 35 K2N2O5 + [M-Br] + 629.1815;found 629.1794;(3)Calcd.for C 34 H 35 K3N2O5 2 + [M-Br+K] 2+ 334.0723;found 334.0718;(4)Calcd.for C 34 H 35 K2N2NaO5 + [M-Br+Na] 2+ 326.0853;found 326.0838;(5)Calcd.for C 34 H 35 KN2NaO5 + [M-Br-K+Na] + 613.2075;found613.2055;(6)Calcd.for C 34 H 35 KN2Na2O5 2+ [M-Br-K+2Na] 2+ 318.0984;found 318.0961;(7)Calcd.for C 34 H 36 K2N2O5 2+ [M-Br+H] 2+ 315.0944;found,315.0933;(8)Calcd.for C 34 H 36 KN2O5 + [M-Br-K+H] + 591.2256;found 591.2241;(9)Calcd.for C 34 H 37 KN2O5 2+ [M-Br-K+2H] 2+ 296.1164;found,2+096.1151;(10)Calcd.for C 34 H 37 N2O5 + [M-Br-2K+2H] + 553.2697;found553.2685.

[0048] Single-photon properties of target product L:

[0049] The prepared L aqueous solution (concentration 2.0×10 -5 mol·L -1 ) was placed in a 1 cm quartz cuvette, and the UV-visible absorption spectrum of the sample was collected by EVOLUTION 220 UV-visible spectrophotometer, with the scanning wavelength range set to 190-1100 nm. Figure 2 a It can be seen that the target product L aqueous solution has no linear absorption above 450nm. There is charge transfer from the naphthalene ring to the pyridine group through the bridging π bond in the target product L molecule. -6 mol·L -1 ) were recorded using an LS 55 fluorescence spectrophotometer. Figure 2 b and Figure 2 c It can be seen that the maximum excitation wavelength and emission wavelength are 328nm and 459nm respectively, and the Stokes shift is relatively large, reaching 131nm. It is speculated that the emission peak of L can be attributed to the emission caused by intramolecular charge transfer transition.

[0050] Two-photon properties of target product L:

[0051] The two-photon induced fluorescence test uses femtosecond laser pulses and a Ti sapphire system (excitation wavelength range: 680-1080nm, 80MHz, 140fs, Chameleon UltraⅡ) as the pump light source to measure the two-photon excited fluorescence spectrum. All measurements were performed in air at room temperature (below 20℃). The integration time was set to 300ms and the power was set to 0.5W, and the two-photon absorption cross section was tested using the two-photon induced fluorescence measurement technique.

[0052] The concentration of the aqueous solution of the target product L in the experiment was 0.1 mmol·L -1 Preparation of fluorescein reference stock solution: weigh 0.0017 g of fluorescein solid into a centrifuge tube, pipette 5 mL of NaOH standard solution (1.000 mol·L -1 ) dissolve the solid fluorescein to prepare 1 mmol·L -1 Fluorescein reference stock solution. Then aspirate 300 μL and dilute to 3 mL to obtain a concentration of 0.1 mmol·L -1 Fluorescein NaOH aqueous solution (where the NaOH concentration is 1 mol·L -1 ) as a reference. The sample pool is a 1.0×1.0 cm four-sided transparent quartz cuvette. The laser light source power is controlled at 300 mW, the wavelength range is 700-900 nm, and the wavelength interval is 20 nm. The two-photon induced fluorescence spectrum is measured. The results are shown in Figure 3 As shown in a. As can be seen from the figure, as the excitation wavelength increases, the fluorescence intensity increases, and the intensity begins to weaken after the excitation wavelength increases to 780nm, so there is a maximum fluorescence intensity at 780nm, that is, 780nm is the best excitation wavelength (see Figure 3 a), and Figure 2 a shows that 8 has no single-photon absorption above 450nm, so L has maximum two-photon absorption at 780nm.

[0053] At the optimal excitation wavelength of 780nm for the target product L, by adjusting the laser power I in (mW) by changing the intensity of the excitation light and measuring the two-photon induced fluorescence intensity I of L under different intensities of excitation light out (au) further verifies the two-photon characteristics of L. The output / input intensity power logarithm ratio of the target product L in aqueous solution lgI out / lgI in (Slope) is around 2 (see Figure 3 b), which is consistent with the characteristics of two-photon excitation, further proving that L has two-photon activity.

[0054] The two-photon induced fluorescence method was used to calculate the two-photon absorption cross section (δ) of the compound. The reference reagent used was a fluorescein aqueous solution at pH = 14. The calculation formula is as follows:

[0055] δ s =δ r (C r n r Φ r F s ) / (C s n s Φ s F r )

[0056] Where s represents the sample, r represents the reference, δ is the two-photon absorption cross section, and the reference sample fluorescein δ r =43GM (wavelength 790nm), C is the concentration of the solution, and the reference sample fluorescein is prepared in NaOH aqueous solution with a concentration of 0.1mmol·L -1 , n is the refractive index of the solvent, Ф is the two-photon quantum yield, Ф r =0.97 comes from the literature, and F is the integrated area value of the two-photon fluorescence intensity corresponding to different excitation wavelengths.

[0057] The calculated results of the two-photon absorption cross section of the target product L in water are as follows: Figure 4 As shown, the two-photon absorption cross section of the target product L in aqueous solution reaches a maximum value at around 780 nm, and the two-photon absorption cross section value is about 86 GM.

[0058] Target product L fluorescent cell microscopy imaging:

[0059] 4T1 cells were fixed with formaldehyde and loaded with L in PBS (phosphate buffered saline for cell culture) solution for fluorescent cell microscopy imaging. First, 4T1 cells were seeded on a 35 mm diameter cell culture dish and placed in an incubator containing 5% CO2 and 95% oxygen. After culturing with culture medium at 37°C for 24 h, 4T1 cells were washed three times with PBS, and after washing away the culture medium, PBS solution containing L (10 μmol·L -1 ) 4T1 cells were incubated for 4 h, and then washed 3 times with PBS to remove excess L. The cells were then fixed with 4% formaldehyde in PBS. Finally, fluorescent cell imaging was performed on the treated and colored 4T1 cells.

[0060] Single-photon fluorescence cell microscopy imaging was performed on a FV1200 / FV1200MPE laser confocal microscope using CY3, AF488, and DAPI dye channels (see Figure 5 a), their excitation wavelengths and maximum emission wavelengths are 550nm and 570nm, 488nm and 519nm, 405nm and 488nm respectively. Figure 5 a As can be seen, single-photon fluorescence cell microscopy imaging exhibits blue light emission.

[0061] Two-photon fluorescence cell microscopy imaging was performed on a FV1200 / FV1200MPE laser confocal microscope. The excitation wavelength range of 950-700nm can be used for imaging, but 800nm ​​is the best. Therefore, the excitation wavelength was selected as 800nm, and the fluorescence of two channels at 426-460nm and 495-540nm was collected. The results are shown in Figure 5 b. By Figure 5 As can be seen from b, two-photon fluorescence exhibits strong and bright green light emission, and the resolution of two-photon fluorescence cell microscopy imaging is significantly increased compared to single-photon imaging.

[0062] from Figure 5 b It can be seen that the target product L penetrates the cell membrane of 4T1 cells, enters the cytoplasm, and is well dispersed in the cytoplasm, indicating that the target product has a high localization ability to the cytoplasm of 4T1 cells. In addition, several bright spots were observed in the cytoplasm, indicating that L preferentially targets organelles in the cytoplasm.

[0063] Target product L cytotoxicity

[0064] Cell Counting Kit-8 (CCK-8) was used to detect cell viability (CCK-8 method). 4T1 cells in logarithmic phase were collected and the cell density was adjusted to 5×10 4 Cells·mL-1 , inoculated in a 96-well cell culture plate, placed in a constant temperature incubator at 37°C and 5% CO2, and after the cells adhered to the wall, replaced with a culture medium containing different concentrations of drugs, incubated for 24 hours, added CCK-8 solution, protected from light, placed in a 37°C incubator for 2 hours, and measured the absorbance at 450nm with an enzyme marker. Cell survival rate was calculated as follows:

[0065] CSR=[(A s -A b ) / (A c -A b )]×100%

[0066] In the formula, A s is the absorbance of the experimental group (including cells, culture medium, CCK-8 solution and drug solution), A c is the absorbance of the control group (containing cells, culture medium, CCK-8 solution, but no drugs), A b It is the absorbance of the blank group (containing culture medium and CCK-8 solution, but not cells or drugs).

[0067] Test results see Figure 6 .Depend on Figure 6 It can be seen that the target product L is basically non-toxic to 4T1 cells, and the cell survival rate is above 89%. -1 Within the range, it can be used to detect targets in cells and organisms.

Claims

1. A two-photon fluorescent dye, characterized in that: Its general structure is as follows: Wherein X is a halogen; R-, R', R", R'" are hydrocarbon groups; and M is a metal ion.

2. The method for preparing a two-photon fluorescent dye according to claim 1, characterized in that: The following steps are involved: (1) Preparation of intermediate S1: The monohalogenated hydrocarbon aromatic ether obtained by reacting 6-hydroxy-2-naphthaldehyde with a dihalogenated hydrocarbon under the action of an alkaline substance is then reacted with an aqueous solution of trimethylamine to prepare S1: Wherein, X is a halogen; R-, R', R", R'" are hydrocarbon groups; The molar ratio of 6-hydroxy-2-naphthaldehyde to dihalogenated hydrocarbon is 1:1.5-3.5, the reaction temperature is 20-115°C, the solvent is selected from one of methanol, ethanol, propanol, butanol, acetonitrile, tetrahydrofuran or a mixture thereof, the alkaline substance is carbonate or hydroxide, and the reaction time is 6-30h; when reacting with trimethylamine, the temperature is 25-120°C, the reaction time is 12-48h, the concentration of trimethylamine is 25%-40%, and the molar volume ratio of 6-hydroxy-2-naphthaldehyde to trimethylamine mol:L is 1:0.25-1. (2) Preparation of intermediate S2: The brominated product obtained by the reaction of diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate with N-bromosuccinimide is then reacted with triphenylphosphine to prepare S2: The molar ratio of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester to N-bromosuccinimide is 1:1-1:3, benzoyl peroxide or azobisisobutyronitrile is used as an initiator, wherein the initiator accounts for 10%-30% of the amount of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester, carbon tetrachloride is used as a solvent, and the reaction is carried out at reflux temperature for 12h-80h; when reacting with triphenylphosphine, the molar ratio of 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester to triphenylphosphine is 1:0.8-3.0, toluene is used as a solvent, and the reaction is carried out at reflux for 3-10h. (3) Preparation of target product L two-photon fluorescent dye substituted pyridine dicarboxylate: After the intermediate S1 reacts with S2 under the action of strong alkali, it is hydrolyzed to prepare the target product L: Wherein X is a halogen; R-, R', R", R'" are hydrocarbon groups; M is a metal ion; The molar ratio of S1 to S2 is 1:0.7 to 1:1.3, the strong base is selected from alcohol salts or hydroxides, the reaction temperature is -20 to 40°C, the solvent is selected from one of methanol, ethanol, propanol, acetonitrile or a mixture thereof, and the reaction time is 4 to 36 hours; the alkaline substance selected for hydrolysis is carbonate or hydroxide, and the reaction time is 10 to 60 hours.

3. The method for preparing a two-photon fluorescent dye according to claim 2, characterized in that: In the step (1), the alkaline substance is selected from one of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide or a mixture thereof.

4. The method for preparing a two-photon fluorescent dye according to claim 2, characterized in that: The strong base in step (3) is selected from one of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, potassium tert-butoxide, or a mixture thereof.

5. The method for preparing a two-photon fluorescent dye according to claim 2, characterized in that: The alkaline substance selected for hydrolysis in step (3) is one of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide, etc., or a mixture thereof.

6. Use of a two-photon fluorescent dye according to claim 1 in preparing a cell imaging reagent.

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