A two-photon fluorescent dye, its preparation method and application

Abstract

This invention discloses a two-photon fluorescent dye, prepared as follows: 6-hydroxy-2-naphthaldehyde reacts with a dihaloalkane under alkaline conditions to obtain a monohaloalkane-based aryl ether, which is then reacted with an aqueous trimethylamine solution to prepare S1; diethyl 4-(4-tolyl)pyridine-2,6-dicarboxylate reacts with N-bromosuccinimide to obtain a brominated product, which is then reacted with triphenylphosphine to prepare S2. After intermediates S1 and S2 react under strongly alkaline conditions, the product is then hydrolyzed to prepare the target product. This dye exhibits good cell membrane permeability, good dispersion in the cytoplasm, preferentially targets organelles in the cytoplasm, is water-soluble, has very low toxicity, and a high two-photon absorption interface, making it suitable for two-photon fluorescence confocal microscopy imaging.

Description

Technical Field

[0001] This invention relates to a two-photon fluorescent dye, its preparation method, and its application. Background Technology

[0002] Two-photon excitation fluorescence (TPEF) typically uses an excitation wavelength longer than the emission wavelength, shifting it to the near-infrared region. This avoids the influence of ultraviolet lasers, resulting in low optical damage and significantly reducing harm to living systems. Furthermore, TPEF is unaffected by background interference from Rayleigh and Raman scattering at wavelengths far from the near-infrared region (J Photochem Photobiol C Photochem Rev. 2022, 52:100529). Two-photon excitation fluorescence probes (TPEFP) can also overcome some limitations of single-photon excitation for imaging (such as out-of-plane bleaching and scattering). They exhibit strong penetration in scattering media (>500 μm), low tissue autofluorescence self-absorption, and high resolution. Two-photon microscopy (TPM) uses two photons with longer wavelengths and lower energy as the excitation source. Combined with an appropriate TPEFP, it can achieve long-term imaging within intact tissues without producing tissue artifacts. TPEF imaging technology can perform functional imaging of biological tissues at the subcellular level, enabling the detection of living tissues and cells. It can be used to directly observe metabolism within living cells, cancer diagnosis, and other applications, and is quite 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 in living organisms are primarily manifested and accomplished through cells; diseases are the result of pathological changes in cells within local tissue structures. Fluorescence microscopy has become a crucial visual tool for cellular research, and fluorescent dyes have become the most important supporting tool in microscopic imaging studies. However, most fluorescent dyes used for fluorescence imaging have poor water solubility and low two-photon absorption cross-sections (δ), which limits their use in total photomicroscopy (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 with a large two-photon absorption cross section, strong induced luminescence and good water solubility, which gives it good cell membrane permeability and can be used for bioimaging.

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

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

[0007] To solve the first technical problem mentioned above, the present invention provides a two-photon fluorescent dye with the following general structural formula:

[0008]

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

[0010] A benzene ring and a naphthalene ring are added to the pyridine dicarboxylic acid unit. The naphthalene ring has an alkyl group as an electron-donating group, and the pyridine ring is an electron-withdrawing group. The naphthalene ring is bridged to the benzene ring by a π bond, thus possessing a electron-donating-π-acceptor structure (donor-π-A). This increases the conjugation system, the number of rigid planar structures, the intramolecular charge transfer, the absorption of light radiation, the two-photon absorption cross section, and the fluorescence intensity. The presence of two hydrophilic carboxylate groups and one strongly hydrophilic quaternary ammonium group in its structure gives it good water solubility. Its large two-photon absorption cross section, strong induced luminescence, and good water solubility make it suitable for cell membrane permeability and bioimaging applications.

[0011] The two-photon fluorescent dye described in this invention has excellent single-photon fluorescence properties. Its aqueous solution has no linear absorption above approximately 450 nm, with maximum excitation and emission wavelengths of approximately 330 nm and 460 nm, respectively. It exhibits a large Stokes shift of approximately 130 nm. It also has good two-photon fluorescence properties around 510 nm and a large absorption cross-section in the range of 680 nm to 820 nm. The two-photon absorption cross-section is largest at an excitation wavelength of 780 nm, reaching approximately 85 GM, thus having a wide range of two-photon applications.

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

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

[0014] (1) Preparation of intermediate S1:

[0015] The monohaloalkyl aryl ether obtained by reacting 6-hydroxy-2-naphthaldehyde with a dihaloalkyl group under alkaline conditions is then reacted with an aqueous trimethylamine solution to prepare S1:

[0016]

[0017] Where X is a halogen; R-, R', R”, R”' are hydrocarbon groups;

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

[0019] (2) Preparation of intermediate S2:

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

[0021]

[0022] The molar ratio of 4-(4-tolyl)pyridine-2,6-dicarboxylate to N-bromosuccinimide is 1:1 to 1:3, with benzoyl peroxide or azobisisobutyronitrile as the initiator, wherein the initiator accounts for 10% to 30% of the molar amount of 4-(4-tolyl)pyridine-2,6-dicarboxylate, and carbon tetrachloride as the solvent. The reaction is carried out at reflux for 12 to 80 hours. When reacting with triphenylphosphine, the molar ratio of 4-(4-tolyl)pyridine-2,6-dicarboxylate to triphenylphosphine is 1:0.8 to 3.0, with toluene as the solvent, and the reaction is carried out at reflux for 3 to 10 hours.

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

[0024] After intermediates S1 and S2 react under strong alkaline conditions, hydrolysis is then performed to prepare the target product L.

[0025]

[0026] Where 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 an alkoxide or hydroxide. The reaction temperature is -20 to 40°C. The solvent is selected from one or a mixture of methanol, ethanol, propanol, and acetonitrile. The reaction time is 4 to 36 hours. The alkaline substance used for hydrolysis is a carbonate or hydroxide. The reaction time is 10 to 60 hours.

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

[0029] Preferably, the strong alkali in step (3) is selected from one or a mixture of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide, and potassium tert-butoxide.

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

[0031] The preparation method of the two-photon fluorescent dye described in this invention is reasonably designed, simple in steps, uses readily available raw materials, and has relatively relaxed reaction conditions.

[0032] To address the third technical problem mentioned above, this invention provides the application of the aforementioned two-photon fluorescent dye in the preparation of cell imaging reagents.

[0033] In fluorescence confocal microscopy, the two-photon fluorescent dye provided by this invention can be used to stain mouse breast cancer cells (4T1). After staining with the two-photon fluorescent dye of this invention, it can be observed that the two-photon fluorescent dye is located in the cytoplasm and organelles of 4T1 cells. Single-photon fluorescence shows blue emission, while two-photon fluorescence shows strong and bright green emission. The resolution of two-photon fluorescence cell microscopy is significantly increased compared with single-photon fluorescence.

[0034] The two-photon fluorescent dye described in this invention is a water-soluble two-photon induced fluorescent material with very low toxicity, and is effective at concentrations of 0.02-25 μmol·L⁻¹. -1 Within this range, it has virtually no significant toxicity to 4T1 cells, and the cell survival rate is above 89%, making it suitable for the detection of targets in cells and organisms within this range. Attached Figure Description

[0035] Figure 1 This is a flowchart illustrating the preparation method of the two-photon fluorescent dye-substituted pyridine dicarboxylate of this invention.

[0036] Figure 2 a, Figure 2 b and Figure 2In the diagram, c represents the UV-Vis absorption spectrum, fluorescence excitation spectrum, and fluorescence emission spectrum of the target product L aqueous solution, respectively.

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

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

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

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

[0041] For the preparation method of two-photon fluorescent dyes, see [link to documentation]. Figure 1 This includes the following steps:

[0042] (1) Preparation of intermediate S1:

[0043] Under 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). The mixture was refluxed with stirring for 24 h, cooled, filtered, and the residue was washed three times with acetonitrile. The solvent was removed from the filtrate under reduced pressure. The crude product was eluted by column chromatography with petroleum ether and ethyl acetate and petroleum ether (1:9, v / v) to give 6.71 g of a pale yellow solid. The pale yellow solid (0.63 g, 1.88 mmol), acetonitrile (45 mL), and 30% trimethylamine aqueous solution (18 mL) were added to each of five 90 mL polytetrafluoroethylene pressure vessels, and the mixtures were reacted at 90 °C for 30 h. After cooling, the reaction mixtures were combined and the solvent was removed under reduced pressure. The mixture was recrystallized with acetonitrile as the solvent to give 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 H NMR(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] Under a nitrogen atmosphere, diethyl 4-(4-tolyl)pyridine-2,6-dicarboxylate (3.89 g, 12.41 mmol), N-bromosuccinimide (NBS, 2.31 g, 12.98 mmol), and dibenzoyl peroxide (BPO, 0.71 g, 2.93 mmol) were added to anhydrous carbon tetrachloride (800 mL). The mixture was refluxed with stirring for 18 h. Then, NBS (0.77 g, 4.33 mmol) was added in three separate additions, and the mixture was refluxed with stirring for 24 h each time. The mixture was cooled, filtered, and the solvent was removed from the filtrate under reduced pressure. The residue (approximately 200 mL) was washed five times with distilled water (150 mL) and dried over anhydrous magnesium sulfate. The residue was filtered again, the solvent was removed from the filtrate under reduced pressure, and the mixture was dried under vacuum at 50 °C for 8 h. The sample was eluted with ethyl acetate and petroleum ether (1:11, v / v) to give 4.25 g of a yellow solid. Under stirring and a nitrogen atmosphere, the obtained yellow solid (3.93 g, 10.02 mmol) and triphenylphosphine (6.59 g, 25.13 mmol) were added to toluene (50 mL), and the mixture was gradually heated and refluxed for 7 h. After cooling, the mixture was filtered, and the residue was washed three times with toluene. The crude product was dried under vacuum at 60 °C for at least 6 h, and then recrystallized from acetonitrile and ethyl acetate to give 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).13C NMR(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 a nitrogen atmosphere at -10°C, with stirring, 45 mL of anhydrous methanol solution containing 0.71 g (6.33 mmol) of potassium tert-butoxide was slowly added dropwise to 30 mL of anhydrous methanol solution containing S1 (0.61 g, 1.55 mmol) and S2 (1.05 g, 1.60 mmol). After the addition was complete, the reaction was continued at -5°C for approximately 8 h with stirring, followed by a further reaction at room temperature for 24 h. After the reaction was complete, the solvent was removed under reduced pressure, and the resulting yellow solid was added to 70 mL of tetrahydrofuran and stirred for 8 h. The mixture was then filtered, and the residue was washed three times with tetrahydrofuran. This process was repeated three times. The residue was dried under vacuum at 30°C for 8 h, yielding 1.01 g of yellow solid. This yellow solid was then added to 12 mL of an aqueous solution of KOH (0.27 g, 4.81 mmol) at room temperature, and the mixture was stirred at room temperature for 48 h. After the reaction was complete, acetonitrile (200 mL) was added, mixed well, and allowed to stand for 6 h. The mixture was then filtered, and the residue was washed three times with acetonitrile and dried under vacuum at 30 °C for 8 h. Recrystallization was performed with distilled water and isopropanol, followed by drying under vacuum at 30 °C for 48 h. The target product L (0.63 g, 0.89 mmol) was obtained as a pale yellow solid, with a yield of 61%. FT-IR (KBr), v / 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),7.08-7.86(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),1.43-1.87(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 of 2.0 × 10⁻⁶) -5 mol·L -1 The sample was placed in a 1 cm quartz cuvette, and the UV-Vis absorption spectrum was collected using an EVOLUTION 220 UV-Vis spectrophotometer, with the scanning wavelength range set to 190–1100 nm. Figure 2 As can be seen from Figure a, the aqueous solution of the target product L exhibits no linear absorption above 450 nm. The target product L molecule contains charge transfer from the naphthalene ring to the pyridine group via bridging π bonds. Its aqueous solution (8.33 × 10⁻⁶ nm) shows... -6 mol·L -1 The steady-state excitation and emission spectra of the sample were recorded using an LS 55 fluorescence spectrophotometer. Figure 2 b and Figure 2 As can be seen from c, the maximum excitation wavelength and emission wavelength are 328 nm and 459 nm, respectively. The Stokes shift is relatively large, reaching 131 nm. 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] Two-photon induced fluorescence (2PS) measurements were performed using a femtosecond laser pulse and a Ti sapphire system (excitation wavelength range: 680–1080 nm, 80 MHz, 140 fs, Chameleon Ultra II) as the pump source. The 2PS fluorescence spectra were measured. All measurements were conducted in air at room temperature (below 20 °C). With an integration time of 300 ms and a power of 0.5 W, the 2PS absorption cross-section was measured using 2PS measurement technology.

[0052] The concentration of the target product L in aqueous solution was 0.1 mmol·L⁻¹ during the experiment. -1 Preparation of the fluorescein reference stock solution: Weigh 0.0017 g of fluorescein solid into a centrifuge tube, and transfer 5 mL of NaOH standard solution (1.000 mol·L⁻¹) to a centrifuge tube. -1 Dissolve the solid fluorescein to prepare a 1 mmol·L⁻¹ solution. -1 The fluorescein reference stock solution was further diluted with 300 μL to 3 mL, yielding a concentration of 0.1 mmol·L⁻¹. -1 Fluorescein NaOH aqueous solution (where the NaOH concentration is 1 mol·L⁻¹) -1(A sample was used as a reference.) The sample cell was a 1.0 × 1.0 cm four-sided transparent quartz cuvette. The laser source power was controlled at 300 mW, the wavelength range was 700-900 nm, and the wavelength interval was 20 nm. The two-photon induced fluorescence spectrum was measured, and the results are shown in [Figure number missing]. Figure 3 As shown in Figure a, the fluorescence intensity increases with increasing excitation wavelength, but begins to decrease after reaching 780 nm. Therefore, the maximum fluorescence intensity is found at 780 nm, which is the optimal excitation wavelength (see Figure a). Figure 3 a), and Figure 2 The value of 'a' indicates that 8 has no single-photon absorption above 450nm, therefore L has maximum two-photon absorption at 780nm.

[0053] At the optimal excitation wavelength of 780 nm for the target product L, the laser power I was adjusted. in (mW) By changing the excitation light intensity, the two-photon induced fluorescence intensity I under different excitation light intensities was measured. out (au) Further verification of the two-photon characteristics of L. The logarithmic ratio of the output / input intensity power of the target product L in aqueous solution, lgI. out / lgI in (Slope) is around 2 (see...) Figure 3 (b) shows that L is well-suited to the characteristics of two-photon excitation, further proving that L has two-photon activity.

[0054] The two-photon absorption cross section (δ) of the compound was calculated using two-photon induced fluorescence method, with a fluorescein aqueous solution at pH 14 as the reference reagent. 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] In the formula, s represents the sample, r represents the reference, δ is the two-photon absorption cross section, and the fluorophore of the reference sample is δ. r = 43GM (wavelength 790nm), C is the concentration of the solution, and the reference sample fluorescein was prepared in NaOH aqueous solution with a concentration of 0.1 mmol·L⁻¹. -1 n is the refractive index of the solvent, Ф is the two-photon quantum yield, and Ф r =0.97 is from the literature, and F is the integral 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, with a two-photon absorption cross section value of approximately 86 GM.

[0058] L-fluorescence cell microscopy of the target product:

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

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

[0061] Two-photon fluorescence cell imaging was performed using an FV1200 / FV1200MPE laser confocal microscope. Imaging was possible with excitation wavelengths in the range of 950-700 nm, but 800 nm was optimal. Therefore, an excitation wavelength of 800 nm was selected, and fluorescence was collected from two channels at 426-460 nm and 495-540 nm. The results are shown in [Figure number missing]. Figure 5 b. From Figure 5 As can be seen in b, two-photon fluorescence exhibits strong and bright green light emission, and the resolution of two-photon fluorescence cell microscopy is significantly increased compared to single-photon fluorescence.

[0062] from Figure 5 As shown in Figure b, the target product L penetrates the cell membrane of 4T1 cells and enters the cytoplasm, where it disperses well, indicating that the target product has a high localization ability in the cytoplasm of 4T1 cells. Furthermore, several bright spots were observed in the cytoplasm, suggesting that L preferentially targets organelles within the cytoplasm.

[0063] Target product L cytotoxicity

[0064] Cell viability was determined using the Cell Counting Kit-8 (CCK-8 assay). Stable 4T1 cells in the logarithmic growth phase were collected and the cell density was adjusted to 5 × 10⁶ cells / cells. 4 cells·mL -1 Cells were seeded in 96-well cell culture plates and incubated at 37°C with 5% CO2. After cell attachment, the culture medium was replaced with different concentrations of drug, and incubated for 24 hours. Then, CCK-8 solution was added, and the plates were incubated in the dark at 37°C for 2 hours. The absorbance at 450 nm was measured using a microplate reader. Cell survival rate was calculated using the following formula:

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

[0066] In the formula, A s The absorbance of the experimental group (including cells, culture medium, CCK-8 solution, and drug solution), A c The absorbance of the control group (containing cells, culture medium, and CCK-8 solution, but without drugs) is A. b The absorbance is for the blank control group (containing culture medium and CCK-8 solution, but excluding cells and drugs).

[0067] Test results are shown Figure 6 .Depend on Figure 6 It can be seen that the target product L is essentially non-toxic to 4T1 cells, with cell viability exceeding 89% at concentrations of 0.2-25 μmol·L⁻¹. -1 Within its range, it can be used to detect targets in cells and organisms.

Claims

1. A two-photon fluorescent dye, characterized in that, Its structural formula is as follows:

2. The method for preparing a two-photon fluorescent dye according to claim 1, characterized in that, Includes the following steps: (1) Preparation of intermediate S1: Under a nitrogen atmosphere, 7.11 g of 41.29 mmol of 6-hydroxy-2-naphthaldehyde, 19 mL of 1,6-dibromohexane, and 9.69 g of 70.11 mmol of anhydrous potassium carbonate were added to 100 mL of acetonitrile. The mixture was refluxed with stirring for 24 h, cooled, filtered, and the residue was washed three times with acetonitrile. The solvent was removed from the filtrate under reduced pressure. The crude product was eluted by column chromatography with petroleum ether and a mixture of ethyl acetate and petroleum ether to obtain 6.71 g of a pale yellow solid, in which the volume ratio of ethyl acetate and petroleum ether was 1:

9. 0.63 g of the pale yellow solid (1.88 mmol), 45 mL of acetonitrile, and 18 mL of 30% trimethylamine aqueous solution were added to each of five 90 mL polytetrafluoroethylene pressure vessels, and the mixture was reacted at 90 °C for 30 h. After cooling, the reaction mixtures were combined and the solvent was removed under reduced pressure. Recrystallization was performed using acetonitrile as the solvent to obtain 2.01 g of a white solid, S1, with a molar amount of 5.10 mmol. The structural formula of S1 is: (2) Preparation of intermediate S2: Under a nitrogen atmosphere, 3.89 g of 12.41 mmol of diethyl 4-(4-tolyl)pyridine-2,6-dicarboxylate, 2.31 g of 12.98 mmol of N-bromosuccinimide (NBS), and 0.71 g of 2.93 mmol of benzoyl peroxide (BPO) were added to 800 mL of anhydrous carbon tetrachloride. The mixture was refluxed with stirring for 18 h. Then, 4.33 mmol of NBS was added in three separate additions, and the mixture was refluxed with stirring for 24 h each time. Cool, filter, remove solvent from filtrate under reduced pressure, wash residue five times with 150 mL distilled water, and dry with anhydrous magnesium sulfate; filter, remove solvent from filtrate under reduced pressure, and dry under vacuum at 50 °C for 8 h; elute with a mixture of ethyl acetate and petroleum ether to obtain 4.25 g of yellow solid, the volume ratio of ethyl acetate and petroleum ether being 1:11; under stirring and a nitrogen atmosphere, add 3.93 g (10.02 mmol) of the obtained yellow solid and 6.59 g (25.13 mmol) of triphenylphosphine to 50 mL of toluene, gradually heat and reflux for 7 h; cool and filter, wash residue three times with toluene, dry crude product under vacuum at 60 °C for more than 6 h, and recrystallize from acetonitrile and ethyl acetate to obtain 3.70 g (5.65 mmol) of yellow solid S2, the structural formula of S2 is: (3) Preparation of the target product L-two-photon fluorescent dye substituted pyridine dicarboxylate: Under stirring and in a nitrogen atmosphere at -10°C, 45 mL of anhydrous methanol solution containing 6.33 mmol potassium tert-butoxide was slowly added dropwise to 30 mL of anhydrous methanol solution containing 1.55 mmol S1 and 1.60 mmol S2. After the addition was complete, the reaction was continued at -5°C for approximately 8 hours, followed by a further reaction at room temperature for 24 hours. Upon completion of the reaction, the solvent was removed under reduced pressure. The resulting yellow solid was added to 70 mL of tetrahydrofuran and stirred for 8 hours. The mixture was then filtered, and the residue was washed three times with tetrahydrofuran. This process was repeated three times. The residue was then dried under vacuum at 30°C for 8 hours to obtain 1.01 g of the yellow solid. This yellow solid was added to 12 mL of a KOH aqueous solution containing 4.81 mmol of KOH at room temperature, and the mixture was stirred for 48 hours. Upon completion of the reaction, 200 mL of acetonitrile was added, mixed thoroughly, and allowed to stand for 6 hours. The mixture was then filtered, and the residue was washed three times with acetonitrile and dried under vacuum at 30°C for 8 hours. The residue was then recrystallized from distilled water and isopropanol, and dried under vacuum at 30°C for 48 hours to obtain a pale yellow solid, the target product L, with a molar amount of 0.89 mmol. The structure of L is as follows:

3. The application of the two-photon fluorescent dye according to claim 1 in the preparation of cell imaging reagents.

Images