Two-photon fluorescent dyes containing pyridinedicarboxylic acid units, methods of making and uses thereof

Abstract

The application discloses a two-photon fluorescent dye containing pyridine dicarboxylic acid units, and a preparation method thereof. The dye has the following preparation method: 6-hydroxy-2-naphthaldehyde reacts with a sulfonic acid lactone under the action of an alkaline substance, or reacts with a dihalogenated hydrocarbon to obtain a monohalogenated hydroxyl aryl ether, and then the monohalogenated hydroxyl aryl ether reacts with a sulfite to prepare S1; 4-(4-methylphenyl) pyridine-2,6-dicarboxylic acid diethyl ester reacts with N-bromosuccinimide to obtain a bromide, and then the bromide reacts with triphenylphosphine to prepare S2; intermediates S1 and S2 react under the action of a strong base, and then hydrolysis is carried out to prepare a target product. The dye has good cell membrane permeability, preferentially targets a cell nucleus, is well dispersed in the cell nucleus, has water solubility, has very low toxicity, has a high two-photon absorption interface, and can be applied to two-photon fluorescence confocal microscopic imaging.

Description

TECHNICAL FIELD

[0001] The present application relates to a two-photon fluorescent dye containing a pyridinedicarboxylic acid unit and a preparation method and application thereof. BACKGROUND

[0002] The excitation wavelength of two-photon excitation fluorescence (TPEF) is generally longer than the emission wavelength, moving to the near-infrared region, avoiding the influence of ultraviolet laser, low photo damage, and can reduce the harm to the life system to a large extent, and TPEF will not be disturbed by Rayleigh and Raman scattering and other backgrounds far away from the near-infrared region (J Photochem Photobiol C Photochem Rev.2022, 52:100529). Two-photon excitation fluorescent probes (TPEFP) can also overcome some limitations of single-photon excitation on imaging (such as out-of-plane photobleaching and scattering), have strong penetration ability in scattering media (>500 μm), low tissue autofluorescence and self-absorption, and high resolution. The excitation source of two-photon microscopy (TPM) uses two photons with larger wavelength and lower energy, combined with appropriate TPEFP, can realize long-time imaging inside the intact tissue without tissue artifacts. The TPEF imaging technology can realize functional imaging of biological tissues at the subcellular level, realize the detection of living tissues and living cells, and can be used for direct observation of metabolism in living cells, cancer diagnosis, etc., and is 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, and various life phenomena of biological organisms are mainly manifested and completed by cells. Diseases are the result of cell lesions in local tissue structures of the human body. Fluorescence microscopic imaging has become a crucial visual tool for studying cells, and fluorescent dyes have become the most important tool 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 the progress in this field (Chem-An Asian J.2011, 6:58-69.). SUMMARY

[0004] The first technical problem to be solved by the present application is to provide a two-photon fluorescent dye containing a pyridinedicarboxylic acid unit, which has a large two-photon absorption cross-section, strong induced luminescence and good water solubility, and good cell membrane permeability, and can be used for biological imaging.

[0005] The second technical problem to be solved by the present application is to provide a preparation method of the two-photon fluorescent dye containing the pyridinedicarboxylic acid unit.

[0006] The third technical problem to be solved by the present application is to provide an application of the two-photon fluorescent dye containing the pyridinedicarboxylic acid unit in preparing a cell imaging reagent.

[0007] To solve the first technical problem, the present application provides a two-photon fluorescent dye containing a pyridinedicarboxylic acid unit, and the structural general formula is as follows:

[0008]

[0009] wherein R- is a hydrocarbon group; M and M' are metal ions.

[0010] The benzene ring and the naphthalene ring are added to the pyridinedicarboxylic acid unit, the naphthalene ring has a hydrocarbon oxy group as an electron-donating group, the pyridine ring is an electron-withdrawing group, and the naphthalene ring is bridged to the benzene ring through a pi bond, that is, a donor-π-acceptor (D-π-A) structure is formed, the conjugated system is increased, the rigid planar structure is increased, the charge transfer in 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. The two-photon fluorescent dye contains two hydrophilic carboxylate groups and one strong hydrophilic sulfonate group in the structure, and thus has good water solubility. The large two-photon absorption cross section, the strong induced luminescence, and the good water solubility enable the two-photon fluorescent dye to have good cell membrane permeability, and thus can be used for biological imaging.

[0011] The two-photon fluorescent dye containing the pyridinedicarboxylic acid unit has good single-photon fluorescence properties. The aqueous solution of the two-photon fluorescent dye has no linear absorption above about 450 nm, the maximum excitation wavelength and the emission wavelength are about 330 nm and 460 nm respectively, the Stokes shift is large, about 130 nm, the two-photon fluorescence properties are good at about 510 nm, and the absorption cross section is large in the range of 680 nm to 820 nm. The two-photon absorption cross section is the largest when the excitation wavelength is 780 nm, and thus the two-photon fluorescent dye has a wide application range.

[0012] The two-photon fluorescent dye containing the pyridinedicarboxylic acid unit has the characteristics of high fluorescence intensity, good water solubility, and low fluorescence background. The two-photon fluorescent dye can be used as a cytoplasm and organelle fluorescent dye, and can realize single-photon and two-photon imaging of cells and tissues.

[0013] To solve the second technical problem, the present application provides a preparation method of the two-photon fluorescent dye containing the pyridinedicarboxylic acid unit, and the preparation method comprises the following steps:

[0014] (1) Preparation of the intermediate S1;

[0015] 6-hydroxy-2-naphthaldehyde reacts with sultone in the presence of a base to produce S1, the structure of which is as follows:

[0016]

[0017] wherein R- is a hydrocarbon group;

[0018] The molar ratio of 6-hydroxy-2-naphthaldehyde to sultone is 1:1.5-3.5, the reaction temperature is 20-115°C, the solvent is selected from one or a mixture of methanol, ethanol, propanol, butanol, acetonitrile, tetrahydrofuran, and the base is selected from a carbonate or a hydroxide, and the reaction time is 6-30h;

[0019] (2) Preparation of intermediate S2;

[0020] S2 is prepared by reacting diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate with N-bromosuccinimide and then with triphenylphosphine, and the structure of S2 is as follows:

[0021]

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

[0023] (3) Preparation of target product L;

[0024] The target product L is prepared by reacting intermediate S1 with S2 in the presence of a strong base and then hydrolyzing, and the structure of L is as follows:

[0025]

[0026] wherein R- is a hydrocarbon group; M and M' are metal ions;

[0027] The molar ratio of S1 to S2 is 1:1-1.3, the strong base is selected from an alkoxide or a hydroxide, the reaction temperature is -20-40°C, the solvent is selected from one or a mixture of methanol, ethanol, propanol and acetonitrile, the reaction time is 4-36h, the base used for hydrolysis is a carbonate or a hydroxide, and the reaction time is 10-60h.

[0028] As preferred, the basic 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] As preferred, 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.

[0030] As preferred, the basic substance for hydrolysis in step (3) is selected from one of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide, cesium hydroxide or a mixture thereof.

[0031] To solve the above-mentioned second technical problem, the application further provides another method for preparing a two-photon fluorescent dye containing a pyridine dicarboxylic acid unit, comprising the following steps:

[0032] (1) preparation of intermediate S1;

[0033] S1 is prepared by reacting 6-hydroxy-2-naphthaldehyde with a dihalogenated hydrocarbon to obtain a monohalogenated hydrocarbon aryl ether, and then reacting the aryl ether with a sulfite, and the structure of S1 is as follows:

[0034]

[0035] wherein R- is a hydrocarbon group;

[0036] The molar ratio of 6-hydroxy-2-naphthaldehyde to dihalogenated hydrocarbon is 1:1.5-3.5, the reaction temperature is 20-115℃, the solvent is selected from one of methanol, ethanol, propanol, butanol, acetonitrile, tetrahydrofuran or a mixture thereof, and the reaction time is 6-30h; when reacting with the sulfite, the molar ratio of the sulfite to 6-hydroxy-2-naphthaldehyde is 0.5-3:1, the temperature is 25-180℃, and the reaction time is 20-72h;

[0037] (2) preparation of intermediate S2;

[0038] S2 is prepared by reacting diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate with N-bromosuccinimide to obtain a bromide, and then reacting the bromide with triphenylphosphine, and the structure of S2 is as follows:

[0039]

[0040] 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester and N-bromosuccinimide are reacted at a molar ratio of 1:1-3, with benzoyl peroxide or azobisisobutyronitrile as an initiator, the initiator accounts for 10%-30% of the amount of substance 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 a reflux temperature for 12-80 hours; when reacted 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 a reflux temperature for 3-10 hours;

[0041] (3) preparation of the target product L;

[0042] After the intermediates S1 and S2 are reacted under the action of a strong base, the target product L is prepared by hydrolysis, and the structural formula of L is as follows:

[0043]

[0044] wherein R- is a hydrocarbon group; M and M' are metal ions;

[0045] The molar ratio of S1 to S2 is 1:1-1.3, a strong base is selected from an alkoxide or a hydroxide, the reaction temperature is -20-40°C, a solvent is selected from one or a mixture of methanol, ethanol, propanol and acetonitrile, and the reaction time is 4-36 hours; when hydrolysis is performed, a basic substance selected is a carbonate or a hydroxide, and the reaction time is 10-60 hours.

[0046] Preferably, in step (3), the strong base is selected from one or a mixture of sodium methoxide, potassium methoxide, sodium ethoxide, potassium ethoxide, sodium tert-butoxide and potassium tert-butoxide;

[0047] Preferably, when hydrolysis is performed in step (3), the basic substance selected is one or a mixture of sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, potassium hydroxide and cesium hydroxide.

[0048] The preparation method of the two-photon fluorescent dye containing a pyridine dicarboxylic acid unit has reasonable design, simple steps, easily available raw materials and relatively loose reaction conditions.

[0049] To solve the third technical problem, the application provides an application of the two-photon fluorescent dye containing a pyridine dicarboxylic acid unit in the preparation of a cell imaging reagent.

[0050] The two-photon fluorescent dye provided by the application can be used for observing the location of the two-photon fluorescent dye in the cytoplasm and organelles of 4T1 cells after the two-photon fluorescent dye is used to dye the mouse breast cancer cells (4T1), the single-photon fluorescence of the two-photon fluorescent dye is blue emission, and the two-photon fluorescence of the two-photon fluorescent dye is strong and bright green emission, and the resolution of the two-photon fluorescence cell microscopic imaging is significantly increased compared with that of the single-photon fluorescence.

[0051] The two-photon fluorescent dye provided by the application is a water-soluble two-photon induced fluorescent material, has very low toxicity, and has no obvious toxicity to 4T1 cells at a concentration of 0.04-50 μmol·L -1 -1, and the cell survival rate of the 4T1 cells is above 86%, so the two-photon fluorescent dye can be used for detecting target objects in cells and organisms in the range. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a preparation method route map of the two-photon fluorescent dye containing a pyridine dicarboxylic acid unit.

[0053] Figure 2 a and b in the formula (I) are respectively the target product L. Figure 2 a and b in the formula (I) are respectively the target product L.

[0054] Figure 3 a and b in the formula (I) are respectively the target product L. Figure 3 a and b in the formula (I) are respectively the target product L.

[0055] Figure 4 is the two-photon absorption cross-section value (GM) of the target product L in water.

[0056] Figure 5 is the single-photon and two-photon fluorescence confocal microscopic imaging diagram of the target product L on 4T1 cells. Figure 5 a is the single-photon fluorescence confocal microscopic photograph of the 4T1 cells dyed by the target product L. Figure 5 b is the two-photon fluorescence confocal microscopic photograph of the 4T1 cells dyed by the target product L.

[0057] Figure 6 is the cell survival rate diagram of the 4T1 cells in the target product L aqueous solution in the concentration range of 0.04-50 μmol·L -1 -1. DETAILED DESCRIPTION

[0058] Referring to Figure 1 , the preparation method of the two-photon fluorescent dye containing a pyridine dicarboxylic acid unit comprises the following steps:

[0059] (1) Preparation of intermediate S1:

[0060] To a stirred suspension of 6-hydroxynaphthalene-2-carboxaldehyde (1.73 g, 10.05 mmol) in methanol (80 mL) at -5 °C was added dropwise a solution of potassium tert-butoxide (2.56 g, 22.81 mmol) in methanol (40 mL). After the addition was complete, the cooling was removed and the reaction was allowed to warm to room temperature and stirred for an additional 2 h. The methanol was removed under reduced pressure and the residue was taken up in acetonitrile (80 mL). After sonication, 1,4-butane sultone (3.11 g, 22.84 mmol) was added and a large amount of yellow solid precipitated. The reaction was refluxed for an additional 6 h with stirring. The reaction was cooled, filtered and the residue was washed with acetonitrile (3x). The residue was taken up in THF (50 mL) and stirred for 8 h. The residue was filtered and redispersed in THF (50 mL) and stirred for 8 h. This procedure was repeated once more. THF was replaced with acetone and the procedure was repeated 3 times. The residue was dried under vacuum at 50 °C for 4 h and recrystallized from ethanol and water to give yellow solid S1 (2.54 g, 7.33 mmol) in 73% yield. m.p. 237-239 °C. FT-IR (KBr), v / cm"1: 3060, 2947, 2875, 1682, 1627, 1472, 1391, 1271, 1180, 1047, 1008, 734. -1 :3060,2947,2875,1682,1627,1472,1391,1271,1180,1047,1008,734. 1 HNMR (600 MHz, D2O, d ppm): 9.26 (s, 1H, -CHO), 6.26-7.33 (6H, Naph-H), 3.37 (t, 2H, -OCH2-), 2.77 (t, 2H, -CH2-SO3K), 1.65-1.49 (m, 4H, -CH2CH2-). 13 C NMR (600 MHz, D2O, d ppm): 195.24, 158.77, 137.84, 135.35, 131.13, 130.93, 127.49, 127.16, 122.39, 119.17, 106.92, 67.60, 50.74, 27.29, 20.97. HRMS (ESI, m / z): Calcd. for C 15 H 15 O5S - [M-K] - 307.0646; found 307.0654.

[0061] (2) Preparation of intermediate S2:

[0062] To anhydrous carbon tetrachloride (450 mL) was added 4-(4-methylphenyl)pyridine-2,6-dicarboxylic acid diethyl ester (2.33 g, 7.44 mmol), N-bromosuccinimide (NBS, 1.39 g, 7.81 mmol), dibenzoyl peroxide (BPO, 0.45 g, 1.86 mmol) under nitrogen atmosphere, after refluxing for 16 h with stirring, NBS (0.46 g, 2.58 mmol) was added in three portions and refluxed for 24 h with stirring. Cooled, filtered, the filtrate was evaporated under reduced pressure, the residue (about 120 mL) was washed with distilled water (100 mL) for 5 times and dried over anhydrous magnesium sulfate. Filtered, the filtrate was evaporated under reduced pressure and dried under vacuum at 50 °C for 8 h. Eluted with ethyl acetate and petroleum ether (1 : 11, v / v) to give yellow solid 2.57 g. To toluene (50 mL) was added the above obtained yellow solid (2.60 g, 6.63 mmol), triphenylphosphine (4.35 g, 16.59 mmol) under nitrogen atmosphere with stirring, gradually heated and refluxed for 7 h. After cooling, filtered, the filter residue was washed with toluene for 3 times, the crude product was dried under vacuum at 60 °C for 6 h above, recrystallized with acetonitrile and ethyl acetate to give yellow solid S2 (2.48 g, 3.79 mmol), yield 57%. m.p. 233-235 °C. FT-IR (KBr), v / cm -1 : 3054, 3040, 3000, 2982, 2961, 2873, 2836, 2770, 1745, 1722, 1597, 1437, 1343, 1245, 1165, 1110, 1024, 850. 1 H NMR (400 MHz, CD3OD, d ppm): 8.42 (s, 2H, Py-H), 7.94-7.81 (19H, Ph-H), 5.18 (2H, Ph-CH2-), 4.42-4.47 (m, 4H, -CH2-), 1.43 (t, 6H, -CH3). 13 C NMR (400 MHz, 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, 29.18, 13.22. HRMS (ESI, m / z): Calcd. for C 36 H 33 NO4P + [M-Br] + 574.2142; found 574.2120.

[0063] (3) Preparation of target product L:

[0064] To a solution of S1 (0.60 g, 1.73 mmol), S2 (1.21 g, 1.85 mmol) in 100 mL of anhydrous methanol under nitrogen atmosphere at -10 °C with stirring, a solution of potassium tert-butoxide (0.86 g, 7.66 mmol) in 35 mL of anhydrous methanol was added dropwise slowly. After the addition, the reaction was continued to stir at -5 °C for about 10 h, and then at room temperature for 24 h. After the reaction was completed, filtration was performed, the filter residue was washed with methanol for 3 times, the solvent in the filtrate was removed under reduced pressure, the obtained yellow solid was stirred in anhydrous ethanol (60 mL) for 8 h, filtration was performed, and the filter residue was washed with anhydrous ethanol for 3 times. The above operation was repeated for 3 times. The filter residue was dried at 30 °C under vacuum for 8 h. Yellow solid 7 (0.93 g, 1.45 mmol) was obtained with a yield of 84%. 7 (0.65 g, 1.01 mmol) was added to a solution of KOH (0.21 g, 3.74 mmol) in water (7 mL) at room temperature, and the reaction was stirred at room temperature for 48 h. After the reaction was completed, anhydrous ethanol (100 mL) was added to mix, and then the mixture was allowed to stand for 6 h. Filtration was performed, the filter residue was washed with anhydrous ethanol for 3 times, and dried at 30 °C under vacuum for 8 h. The product was recrystallized from distilled water and isopropyl alcohol, and dried at 30 °C under vacuum for 48 h. Light yellow solid (0.34 g, 0.51 mmol) was obtained with a yield of 50%. FT-IR (KBr), v / cm-1: 3057, 2945, 1648, 1623, 1584, 1400, 1350, 1181, 1046, 1008, 832. -1 :3057, 2945, 1648, 1623, 1584, 1400, 1350, 1181, 1046, 1008, 832. 1 H NMR (600 MHz, D2O, d ppm): 7.83 (s, 2H), 6.56-7.16 (10H, Naph-H and Ph-H), 6.35 (t, J = 15.3 Hz, 1H, -HC=C=), 6.27 (t, J = 18.0 Hz, 1H, =C=CH-), 3.24 (2H, -OCH2-), 2.57 (2H, -CH2-SO3K), 1.28-1.49 (4H, -CH2CH2-). 13 C NMR (D2O, d ppm): 172.29, 161.05, 155.98, 153.32, 148.77, 137.97, 134.77, 133.68, 131.95, 129.48, 128.53, 127.21, 126.80, 126.62, 126.27, 123.32, 121.93, 118.23, 106.86, 67.33, 50.62, 27.36, 20.88. HRMS (ESI, m / z): (1) Calcd. for C 29 H 22 K2NO8S- [M-K] - 622.0346; found 622.0337; (2) Calcd. for C 29 H 22 KNO8S 2- [M-2K] 2- 291.5357; found 291.5346; (3) Calcd. for C 29 H 22 NO8S 3- [M-3K] 3- 181.3694; found 181.3681; (4) Calcd. for C 29 H 23 NO8S 2- [M-3K+H] 2- 272.5578; found 272.5579; (5) Calcd. for C 29 H 24 NO8S - [M-3K+2H] - 546.1228; found 546.1229; (6) Calcd. for C 29 H 23 KNO8S - [M-2K+H] - 584.0787; found 584.0787; (7) Calcd. for C 29 H 23 K3NO8S + [M+H] + 662.0050; found 662.0011; (8) Calcd. for C 29 H 24 K2NO8S + [M-K+2H] + 624.0491; found 624.0488; (9) Calcd. for C 29 H 22 K4NO8S + [M+K] + 699.9609; found 609.9598; (10) Calcd. for C 29 H 22 K3NNaO8S + [M+Na] + 683.9870; found 683.9844; (11) Calcd. for C29 H 23 K2NNaO8S + [M+Na-K+H] + 646.0311; found 646.0293; (12)Calcd.for C 29 H 22 K2NNa2O8S + [M+2Na-K] + 668.0130; found668.0134.

[0065] Determination of single-photon properties of target product L

[0066] The prepared L aqueous solution (concentration 2.5×10 -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 As can be seen in Figure a, 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 bridged π bond within the target product L molecule. Its aqueous solution (7.55×10 -6 mol·L -1 ) were recorded using an LS 55 fluorescence spectrophotometer. Figure 2 As can be seen in b, the maximum excitation wavelength and emission wavelength are 328 nm and 471 nm, respectively, and the Stokes shift is large, reaching 143 nm. It is speculated that the emission peak of L can be attributed to the emission caused by intramolecular charge transfer transition.

[0067] Determination of the two-photon properties of the target product L

[0068] Two-photon-induced fluorescence (TPIF) measurements were performed using femtosecond laser pulses and a Ti-sapphire system (excitation wavelength range: 680–1080 nm, 80 MHz, 140 fs, Chameleon Ultra II) as the pump light source. All measurements were performed in air at room temperature (below 20°C). TPIF measurements were used to measure the TPA cross-section using an integration time of 300 ms and a power of 0.5 W.

[0069] The concentration of the aqueous solution of the target product L during 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 fluorescein solid to prepare 1mmol·L -1 Fluorescein reference stock solution. Pipette 300 μL and dilute to 3 mL to obtain a concentration of 0.1 mmol·L -1 Fluorescein NaOH aqueous solution (where the concentration of NaOH is 1 mol·L -1 ) was used as a reference. The sample cell was a 1.0×1.0 cm four-sided transparent quartz cuvette. The laser light source power was controlled at 300 mW, the wavelength range was 680-880 nm, and the wavelength interval was 20 nm. The two-photon induced fluorescence spectrum was 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 760nm. Therefore, the maximum fluorescence intensity is at 760nm, that is, 760nm is the optimal excitation wavelength (see Figure 3 In a), Figure 2 In the figure a, it is shown that 8 has no single-photon absorption above 450nm, so L has maximum two-photon absorption at 760nm.

[0070] At the optimal excitation wavelength of 760 nm for the target product L, the excitation light intensity was changed by adjusting the laser power, and the two-photon induced fluorescence intensity of L under different excitation light intensities was measured to further verify the two-photon characteristics of L. The output / input intensity power logarithm ratio of the target product L in aqueous solution is lgI out / lgI in (Slope) is around 2 (see Figure 3 Middle b) is consistent with the characteristics of two-photon excitation, further proving that L has two-photon activity.

[0071] 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:

[0072] δ s =δ r (C r n r Φ r F s ) / (C s n s Φ s F r )

[0073] 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, Фr = 0.97 from literature, F is the integrated area value of the corresponding two-photon fluorescence intensity at different excitation wavelengths.

[0074] The calculated results of two-photon absorption cross section of target product L in water are shown in Figure 4 The two-photon absorption cross section of target product L in aqueous solution has a maximum value at about 760 nm, and the two-photon absorption cross section value is about 100 GM.

[0075] Fluorescent cellular microscopy of target product L

[0076] 4T1 cells were fixed with formaldehyde and loaded with L in PBS (phosphate buffered solution for cell culture) solution for fluorescent cellular microscopy. First, 4T1 cells were seeded on a 35 mm diameter cell culture dish and placed in a culture box containing 5% CO2 and 95% oxygen, and incubated at 37°C for 24 h. After washing the 4T1 cells with PBS three times to remove the culture medium, the 4T1 cells were incubated with PBS solution containing L (10 μmol·L -1 ) for 4 h, and then washed with PBS three times to remove excess L. Then the cells were fixed with 4% formaldehyde PBS solution. Finally, the treated and colored 4T1 cells were subjected to fluorescent cellular microscopy.

[0077] Single-photon fluorescent cellular microscopy was performed on an FV1200 / FV1200MPE laser confocal microscope, and CY3, AF488, and DAPI dye channels were used for imaging (see Figure 5 a). Their excitation wavelengths and maximum emission wavelengths were 550 nm and 570 nm, 488 nm and 519 nm, and 405 nm and 488 nm, respectively. As can be seen from Figure 5 a, single-photon fluorescent cellular microscopy showed blue light emission.

[0078] Two-photon fluorescent cellular microscopy was performed on an FV1200 / FV1200MPE laser confocal microscope, and excitation wavelengths in the range of 700-1000 nm could be imaged, but 800 nm was the best. Therefore, an excitation wavelength of 800 nm was selected, and the fluorescence of two channels, 426-460 nm and 495-540 nm, was collected. The results are shown in Figure 5 b. As can be seen from Figure 5 b, two-photon fluorescence showed strong and bright green light emission, and the resolution of two-photon fluorescent cellular microscopy was significantly increased compared to single-photon.

[0079] As can be seen from Figure 5 b, target product L penetrates the cell membrane of 4T1 cells and preferentially targets the nucleus, and is well dispersed in the nucleus, indicating that the target product has a high localization ability for the nucleus of 4T1 cells.

[0080] Determination of cytotoxicity of target product L

[0081] Cell Counting Kit-8 cell counting reagent (CCK-8) was used to detect cell survival rate (CCK-8 method). 4T1 cells stably growing 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, and placed in a constant temperature incubator at 37°C and 5% CO2. After the cells adhered, the culture medium containing different concentrations of drugs was replaced, and after incubation for 24 h, CCK-8 solution was added, protected from light, and incubated in a 37°C incubator for 2 h. The absorbance at 450 nm was measured by an enzyme-labeled instrument. The cell survival rate was calculated according to the following formula:

[0082] CSR = [(A s -A b ) / (A c -A b )] × 100%

[0083] In the formula, A s is the absorbance of the experimental group (containing 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, and no drug), and A b is the absorbance of the blank group (containing culture medium, CCK-8 solution, and no cells or drugs).

[0084] The detection results are shown in Figure 6 . It can be seen from Figure 6 that target product L has no toxicity to 4T1 cells, and the cell survival rate is above 86%. In the concentration range of 0.4-50 μmol·L -1 , it can be used for detection of target substances in cells and organisms.

Claims

1. A two-photon fluorescent dye containing a pyridinedicarboxylic acid unit, characterized in that: The structural formula is as follows:

2. The method for preparing a two-photon fluorescent dye containing a pyridinedicarboxylic acid unit according to claim 1, wherein: The following steps are involved: (1) Preparation of intermediate S1; To 80 mL of a methanol suspension containing 10.05 mmol of 6-hydroxynaphthalene-2-carboxaldehyde was added dropwise 40 mL of a methanol solution containing 22.81 mmol of potassium tert-butoxide at -5°C with stirring. After the addition was completed, cooling was stopped, the mixture was naturally warmed to room temperature, and stirred for 2 h. The methanol was removed from the reaction mixture under reduced pressure, and 80 mL of acetonitrile was added to the residue. After ultrasonic dispersion, 22.84 mmol of 1,4-butane sultone was added, and a large amount of yellow solid was rapidly precipitated. The mixture was refluxed under stirring for 6 h, cooled, filtered, and the filter residue was washed three times with acetonitrile. The filter residue was dispersed in 50 mL of THF and stirred for 8 h. The filter residue was then filtered and redispersed in 50 mL of THF and stirred for 8 h. The above operation was repeated once, and the tetrahydrofuran was replaced with acetone and the above operation was repeated three times. The filter residue was dried in vacuo at 50°C for 4 h and then recrystallized from ethanol and water to obtain 2.54 g of yellow solid S1 with a substance amount of 7.33 mmol. The structural formula of S1 is as follows: (2) Preparation of intermediate S2; Under nitrogen atmosphere, 7.44 mmol of diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate, 7.81 mmol of N-bromosuccinimide NBS, and 1.86 mmol of dibenzoyl peroxide were added to 450 mL of anhydrous carbon tetrachloride. After stirring and reflux reaction for 16 h, 2.58 mmol of diethyl 4-(4-methylphenyl)pyridine-2,6-dicarboxylate were added in three portions. NBS and reflux under stirring for 24 hours, cool, filter, remove the solvent from the filtrate under reduced pressure, wash the residue with distilled water 5 times, dry over anhydrous magnesium sulfate, filter, remove the solvent from the filtrate under reduced pressure, dry in vacuo at 50°C for 8 hours, and elute with ethyl acetate and petroleum ether in a volume ratio of 1:11 to obtain 2.57 g of a yellow solid. To 50 mL of toluene under a nitrogen atmosphere were added 2.60 g of the yellow solid obtained above, the amount of substance was 6.63 mmol, and 16.59 mmol of triphenylphosphine. The temperature was gradually increased and refluxed for 7 hours. After cooling, filter, wash the filter residue with toluene 3 times, and the crude product was dried in vacuo at 60°C for more than 6 hours, and then recrystallized from acetonitrile and ethyl acetate to obtain 2.48 g of S2 as a yellow solid, the amount of substance was 3.79 mmol; the structural formula of S2 is as follows: (3) Preparation of target product L; To a 100 mL anhydrous methanol solution containing 1.73 mmol S1 and 1.85 mmol S2 was slowly added dropwise 35 mL anhydrous methanol solution containing 7.66 mmol potassium tert-butoxide at -10°C in a nitrogen atmosphere with stirring. After the addition, the reaction was continued at -5°C with stirring for 10 h, and then at room temperature for another 24 h. After the reaction was completed, the solution was filtered, and the filter residue was washed three times with methanol. The solvent in the filtrate was removed under reduced pressure. The obtained yellow solid was added to 60 mL anhydrous ethanol and stirred for 8 h. The solution was filtered and the filter residue was washed three times with anhydrous ethanol. The above operation was repeated three times. The filter residue was vacuum dried at 30°C for 8 h to obtain 0.93 g of a yellow solid. 0.65 g of the yellow solid was added to 7 mL of a solution containing 3.74 mmol at room temperature. The reaction mixture was stirred in a KOH aqueous solution at room temperature for 48 h. After the reaction was completed, 100 mL of anhydrous ethanol was added and mixed, and the mixture was allowed to stand for 6 h. The residue was filtered, and the filter residue was washed three times with anhydrous ethanol and dried in vacuum at 30°C for 8 h. The residue was recrystallized with distilled water and isopropanol, and dried in vacuum at 30°C for 48 h to obtain 0.34 g of light yellow target product L. The amount of substance was 0.51 mmol. The structural formula of L is as follows:

3. Use of the two-photon fluorescent dye containing a pyridinedicarboxylic acid unit according to claim 1 in the preparation of a cell imaging reagent.

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