CN107382991B - Two-photon fluorescent material benzoxazolyl pyridine salt and preparation method and application thereof - Google Patents

Two-photon fluorescent material benzoxazolyl pyridine salt and preparation method and application thereof Download PDF

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CN107382991B
CN107382991B CN201710671589.1A CN201710671589A CN107382991B CN 107382991 B CN107382991 B CN 107382991B CN 201710671589 A CN201710671589 A CN 201710671589A CN 107382991 B CN107382991 B CN 107382991B
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许洪康
周虹屏
刘丹
王海燕
孔林
田玉鹏
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Abstract

The invention discloses a two-photon fluorescent material benzoxazolyl pyridine salt, a preparation method and application thereof. The structural formula of the two-photon fluorescent material benzoxazolyl pyridine salt is as follows:
Figure DDA0001373167060000011

Description

Two-photon fluorescent material benzoxazolyl pyridine salt and preparation method and application thereof
Technical Field
The invention relates to the field of fluorescent materials, in particular to a two-photon fluorescent material benzoxazolyl pyridine salt and a preparation method and application thereof.
Background
In recent years, organic two-photon materials have attracted much attention in many fields, especially in two-photon fluorescence microscopy and imaging. Two-photon confocal microscopes have many advantages over single-photon confocal microscopes: (1) the two-photon confocal microscope can adopt infrared laser with longer wavelength (near infrared region) and stronger penetrability in biological tissues as an excitation light source, can greatly reduce the absorption and scattering of the biological tissues to the excitation light, can obtain stronger sample fluorescence and solves the problem of tomography of deep substances in the biological tissues. (2) Dark field imaging can be achieved because the two-photon fluorescence emission wavelength is far from the excitation light wavelength. (3) Two-photon fluorescence can avoid the fluorescence bleaching problem and the toxicity to biological cells in common fluorescence imaging. (4) The two-photon transition has strong selective excitation, and is beneficial to imaging research on some special substances in biological tissues. (5) The two-photon confocal microscope has higher transverse resolution and longitudinal resolution, the two-photon absorption intensity of the material is related to the square of the excitation light intensity, and under the condition of confocal, the two-photon absorption is only limited in a narrow space volume range at the focal point of the objective lens, so that the design of the confocal microscope is greatly simplified, and the operation is easy.
However, at present, the two-photon absorption cross section of a general material is usually many orders of magnitude smaller than the single-photon absorption cross section, so that the practical application of two-photon absorption is limited. The two-photon absorption cross section increases in magnitude in relation to the type of conjugated chain bridge, and for most organic molecules, their two-photon absorption cross sections exhibit wavelength dependence. The research on the correlation rule between the structure of organic molecules and the absorption of two photons, the fluorescence characteristics of two photons, the energy transmission mechanism in molecules and the like has become a hot problem in the current international research on two-photon materials.
Disclosure of Invention
The invention aims to solve the technical problem of providing a water-soluble and low-toxicity two-photon fluorescent material benzoxazolyl pyridine salt with two-photon induced fluorescence emission capability and a preparation method and application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme: the two-photon fluorescent material benzoxazolyl pyridine salt has the following structural formula:
Figure BDA0001373167040000011
the application provided by the invention is the application of the benzoxazolyl pyridinium serving as the two-photon fluorescent material in cell imaging.
Benzoxazoles have higher fluorescence quantum yield, but have dipole structures which are not enough to generate obvious ICT processes to enhance the absorption of light radiation, and the inventors found that abundant optical properties can be generated by modifying a push-pull electron group on 2-position or 6-position of the benzoxazoles to adjust the molecular polarity. Through repeated demonstration and experiments, the inventor selects pyridine cations for modification, the pyridine cations are used as strong electron-withdrawing groups, the pyridine cations have excellent membrane penetrability and can penetrate cell membranes into cells in a short time, and the pyridine salt compounds generally have good water solubility and have the prerequisite condition of biological imaging.
The preparation method of the benzoxazolyl pyridine salt serving as the two-photon fluorescent material comprises the following steps:
A. preparation of intermediate M1(2- (p-tolyl) benzoxazole)
Mixing p-methylbenzoic acid, o-aminophenol and polyphosphoric acid, refluxing for 4-7 hours at 210-240 ℃, and extracting, drying and recrystallizing the obtained reaction liquid to obtain an intermediate M1;
B. preparation of intermediate M2(2- (4- (bromomethyl) phenyl) benzoxazole)
Dissolving M1 and N-bromosuccinimide in benzene, adding an initiator benzoyl peroxide, reacting at 60-80 ℃ for 6-8 h, and standing, filtering, washing and drying the obtained reaction solution to obtain an intermediate M2;
C. preparation of intermediate M3(2- (4- ((triphenylphosphine) methyl) phenyl) benzoxazole)
Dissolving M2 in benzene, and adding PPh3Reacting for 6-8 h at 40-80 ℃, and filtering and drying the obtained reaction solution while the reaction solution is hot to obtain an intermediate M3;
D. preparation of intermediate M4(E-2- (4- (2- (pyridin-4-yl) ethenyl) phenyl) benzoxazole)
Adding M3, 4-pyridylaldehyde and potassium carbonate into N, N-dimethylacetamide, reacting at 140 ℃ for 10-14 h, adding dichloromethane into the obtained reaction liquid, performing suction filtration, washing the filtrate with water, and drying to obtain an intermediate M4;
E. preparation of intermediate M5(E-4- (4- (2-benzoxazolyl) -2-styryl) -N-methylpyridine iodide salt)
Dissolving M4 in tetrahydrofuran, and adding CH3I, reacting for 3-6 h at 40-60 ℃, and carrying out suction filtration on the obtained reaction liquid while the reaction liquid is hot to obtain an intermediate M5;
F. preparation of target product L two-photon fluorescent material benzoxazolyl pyridine salt
Dissolving M5 in ethanol, and adding NaBPh4And reacting for 4-7 h at 60-90 ℃, and carrying out suction filtration on the obtained reaction solution while the reaction solution is hot to obtain a target product L.
Further, in the step A, the molar ratio of the p-methyl benzoic acid to the o-aminophenol is 1: 2.2.
Further, in step B, intermediate M1 and N-bromosuccinimide were used in a molar ratio of 13.3: 30.
Further, in step C, intermediates M2 and PPh3The molar ratio of (A) to (B) is 32.3: 50.
Further, in step D, the molar ratio of intermediate M3, 4-pyridinecarboxaldehyde and potassium carbonate was 9.4:13.5: 36.
Further, in step E, intermediates M4 and CH3The molar ratio of I used is 3.1: 5.5.
Further, in step F, intermediate M5 and NaBPh4The molar ratio of (A) to (B) is 1:2.
In the process of implementing the invention, the inventor finds that the yield of each intermediate product and the target product is higher by adopting the molar use ratio of each raw material.
The invention has the beneficial effects that:
1. the benzoxazolyl pyridinium salt of the two-photon fluorescent material has good single-photon fluorescence property at about 510nm and good two-photon fluorescence property at about 530nm (see figures 2 and 3), the two-photon absorption cross section value can reach 103GM, and in fluorescence confocal microscopy imaging, after Hela cells are dyed by the benzoxazolyl pyridinium salt of the two-photon fluorescent material, the benzoxazolyl pyridinium salt of the two-photon fluorescent material can be clearly observed to be positioned in cytoplasm of the Hela cells, and the research result has great significance for the design, preparation and life science research of the two-photon material.
2. The two-photon fluorescent material benzoxazolyl pyridine salt is a water-soluble two-photon induced fluorescent material, has low toxicity and good membrane penetrability, can enter cells, and can be applied to two-photon fluorescence confocal microscopic imaging.
3. The preparation method has the advantages of easily available raw materials, low cost, simple synthesis steps and easy operation.
4. The benzoxazolyl pyridinium with strong photochemical photophysical stability and special asymmetric dipole structure is prepared by taking a benzoxazole group with strong photochemical photophysical stability as a main body, introducing a 4-position pyridine group through a Wittig reaction, forming pyridinium through a methylation reaction, and finally preparing the tetraphenyl borate pyridinium through an anion exchange reaction.
Drawings
FIG. 1 is a schematic diagram of a preparation method of benzoxazolyl pyridine salt as a two-photon fluorescent material.
FIGS. 2a and 2b are UV-VIS absorption spectra and fluorescence spectra of the target product L in different solvents.
FIGS. 3a and 3b show the two-photon induced fluorescence spectrum and the output fluorescence energy (I) of the target product L in waterout) With input of laser energy (I)in) Is fitted to the logarithm of the graph.
Fig. 4 is a two-photon absorption cross-section value (GM) of the target product L in water.
FIG. 5 is the single-photon and two-photon fluorescence confocal microscopic imaging of the target product L on Hela cells: (a) a single-photon fluorescence confocal micrograph of Hela cells stained by a target product L; (b) bright field effect diagram; (c) two-photon fluorescence confocal micrographs of Hela cells stained with the target product L; (d) a superimposed photograph.
Detailed Description
This section generally describes the materials used in the experiments of the present invention, as well as the experimental methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is described herein in detail as much as possible. It will be apparent to those skilled in the art that the materials, equipment, and methods of operation used in the present invention are well known in the art to which the invention pertains, unless otherwise specified.
Example 1
The preparation method of the two-photon fluorescent material benzoxazolyl pyridine salt is shown in figure 1, and comprises the following steps:
A. preparation of intermediate M1
Weighing 20.4g (0.15mol) of p-toluic acid and 35.97g (0.33mol) of o-aminophenol, placing the p-toluic acid and 35.97g (0.33mol) of o-aminophenol in a 250mL three-neck flask, adding 1500mL of polyphosphoric acid, refluxing for 6 hours at 220 ℃ under the protection of nitrogen, cooling the obtained reaction liquid to 80 ℃, pouring the reaction liquid into a big beaker filled with ice water, dropwise adding a proper amount of saturated NaOH aqueous solution to adjust the pH of the solution to 7-8, performing multiple extraction with 300mL of ethyl acetate, spin-drying the ethyl acetate, and performing ethanol recrystallization to obtain 9.84g of a solid, namely an intermediate M1, with the yield of 31.
Of intermediate M11H NMR:(400MHz,CD2Cl2):(ppm)8.17(s,2H),7.77(s,1H),7.63(s,1H),7.38(s,4H),2.48(s,2H).13C NMR:(100MHz,CD2Cl2):(ppm)163.58,151.15,142.67,130.05,127.86,125.26,124.81,120.07,110.84,21.75.FT-IR(KBr,cm-1):3058.30(w),2919.16(m),2870.11(w),1621.99(s),1555.47(vs),1345.57(m),1243.21(vs),1198.73(m),1177.50(s),1116.39(m),1055.18(m),1000.15(w),926.47(s),820.77(vs),785.82(w),745.62(s),726.69(m),692.70(w),501.44(vs).MS(ESI):m/z,calcd:209.0841,found:209.0908.
B. Preparation of intermediate M2
2.78g (13.30mmol) of M1 and 5.34g (30mmol) of N-bromosuccinimide (NBS) are weighed and placed in a 250mL round-bottom flask, 100mL of benzene and 0.5g of initiator benzoyl peroxide are added, the temperature is raised to 80 ℃ under the protection of nitrogen, the reaction is carried out for 8 hours at the temperature, the obtained reaction liquid is stood at normal temperature, a large amount of white solid is separated out, then the filtration is carried out, the filter cake is washed by water and dried, and 2.26g of light yellow crystalline solid, namely the intermediate M2, is obtained, and the yield is 59%.
Of intermediate M21H NMR(400MHz,CDCl3):(ppm)8.28–8.26(d,J＝8.00Hz,2H),7.90-7.82(m,1H),7.62–7.59(m,1H),7.58-7.56(d,J＝8.00Hz,2H),7.40-7.36(m,2H),7.55(s,2H).13C NMR(100MHz,CDCl3):(ppm)162.44,150.72,141.71,129.66,128.13,126.93,125.44,124.82,120.05,110.69,32.45.FT-IR(KBr,cm-1):3026.33(w),2924.14(vw),2852.01(vw),1618.40(w),1552.15(s),1497.27(vs),1451.23(vs),1411.89(vs),1244.17(s),1227.47(m),1197.50(m),1169.28(w),1126.28(w),1106.83(w),1053.27(vs),1013.26(m),925.74(s),846.85(w),824.39(s),759.30(s),746.30(vs),699.83(s),647.87(w),609.96(s),499.07(w).MS(APCI):m/z,calcd:286.9946,found:288.9991.
C. Preparation of intermediate M3
9.29g (32.3mmol) of M2 was weighed into a 250mL round-bottom flask, 100mL of benzene was added and dissolved under reflux, and 12.24g (50mmol) of PPh was added3Refluxing for 6h at 80 ℃ in a reaction system, separating out a white solid, carrying out suction filtration on the obtained reaction liquid while the reaction liquid is hot, and carrying out vacuum drying on the obtained solid to obtain 13.71g of the white solid, namely the intermediate M3, wherein the yield is 77%.
Of intermediate M31H NMR(400MHz,CDCl3):(ppm)7.89-7.80(m,9H),7.77-7.73(t,J＝12.0Hz,3H),7.64-7.60(m,7H),7.43-7.41(m,1H),7.32-7.29(d,J＝12.00Hz,2H),7.26-7.24(m,1H),5.81-5.77(d,J＝16.00Hz,2H).13C NMR(100MHz,CDCl3):(ppm)162.18,150.52,141.70,134.90,134.60,132.34,131.29,127.53,125.16,124.48,119.83,118.16,117.31,110.49,30.31.FT-IR(KBr,cm-1):3048.72(m),3004.26(w),2988.26(w),2964.25(w),2842.70(vs),2764.35(vs),1613.10(m),1587.13(m),1550.58(s),1493.63(s),1450.84(w),1435.93(s),1344.59(m),1244.35(s),1179.21(w),1110.32(vs),1064.34(s),1013.20(m),995.73(m),868.64(s),802.10(m),746.15(s),720.87(m),685.74(m),561.73(m),508.50(s).MS(ESI):m/z,calcd:549.0857,found:469.1654(M-Br)+.
D. Preparation of intermediate M4
5.17g (9.41mmol) of M3, 1.45g (13.53mmol) of 4-pyridinecarboxaldehyde and 4.98g (36mmol) of potassium carbonate are weighed out and added to 20mL of N, N-Dimethylacetamide (DMA) in sequence, the mixture is refluxed at 140 ℃ for 12 hours under the protection of nitrogen, cooled to room temperature, 60mL of dichloromethane is added to the obtained reaction solution and stirred, the filtrate is obtained by suction filtration, the filtrate is washed with a large amount of water for a plurality of times, then the dichloromethane layer is dried by anhydrous calcium chloride, and dichloromethane is dried by spinning to obtain 1.71g of white solid, namely the intermediate M4, with the yield of 61%.
Of intermediate M41H NMR:(400MHz,DMSO-d6),(ppm):8.60-8.58(d,J＝8.00Hz,2H),8.26-8.24(d,J＝8.00Hz,2H),7.91-7.89(d,J＝8.00Hz,2H),7.84-7.80(t,J＝8.00Hz,2H),7.69-7.64(d,J＝16.00Hz,1H),7.62-7.61(d,J＝4.00Hz,2H),7.47-7.41(m,3H).13CNMR:(100MHz,DMSO-d6),(ppm):161.96,150.11,143.82,141.55,139.59,131.85,128.30,127.81,127.71,126.00,125.61,124.94,121.04,119.81,110.91.
E. Preparation of intermediate M5
0.93g (3.1mmol) of M4 was weighed into a 100mL round-bottom flask, 50mL of Tetrahydrofuran (THF) was added, and the mixture was dissolved by stirring at room temperature, and 0.78g (5.5mmol) of CH was added3And I, heating to 60 ℃, reacting for 4 hours at the temperature, enabling the solution to become turbid, generating a large amount of yellow solid, and carrying out suction filtration on the obtained reaction solution while the reaction solution is hot to obtain 0.99g of yellow powder, namely an intermediate M5, wherein the yield is 72%.
Of intermediate M51H NMR:(400MHz,DMSO-d6),(ppm):8.92-8.91(d,J＝4.00Hz,2H),8.33-8.27(m,4H),8.12-8.08(d,J＝16.00Hz,1H),7.99-7.97(d,J＝8.00Hz,2H),7.86-7.82(t,J＝8.00Hz,2H),7.72-7.68(d,J＝16.00Hz,1H),7.50-7.43(m,2H),4.28(s,3H).13CNMR(100MHz,DMSO-d6),(ppm):161.66，151.91，150.24，145.25，141.49，139.07，138.30，128.80，127.88，127.40，125.86，125.31，125.05，123.84，119.94，110.99，47.05.MS(APCI):m/z 313.1335[(M-I-)+,calcd 313.1337].
F. Preparation of the target product L
0.3g (0.68mmol) of M5 was weighed into a 250mL round-bottom flask, 150mL of ethanol was added, and the mixture was heated to dissolve itThen 0.46g (1.36mmol) NaBPh was weighed out4Adding the mixture into a reaction system, reacting for 6 hours at 80 ℃, enabling the solution to become turbid, and carrying out suction filtration on the obtained reaction solution while the reaction solution is hot to obtain 0.2743g of yellow solid, namely a target product L, namely a two-photon fluorescent material benzoxazolyl pyridinium salt, wherein the yield is 63%.
Of the target product L1H NMR:(400MHz,DMSO-d6),(ppm):8.90-8.88(d,J＝8.00Hz,2H),8.33-8.31(d,J＝8.00Hz,2H),8.27-8.25(d,J＝8.00Hz,2H),8.11-8.07(d,J＝16.00Hz,1H),7.99-7.97(d,J＝8.00Hz,2H),7.86-7.81(t,J＝10.00Hz,2H),7.71-7.67(d,J＝16.00Hz,1H),7.50-7.43(m,2H),7.18(s,8H),6.94-6.90(t,J＝8.00Hz,8H),6.80-6.77(t,J＝6.00Hz,4H),4.26(s,3H).13C NMR(100MHz,DMSO-d6),(ppm):164.02,163.56,162.56,161.57,151.91,150.26,145.27,141.49,139.09,138.30,135.49,128.804,127.91,127.43,125.88,125.28,123.84,121.47,119.95,111.00,47.02.MS(APCI):m/z 313.1335[(M-NO3 -)+,calcd 313.1333].
Example 2
Preparation method of two-photon fluorescent material benzoxazolyl pyridine salt
This embodiment is similar to embodiment 1, and for the sake of brevity, only the differences will be described. They differ in that: in step A, the reaction was carried out at 210 ℃ for 7h under reflux, the yield of M1 was 30.5%, in step B, the reaction was carried out at 70 ℃ for 6h, the yield of M2 was 58%, in step C, the reaction was carried out at 40 ℃ for 8h, the yield of M3 was 75%, in step D, the reaction was carried out at 140 ℃ for 10h, the yield of M4 was 59%, in step E, the reaction was carried out at 50 ℃ for 3h, the yield of M5 was 70%, in step F, the reaction was carried out at 60 ℃ for 7h, and the yield of the target product L was 61%.
Example 3
Preparation method of two-photon fluorescent material benzoxazolyl pyridine salt
This embodiment is similar to embodiment 1, and for the sake of brevity, only the differences will be described. They differ in that: in step A, the reaction was performed at 240 ℃ for 4h under reflux, the yield of M1 was 30%, in step B, the reaction was performed at 60 ℃ for 7h, the yield of M2 was 56%, in step C, the reaction was performed at 60 ℃ for 7h, the yield of M3 was 76%, in step D, the reaction was performed at 140 ℃ for 14h, the yield of M4 was 58%, in step E, the reaction was performed at 40 ℃ for 6h, the yield of M5 was 69%, in step F, the reaction was performed at 90 ℃ for 4h, and the yield of the target product L was 59%.
Example 4
Performance testing of the target product L
2.1 Single photon Properties of target product L
As can be seen from FIG. 2a, the target product L has two absorption peaks in the ranges of 267-290nm and 362-378nm, the former can be attributed to the pi-pi transition of the benzoxazole ring, and the latter can be attributed to the ICT transition in the whole molecule; the target product L has charge transfer from benzoxazole nucleus to pyridine group in molecule. As can be seen from FIG. 2b, the target product L has a single emission peak between 501nm and 512nm, which is presumed to be attributable to the emission caused by intramolecular charge transfer transitions.
2.2 two-photon Properties of the target product L
(1) Test instruments and conditions
Two-photon induced fluorescence (TPEF) test takes a mode-locked titanium sapphire laser (Coherent Mira 900F, pulse 140fs, frequency 80MHz) as a pumping light source, and adopts a physical interference method to provide 680-1080nm variable-wavelength laser.
The concentration of the solution of the target product L in the experiment was 1X 10-4M, mother liquor for testing is aqueous solution. By 1X 10-4M fluorescein in NaOH aqueous solution (where the NaOH concentration is 1M) was used as reference. The used solvents are chromatographic pure reagents, and the sample cell is a four-side light-transmitting quartz cuvette with the thickness of 1.0 multiplied by 1.0 cm. The power of the laser light source is controlled at 300mW, the wavelength range is 720-980nm, and the wavelength interval is 20 nm.
(2) Determination of optimal excitation wavelength
The optimal excitation wavelength of two-photon induced fluorescence is determined by using laser excited molecules with constant power and different wavelengths to generate two-photon absorption, and recording the fluorescence emission intensity of the two-photon excited molecules, wherein the excitation wavelength with the strongest two-photon fluorescence is the optimal excitation wavelengthWavelength. In order to determine the optimal excitation wavelength of the target product L, the power of the laser light source is controlled at 300mW, the wavelength range of the excitation light is 720-900nm, the interval is 20nm, and the wavelength range is 1 × 10-4The two-photon induced fluorescence spectrum of M was measured in an aqueous solution, and the result is shown in FIG. 3 a.
(3) Two-photon verification
In the experiment, the two-photon verification is performed by measuring the two-photon induced fluorescence intensity under different power excitation lights (the power range of 100-700mW is selected in the experiment, and the measurement is performed at an interval of 100 mW) by changing the excitation light intensity under the optimal excitation wavelength of the compound. The two-photon verification slope of the target product L in the aqueous solution is about 1.80, which better conforms to the characteristics of two-photon excitation, and the result is shown in FIG. 3 b.
(4) Two-photon absorption cross section calculation
The two-photon absorption cross section () was measured by a two-photon induced fluorescence method. Calculating the two-photon absorption cross section of the compound by using a two-photon induced fluorescence method (2PA) The reference reagent is fluorescein (fluorescein) water solution with pH value of 11, and the calculation is disclosed as follows:
Figure BDA0001373167040000081
wherein ref represents a reference (fluorescein herein is prepared in aqueous NaOH solution at a concentration c of 1 × 10-3M, phi is the two-photon quantum yield (usually replaced by single-photon fluorescence quantum yield), and the two-photon absorption cross section of the reference sample (phi)ref0.95) is derived from the literature, c is the concentration of the solution, n is the refractive index of the solvent, F is the integrated area value of the corresponding two-photon fluorescence at different excitation wavelengths,refis the reference two-photon absorption cross-section value.
The two-photon absorption cross section of the target product L in water was calculated, and the result is shown in fig. 4, where the two-photon absorption cross section of the target product L in the aqueous solution all showed a maximum at 760nm, and the two-photon absorption cross section values were: 103 GM.
2.3 testing of the developing effect of the target product L Single photon, two-photon cell
The cleaned and sterilized coverslip was placed in 6-well tissue culture plates, and the liver cancer tissue cells (Hela cells) were 5X 105The density of each well was seeded in a 6-well plate having a diameter of 35mm, and cell culture was performed using DMEM as a cell culture medium containing fetal bovine serum (10%), penicillin (100. mu.g/mL) and streptomycin (100 ug/mL). The cell culture dish is placed in a container containing 5% CO2And 95% O2The temperature of the incubator was maintained at 37 ℃ for 24 hours, and Hela cells were washed three times with PBS (phosphate buffered saline, pH 7.4, produced by Gibco reagent) and the medium was washed off. Then, 4 μ L of each target compound L in DMSO (1mM) was added, the mixture was incubated for 0.5h, and the coverslip was washed 6 to 7 times with PBS buffer (pH 7.4), 1mL of 4% paraformaldehyde/PBS solution was dropped to fix the cells for 10min, and the coverslip was washed 6 to 7 times with distilled water. The coverslip was mounted on a clean slide and placed under a confocal laser microscope (LSM-710, Zeiss, Germany) to observe cell morphology and fluorescence uptake, the results of which are shown in FIG. 5.
As is clear from FIG. 5, the target product L permeated the cell membrane of Hela cells, entered the cytoplasm, and completely and uniformly stained it, indicating that the target product had a high localization ability to the cytoplasm of Hela cells. The two-photon material has important significance for the selection and preparation of cell developing materials, life science, material science and the like.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the present disclosure, and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this disclosure.

Claims (9)

1. The two-photon fluorescent material benzoxazolyl pyridine salt is characterized in that the structural formula is as follows:
Figure FDA0002486278810000011
2. the use of the two-photon fluorescent material benzoxazolyl pyridine salt of claim 1 in preparing a cell imaging reagent.
3. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 1, comprising the following steps:
A. preparation of intermediate M1(2- (p-tolyl) benzoxazole)
Mixing p-methylbenzoic acid, o-aminophenol and polyphosphoric acid, refluxing for 4-7 hours at 210-240 ℃, and extracting, drying and recrystallizing the obtained reaction liquid to obtain an intermediate M1;
B. preparation of intermediate M2(2- (4- (bromomethyl) phenyl) benzoxazole)
Dissolving M1 and N-bromosuccinimide in benzene, adding an initiator benzoyl peroxide, reacting at 60-80 ℃ for 6-8 h, and standing, filtering, washing and drying the obtained reaction solution to obtain an intermediate M2;
C. preparation of intermediate M3(2- (4- ((triphenylphosphine) methyl) phenyl) benzoxazole)
Dissolving M2 in benzene, and adding PPh3Reacting for 6-8 h at 40-80 ℃, and filtering and drying the obtained reaction solution while the reaction solution is hot to obtain an intermediate M3;
D. preparation of intermediate M4(E-2- (4- (2- (pyridin-4-yl) ethenyl) phenyl) benzoxazole)
Adding M3, 4-pyridylaldehyde and potassium carbonate into N, N-dimethylacetamide, reacting at 140 ℃ for 10-14 h, adding dichloromethane into the obtained reaction liquid, performing suction filtration, washing the filtrate with water, and drying to obtain an intermediate M4;
E. preparation of intermediate M5(E-4- (4- (2-benzoxazolyl) -2-styryl) -N-methylpyridine iodide salt)
Dissolving M4 in tetrahydrofuran, and adding CH3I, reacting for 3-6 h at 40-60 ℃, and carrying out suction filtration on the obtained reaction liquid while the reaction liquid is hot to obtain an intermediate M5;
F. preparation of target product L two-photon fluorescent material benzoxazolyl pyridine salt
Dissolving M5 in ethanol, and adding NaBPh4And reacting for 4-7 h at 60-90 ℃, and carrying out suction filtration on the obtained reaction solution while the reaction solution is hot to obtain a target product L.
4. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3, wherein the molar ratio of p-methyl benzoic acid to o-aminophenol in step A is 1: 2.2.
5. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3 or 4, wherein in step B, the molar ratio of intermediate M1 to N-bromosuccinimide is 13.3: 30.
6. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3 or 4, wherein in step C, the intermediate M2 and PPh3The molar ratio of (A) to (B) is 32.3: 50.
7. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3 or 4, wherein in the step D, the molar ratio of the intermediate M3, 4-pyridylaldehyde to potassium carbonate is 9.4:13.5: 36.
8. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3 or 4, wherein in step E, the intermediate M4 and CH3The molar ratio of I used is 3.1: 5.5.
9. The method for preparing benzoxazolyl pyridine salt as two-photon fluorescent material according to claim 3 or 4, wherein in the step F, the intermediate M5 and NaBPh4The molar ratio of (A) to (B) is 1:2.
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