Spirofluorene functionalized near-infrared second-zone fluorescent dye, synthesis method and application

Abstract

The invention discloses a spirofluorene functionalized near-infrared zone II fluorescent dye, a synthesis method and an application, and belongs to the technical field of organic dye synthesis; the spirofluorene functionalized near-infrared zone II fluorescent dye uses 2-spirofluorene borate derivatives as raw materials, reacts with 2-bromo-3-thiophenecarboxylate to obtain 2-spirofluorenyl-3-thiophenecarboxylate; and synthesizes the spirofluorene functionalized near-infrared zone II fluorescent dye by nucleophilic substitution reaction, Friedel-Crafts intramolecular cyclization reaction, borate esterification reaction and palladium-catalyzed cross-coupling reaction. The spirofluorene-enabled near-infrared zone II fluorescent dye of the present invention has a long absorption wavelength, can be excited by a 980-nanometer laser, and has a high fluorescence quantum yield, and has great application potential in the field of near-infrared zone II fluorescence imaging.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic dye synthesis, and specifically relates to a near-infrared II region fluorescent dye functionalized with spirofluorene, a synthesis method and applications thereof. Background Art

[0002] Fluorescence medical imaging has the advantages of low cost, high speed and high sensitivity, and can be dynamically monitored in real time. It is a very promising imaging technology. In fluorescence imaging, according to the emission wavelength range of fluorescent dyes, it can be divided into visible light dyes, near-infrared I region (700 - 1000 nm) fluorescent dyes and near-infrared II region (1000 - 1700 nm) fluorescent dyes. Given that the living body is a complex system, biological tissues have a scattering effect on light, and the strength of the scattering effect is inversely proportional to the wavelength, that is, the longer the wavelength, the deeper the imaging depth. In addition, biological tissues themselves have background fluorescence. Therefore, compared with visible light and near-infrared I region fluorescence imaging, near-infrared II region fluorescence imaging can effectively weaken the interaction between light and tissues, improve imaging resolution, imaging depth and sensitivity, and thus has attracted extensive attention from researchers. The key is to develop near-infrared II region fluorescent dyes with good biocompatibility and high luminous efficiency.

[0003] Currently, organic NIR-II fluorescent probes mainly include cyanine dyes and electron donor-acceptor (D-A) type dyes. Indocyanine green, a cyanine dye approved by the US Food and Drug Administration, has low toxicity and can be used for NIR-II fluorescence imaging. Since the emission peak of indocyanine green is located near 820 nm, the NIR-II fluorescence intensity is low (Sci. Rep. 2018, 8, 14455). Therefore, researchers have chemically modified the multi-conjugated double bond bridge and terminal heteroarene of cyanine dyes, which not only improves the structural stability but also shifts the emission peak red to the NIR-II region (J. Am. Chem. Soc. 2021, 143, 6836-6846). However, the Stokes shift of cyanine dyes is generally small, and interference is likely to occur between the excitation light and the emission light, reducing the sensitivity and resolution of imaging. The electron acceptor units of D-A type dyes are mainly benzobisthiadiazole (BBT) and thiazolodiquinoxaline (TQ) units, and the electron donor units are mostly triarylamine and thiophene derivatives. By utilizing the strong intramolecular charge transfer between the donor and acceptor units, a large Stokes shift is generated, which can effectively extend the emission spectrum of D-A type dyes to the NIR-II region (Acc. Mater. Res. 2021, 2, 170-183). Since the electron acceptor units (BBT and TQ) and the electron donor units (thiophene, phenyl, etc.) have large conjugated planes, π-π stacking is likely to occur, resulting in aggregation-induced quenching of luminescence, reducing the fluorescence quantum yield of the NIR-II probe from higher than 15% (solution state) to less than 0.1% (nanoparticles). To address this issue, researchers have proposed two strategies: 1) introducing long chains at the 3- or 3,4- positions of thiophene, using the steric repulsion effect between the long chain and the acceptor unit to distort the conformation between the donor and acceptor units, inhibit intermolecular π-π stacking, and reduce the interaction between water, oxygen and the luminophore, thereby increasing the quantum yield (Adv. Funct. Mater. 2020, 30, 1908125); 2) introducing units such as tetraphenylethylene, using its aggregation-induced fluorescence enhancement effect to increase the NIR-II fluorescence quantum yield of the probe (Nat. Commun. 2020, 11, 1-10). However, in the aggregated state, the PLQY of current NIR-II probes is not higher than 5%, indicating that the above strategies still cannot effectively inhibit the non-radiative transition rate of the probes. Therefore, it is of great significance to develop organic NIR-II fluorescent probes with high quantum efficiency, high brightness and high stability. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a spirofluorene-functionalized near-infrared second-region fluorescent dye, a synthesis method and an application, which solve the problems in the prior art.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The molecular structure formula of the near-infrared second-zone fluorescent dye functionalized with spirofluorene is:

[0007]

[0008] Ar is a benzene ring or a naphthalene ring; X is oxygen, sulfur or a carbon-carbon single bond; the substituent R is a straight-chain alkyl or alkoxy group having ≤8 carbon atoms.

[0009] Furthermore, the molecular structural formula of the fluorescent dye is:

[0010]

[0011] The substituent R is a straight-chain alkyl group or alkoxy group having 8 or less carbon atoms.

[0012] The synthesis method of the spirofluorene functionalized near-infrared second zone fluorescent dye is as follows: using 2-spirofluorene borate derivatives, 2-bromo-3-thiophenecarboxylate, aryl metal, n-butyl lithium, 2-spirofluorene-3-thiophenecarboxylate, isopropyl pinacol borate, and 4,8-dibromobenzobisthiadiazole as raw materials, the synthesis is carried out according to the following synthesis route:

[0013]

[0014] X is oxygen or a single bond, and the substituent R is a straight-chain alkyl or alkoxy group having 8 or less carbon atoms.

[0015] Further, the synthesis method comprises the following steps:

[0016] S1, under the catalysis of tetrakistriphenylphosphine palladium, 2-spirofluorene borate derivative and 2-bromo-3-thiophenecarboxylate undergo coupling reaction to obtain 2-spirofluorene-3-thiophenecarboxylate;

[0017] S2, the aromatic metal reacts with 2-spirofluorenyl-3-thiophenecarboxylate to obtain a tertiary alcohol derivative, and under the action of acetic acid / sulfuric acid, the tertiary alcohol undergoes an intramolecular Friedel-Crafts cyclization reaction to obtain a spiro-functionalized electron donor molecule;

[0018] S3, the spirofunctionalized electron donor molecule undergoes a lithium hydrogen exchange reaction with n-butyl lithium, and then reacts with isopropanol boronic acid pinacol ester. Under the catalysis of tetrakistriphenylphosphine palladium, the borate ester derivative undergoes a cross-coupling reaction with 4,8-dibromobenzobisthiadiazole to synthesize a spirofunctionalized near-infrared second zone fluorescent dye.

[0019] Furthermore, the 2-spirofluorene borate derivative is 2-spirofluorene xanthene pinacol borate or 2-spirobifluorene pinacol borate.

[0020] Further, the 2-bromothiophenecarboxylate is methyl 2-bromothiophenecarboxylate or ethyl 2-bromothiophenecarboxylate.

[0021] Further, the arylmetal is prepared by a halogen-lithium exchange reaction of 4-bromoalkylbenzene or 4-bromoalkoxybenzene with n-butyllithium at -70 °C, or a corresponding Grignard reagent is obtained by reacting 4-bromoalkylbenzene or 4-bromoalkoxybenzene with metallic magnesium, wherein the alkyl or alkoxy group is an alkyl or alkoxy group with ≤8 carbon atoms.

[0022] Application of the nanoparticles prepared using the above fluorescent dye in fluorescence imaging.

[0023] Further, the method for preparing the nanoparticles includes:

[0024] Dissolve the fluorescent dye and polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer or phospholipid-polyethylene glycol surfactant in a water-soluble organic solvent first. The dosage of the surfactant is 4 to 20 times the mass of the near-infrared fluorescent dye. Prepare the fluorescent nanoparticles by the reprecipitation method, and remove the organic solvent under reduced pressure to obtain an aqueous solution of the nanoparticles.

[0025] A composition for use in bioimaging, fluorescent probes, fluorescent sensors, fluorescent labeling, and tumor treatment, which comprises the above fluorescent dye.

[0026] Advantages of the present invention:

[0027] The spiro-functionalized near-infrared second near-infrared fluorescent dye proposed by the present invention. The spirofluorene unit with a three-dimensional rigid structure can inhibit the non-radiative transition caused by the thermal vibration of excited-state molecules and the fluorescence quenching effect induced by intermolecular aggregation, thereby effectively improving the luminescence efficiency of the near-infrared second near-infrared fluorescent dye. The maximum absorption wavelength of the spirofluorene-functionalized near-infrared second near-infrared fluorescent dye is greater than 900 nm, and the luminescence efficiency of the nanoparticles can reach up to 0.8%. Therefore, the spiro-functionalized dye of the present invention can be used as a fluorescence imaging contrast agent for near-infrared second near-infrared biofluorescence imaging to improve imaging sensitivity and resolution. Description of the drawings

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 is the 1H NMR spectrum of methyl 2-spirofluoreno[9,9-b]xanthene-3-carboxylate in Example 1 of the present invention;

[0030] Figure 21H NMR spectrum of 4,4-dibutylspirofluoreneoxanthienothiophene in Example 1 of the present invention;

[0031] Figure 3 MALDI-TOF mass spectrum of fluorescent dye 1 in Example 1 of the present invention;

[0032] Figure 4 1H NMR spectrum of 4,4-dioctyloxyspirofluoreneoxanthienothiophene in Example 2 of the present invention;

[0033] Figure 5 1H NMR spectrum of fluorescent dye 2 in Example 2 of the present invention;

[0034] Figure 6 Absorption and fluorescence spectra of fluorescent dye 1 in Example 1 of the present invention;

[0035] Figure 7 Fluorescence imaging of fluorescent dye 1 in Example 1 of the present invention;

[0036] Figure 8 Absorption and fluorescence spectra of fluorescent dye 2 in Example 2 of the present invention;

[0037] Figure 9 Fluorescence imaging of fluorescent dye 2 in Example 2 of the present invention. Detailed implementation mode

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art without creative efforts based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0039] The general molecular structure formula of the fluorescent dye is:

[0040]

[0041] Ar is a benzene ring or a naphthalene ring; X is oxygen, sulfur or a carbon-carbon single bond; the substituent R is a straight-chain alkyl or alkoxy group with ≤8 carbon atoms;

[0042] In the present invention, the molecular structural formula of the fluorescent dye can be one of the following two: (1):

[0044]

[0045] The substituent R is a straight-chain alkyl or alkoxy group with ≤8 carbon atoms; (2)

[0047]

[0048] The substituent R is a straight-chain alkyl or alkoxy group with ≤8 carbon atoms;

[0049] Among them, the synthetic routes of the fluorescent dyes corresponding to the above molecular structures (1) and (2) are as follows:

[0050]

[0051] X is oxygen or a single bond, and the substituent R is a straight-chain alkyl or alkoxy group with ≤8 carbon atoms.

[0052] The following specifically introduces the synthesis steps of the fluorescent dye through the following examples;

[0053] Example 1

[0054] In this example, fluorescent dye 1 was synthesized, and the specific steps were as follows:

[0055] S1. Synthesize methyl 2-spirofluoreneoxanthenylthiophene-3-carboxylate;

[0056] The synthetic route is as follows:

[0057]

[0058] 1.9 g (4.0 mmol) of 2-spirofluoreneoxanthene boronic acid pinacol, 0.764 g (3.4 mmol) of methyl 2-bromothiophenecarboxylate, 0.1996 g (0.2 mmol) of tetrakis(triphenylphosphine)palladium, and 3.5 mL of 2 mol / L potassium carbonate solution were respectively added to a two-necked flask, and the flask was evacuated and filled with nitrogen 3 times; then, 20 mL of tetrahydrofuran / toluene solvent was added. After nitrogen protection, the mixture was placed in an oil bath at 90 °C and stirred for reaction for 24 h. After the reaction, dichloromethane-saturated brine was used as the extraction system for extraction, dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by rotary evaporation. Petroleum ether and dichloromethane were used as eluents, and the product methyl 2-spirofluoreneoxanthenylthiophene-3-carboxylate, 1.25 g, was obtained by silica gel column chromatography separation, and the yield was 76.7%. As Figure 1 shown, 1 HNMR(400MHz,CDCl3)δ7.82(d,J=8.8Hz,2H),7.51(dd,J=8.0Hz,1H),7.46-7.37(m,2H),7.31-7.27(m,1H),7.25-7.14(m,8H),6.80(d,J=8.4Hz,2H),6.52-6.44(m,2H),3.26(s,3H). 13CNMR (100 MHz, CDCl3) δ 156.64, 152.77, 131.83, 130.72, 130.25, 129.64, 129.52, 129.46, 129.35, 128.84, 127.32, 125.97, 125.47, 124.75, 124.64, 121.61, 120.97, 118.30, 118.21, 52.55, 2.45.

[0059] S2. Synthesis of 4,4-dibutylspirofluoreneoxanthienothiophene;

[0060] The synthetic route of 4,4-dibutylspirofluoreneoxanthienothiophene is as follows:

[0061]

[0062] First, weigh 2.17 g (0.01 mol) of p-bromobutylbenzene and add it to a dried single-necked flask. Add an appropriate amount of tetrahydrofuran as the solvent. Place the flask in a -70 °C low-temperature reaction kettle and stir. Slowly add 6.2 mL of 1.6 M n-butyllithium and continue to react for 1 h to obtain the corresponding aryllithium. Then, add 1.2 g (2.5 mmol) of methyl 2-spirofluoreneoxanthiene-3-thiophenecarboxylate, stir and react at room temperature for 12 h. After that, quench the reaction with saturated ammonium chloride solution, extract with dichloromethane, dry and rotary evaporate to remove the solvent, and directly carry out the ring-closure reaction. Third, dissolve the reaction product in acetic acid, add a drop of concentrated sulfuric acid while stirring in an oil bath at 120 °C. The reaction ends in about 0.5 h. Extract with dichloromethane-saturated brine, dry with anhydrous magnesium sulfate, rotary evaporate to remove the solvent, and use petroleum ether and dichloromethane as eluents to separate by silica gel column chromatography to obtain 580 mg of 4,4-dibutylspirofluoreneoxanthienothiophene, with a yield of 40%. As Figure 2As shown, 1H NMR (400 MHz, CDCl3) δ 8.10 - 7.98 (m, 1H), 7.71 - 7.38 (m, 10H), 7.21 - 7.06 (m, 3H), 6.79 - 6.63 (m, 2H), 3.23 - 2.78 (m, 4H), 1.96 - 1.85 (m, 7H), 1.74 - 1.59 (m, 6H), 1.29 (t, J = 6.8 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ 157.44, 156.19, 152.46, 146.17, 145.60, 142.97, 141.29, 140.19, 139.64, 136.42, 132.87, 131.42, 129.99, 129.42, 129.31, 129.17, 128.55, 128.23, 126.96, 125.42, 124.64, 123.81, 121.44, 121.12, 118.14, 81.99, 55.39, 36.58, 34.86, 31.09, 23.85, 15.41, 2.41.

[0063] S3, synthesize fluorescent dye 1;

[0064] The synthetic route is as follows:

[0065]

[0066] Weigh 550 mg of 4,4 - dibutylspirofluoreneoxanthiothene and place it in a dried single - necked flask, dissolve it with anhydrous tetrahydrofuran, protect it with nitrogen, put the single - necked flask into a low - temperature reactor at - 40 °C and stir. Then, weigh 1.0 mL of 1.6 M n - butyllithium with a syringe and slowly add it dropwise to the single - necked flask. Continue the reaction for 2 h. After that, transfer it to room temperature and stir. Then, weigh and add 0.03 g (1.6 mmol) of isopropanol pinacol borate with a syringe. Stir and react at room temperature for 12 h, and then end the reaction. Then directly spin - dry to remove the solvent, and use it directly for the next step without purification.

[0067] Weigh 100 mg of 4,8 - dibromobenzobisthiadiazole and 82 mg (0.07 mmol) of tetrakis(triphenylphosphine)palladium into a single - necked flask, add 0.6 mL of 2.0 M potassium carbonate solution, use a toluene - tetrahydrofuran mixed system as the solvent, and protect it with nitrogen. Stir and react at 90 °C in an oil bath for 24 h. Then, use a dichloromethane - saturated brine extraction system for extraction, dry it with anhydrous magnesium sulfate, filter, rotary evaporate to remove the organic solvent, use petroleum ether and dichloromethane as eluents, and separate by silica gel column chromatography to obtain 93 mg of the target product with a yield of 23%. The solubility of the product is poor, so NMR analysis was not performed. The molecular weight was confirmed to be consistent with the target product by mass spectrometry, as Figure 3 shown.

[0068] Example 2

[0069] In this embodiment, fluorescent dye 2 was synthesized, and the specific steps were:

[0070] S1, synthesis of 2-spirofluorene xanthracene thiophene-3-carboxylic acid methyl ester; (this step is the same as S1 in Example 1)

[0071] 1.9 g (4.0 mmol) of 2-spirofluorene oxygen anthracene boronic acid pinacol, 0.764 g (3.4 mmol) of methyl 2-bromothiophenecarboxylate, 0.1996 g (0.2 mmol) of tetrakistriphenylphosphine palladium and 3.5 mL of 2 mol / L potassium carbonate solution were added to a two-necked flask, and the flask was evacuated and filled with nitrogen three times; then, 20 mL of tetrahydrofuran / toluene solvent was added, and the mixture was placed in a 90° C. oil bath pot under nitrogen protection and stirred for 24 h. After the reaction was completed, dichloro-saturated brine was used as an extraction system for extraction, and the mixture was dried over anhydrous magnesium sulfate, filtered, and the solvent was removed by rotary evaporation. Petroleum ether and dichloromethane were used as eluents, and the product was separated by silica gel column chromatography to obtain 1.25 g of methyl 2-spirofluorene oxygen anthracene thiophene-3-carboxylate.

[0072] S2, synthesis of 4,4-dioctyloxyspirofluorene anthothiophene;

[0073] The synthetic route is:

[0074]

[0075] Take 0.22g of magnesium chips and an appropriate amount of iodine, then add 2.67g of p-bromooctyloxybenzene (8.5mmol) and dissolve it in tetrahydrofuran, heat to initiate the reaction, and then continue the reaction at room temperature for 4 hours to prepare the corresponding Grignard reagent. Slowly add 1g of 2-spirofluorene oxygen anthracene-3-thiophene carboxylic acid methyl ester, and react at 70°C for 18 hours. After the reaction is completed, quench the reaction with saturated ammonium chloride aqueous solution, extract with dichloromethane-saturated brine, dry with anhydrous magnesium sulfate, filter, and evaporate to remove the organic solvent. The product is not purified for the next step of the reaction. Dissolve the reaction product with acetic acid, stir in a 120°C oil bath and add a drop of concentrated sulfuric acid. After reacting for 3 hours, terminate the reaction, extract with dichloromethane-saturated brine, dry with anhydrous magnesium sulfate, filter, and evaporate to remove the organic solvent. Separate by silica gel column chromatography to obtain 900mg of 4,4-dioctyloxyspirofluorene oxygen anthracene thiophene with a yield of about 60%. Figure 4As shown, 1H NMR (400 MHz, CDCl3) δ 7.75 - 7.65 (m, 2H), 7.28 (d, J = 5.6 Hz, 6H), 7.25 - 7.11 (m, 17H), 6.94 (q, J = 5.5 Hz, 1H), 6.85 - 6.72 (m, 6H), 6.49 (t, J = 6.4 Hz, 2H), 3.91 (t, J = 6.8 Hz, 4H), 1.80 - 1.70 (m, 4H), 1.55 (m, 16H), 0.93 - 0.82 (m, 6H). 13C NMR (100 MHz, CDCl3) δ 158.11, 156.69, 155.29, 154.91, 154.34, 151.56, 140.80, 140.02, 137.86, 137.39, 136.86, 129.12, 128.30, 128.26, 128.21, 128.00, 127.85, 125.75, 125.10, 123.43, 122.92, 119.74, 117.83, 116.90, 116.67, 114.25, 68.01, 61.97, 54.11, 31.92, 31.61, 30.24, 29.81, 29.46, 29.41, 29.34, 26.18, 22.76, 14.21.

[0076] S3, synthesize fluorescent dye 2;

[0077] The synthetic route is as follows:

[0078]

[0079] Weigh 620 mg of 4,4 - dioctyloxysulfonyloxanthone and place it in a dried single - necked flask, dissolve it with anhydrous tetrahydrofuran, protect it with nitrogen. Place the single - necked flask in a low - temperature reactor at - 40 °C and stir. Weigh 1.0 mL of 1.6 M n - butyllithium with a syringe and slowly add it dropwise to the single - necked flask. Continue the reaction for 2 h, then transfer it to room temperature and stir. Weigh and add 0.03 g (1.6 mmol) of isopropanol pinacol borate with a syringe. Stir at room temperature for 12 h to end the reaction. Then directly evaporate the solvent to dryness and use it directly in the next step without purification.

[0080] Weigh 103 mg of 4,8-dibromobenzobisthiadiazole and 65 mg (0.07 mmol) of tetrakis(triphenylphosphine)palladium into a single-necked flask, add 0.4 ml of 2.0 M potassium carbonate solution, use a toluene-tetrahydrofuran mixed system as the solvent, and protect with nitrogen. Stir and react at 90 °C in an oil bath for 24 h. Then, use dichloromethane-saturated brine as the extraction system for extraction, dry with anhydrous magnesium sulfate, filter, and rotary evaporate to remove the organic solvent. Use petroleum ether and dichloromethane as eluents, and separate by silica gel column chromatography to obtain 47 mg of the target product with a yield of 15%. As Figure 5 shown, 1 HNMR(400MHz,CDCl3)δ7.79 - 7.65(m,7H),7.56 - 7.41(m,8H),7.37 - 7.27(m,15H),7.25 - 7.15(m,7H),6.82(dq,J=9.0,2.1Hz,13H),6.52(dd,J=7.9,1.6Hz,4H),3.90(t,J=6.5Hz,8H),1.73(p,J=6.7Hz,10H),0.92 - 0.80(m,12H). 13 CNMR(100MHz,CDCl3)δ158.23,151.69,136.74,132.43,132.32,131.67,131.64,129.32,129.11,128.68,128.56,128.38,128.06,125.16,123.49,117.05,114.47,68.02,54.22,31.88,31.61,31.54,30.23,29.80,29.45,29.38,29.32,26.17,22.80,22.72,14.23,14.17.

[0081] Example 3

[0082] Use the fluorescent dye 1 prepared in Example 1 to prepare fluorescent dye nanoparticles and conduct characterization;

[0083] The preparation process of the fluorescent dye nanoparticles is as follows:

[0084] Dissolve fluorescent dye 1 (2.0 mg) and poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) copolymer (16.0 mg) in 1 ml of tetrahydrofuran. Under vigorous stirring conditions, slowly add it to a beaker containing 10 ml of deionized water; after all the samples are added to the deionized water, continue stirring for 10 minutes, then transfer the samples to a round-bottomed flask, rotary evaporate under reduced pressure to remove tetrahydrofuran, and then make up the volume to prepare a nanoparticle colloidal solution with a fluorescent dye mass concentration of 500 μg / mL.

[0085] Characterization:

[0086] Transfer 300 μL of the nanoparticle colloidal solution into a 1.5 mL EP tube, add 1200 μL of deionized water, dilute the sample to a concentration of 100 μg / mL, and then place the sample in a near-infrared fluorescence imaging system for testing. The test conditions are: 980 nm laser excitation, power of 20 mW / cm2, exposure time of 50 ms, and a 1000 nm long-pass filter.

[0087] It can be seen from Figure 6 that the absorptions of fluorescent dye 1 in toluene solution and aqueous nanoparticle solution are 883 and 891 nm respectively, and their corresponding fluorescence emission peaks are 1178 and 1124 nm respectively. Using the near-infrared second-region fluorescent material FT-BT (whose luminescence efficiency in toluene is 19%) as a standard sample, the luminescence efficiencies of fluorescent dye 1 in toluene and nanoparticles are measured to be 16.4% and 0.8% respectively. It has a relatively high luminescence efficiency and can be used as a near-infrared second-region fluorescent dye.

[0088] It can be seen from Figure 7 that under the same test conditions, due to the aggregation-induced quenching effect, the luminescence brightness of the nanoparticles of the near-infrared second-region fluorescent material FT-BT is very low, while the luminescence brightness of the nanoparticles of fluorescent dye 1 is high, indicating that the spiro-functionalized near-infrared second-region fluorescent material can significantly improve the luminescence efficiency of the material.

[0089] Example 4

[0090] Use the fluorescent dye 2 prepared in Example 2 to prepare fluorescent dye nanoparticles and conduct characterization;

[0091] The preparation process of the fluorescent dye nanoparticles is as follows:

[0092] Dissolve fluorescent dye 2 (2.0 mg) and distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000, 12.0 mg) in 1 mL of tetrahydrofuran, and slowly add it to a beaker containing 10 mL of deionized water under vigorous stirring; after all the samples are added to the deionized water, continue stirring for 10 minutes, then transfer the sample to a round-bottom flask, remove tetrahydrofuran by rotary evaporation under reduced pressure, and then make up the volume to prepare a nanoparticle colloidal solution with a fluorescent dye mass concentration of 500 μg / mL.

[0093] Characterization:

[0094] Transfer 300 μL of the nanoparticle colloidal solution into a 1.5 mL EP tube, add 1200 μL of deionized water, dilute the sample to a concentration of 100 μg / mL, and then place the sample in a near-infrared fluorescence imaging system for testing. The test conditions are: 980 nm laser excitation, power of 50 mW / cm 2, The exposure time is 100 ms, and a 1000 nm long-pass filter is used.

[0095] It can be seen from Figure 8 that the absorptions of fluorescent dye 2 in toluene solution and aqueous nanoparticle solution are 903 and 925 nm respectively, and their corresponding fluorescence emission peaks are 1045 and 1086 nm respectively. Using the near-infrared II fluorescent material FT-BT (whose luminescence efficiency in toluene is 19%) as a standard sample, the luminescence efficiencies of fluorescent dye 2 in toluene and nanoparticles are measured to be 13.9% and 0.2% respectively. It has a relatively high luminescence efficiency and can be used as a near-infrared II fluorescent dye.

[0096] It can be seen from Figure 9 that under the same test conditions, due to the aggregation-induced quenching effect, the luminescence brightness of the nanoparticles of the near-infrared II fluorescent material FT-BT is very low, while the luminescence brightness of the nanoparticles of fluorescent dye 2 is high, indicating that the spiro-functionalized near-infrared II fluorescent material can significantly improve the luminescence efficiency of the material.

[0097] In the description of this specification, the descriptions referring to terms such as "one embodiment", "example", "specific example", etc. mean that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples.

[0098] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. What is described in the above embodiments and the specification only illustrates the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have various changes and improvements, and these changes and improvements all fall within the scope of the present invention claimed.

Claims

1. A near-infrared second-zone fluorescent dye functionalized with spirofluorene, characterized in that: The molecular structure formula of the fluorescent dye is: Ar is a benzene ring or a naphthalene ring; X is oxygen, sulfur or a carbon-carbon single bond; the substituent R is a straight-chain alkyl or alkoxy group having ≤8 carbon atoms.

2. The spirofluorene functionalized near-infrared zone II fluorescent dye according to claim 1, characterized in that: The molecular structural formula of the fluorescent dye is: The substituent R is a straight-chain alkyl group or alkoxy group having 8 or less carbon atoms.

3. A method for synthesizing a spirofluorene-functionalized near-infrared second-zone fluorescent dye, characterized in that: include: The synthesis was carried out according to the following synthetic route: X is oxygen or a single bond, and the substituent R is a straight-chain alkyl or alkoxy group having ≤ 8 carbon atoms; The synthesis method comprises the following steps: S1, under the catalysis of tetrakistriphenylphosphine palladium, a 2-spirofluorene borate derivative and 2-bromo-3-thiophenecarboxylate undergo a coupling reaction to obtain 2-spirofluorene-3-thiophenecarboxylate; S2, the aryl metal reacts with 2-spirofluorenyl-3-thiophenecarboxylate to obtain a tertiary alcohol derivative, and under the action of acetic acid and sulfuric acid, the tertiary alcohol derivative undergoes an intramolecular Friedel-Crafts cyclization reaction to obtain a spiro-functionalized electron donor molecule; S3, the spirofunctionalized electron donor molecule undergoes a lithium hydrogen exchange reaction with n-butyl lithium, and then reacts with isopropanol pinacol borate to obtain a borate derivative. Under the catalysis of tetrakistriphenylphosphine palladium, the borate derivative undergoes a cross-coupling reaction with 4,8-dibromobenzobisthiadiazole to synthesize a spirofunctionalized near-infrared zone II fluorescent dye.

4. The method for synthesizing the spirofluorene functionalized near-infrared zone II fluorescent dye according to claim 3, characterized in that: The 2-spirofluorene borate derivative is 2-spirofluorene xanthene pinacol borate or 2-spirobifluorene pinacol borate.

5. The method for synthesizing the spirofluorene functionalized near-infrared zone II fluorescent dye according to claim 3, characterized in that: The 2-bromo-3-thiophenecarboxylic acid ester is methyl 2-bromo-3-thiophenecarboxylate or ethyl 2-bromo-3-thiophenecarboxylate.

6. The method for synthesizing the spirofluorene functionalized near-infrared zone II fluorescent dye according to claim 3, characterized in that: The aryl metal is prepared by reacting 4-bromoalkylbenzene or 4-bromoalkoxybenzene with n-butyl lithium at -70°C through a halogen-lithium exchange reaction, or by reacting 4-bromoalkylbenzene or 4-bromoalkoxybenzene with metal magnesium to obtain the corresponding Grignard reagent, wherein the alkyl or alkoxy group is an alkyl or alkoxy group with a carbon number of ≤8.

7. Use of nanoparticles prepared using the fluorescent dye according to claim 1 or 2 in fluorescence imaging, wherein the use is for non-diagnostic purposes.

8. The application according to claim 7, wherein The preparation method of the nanoparticles comprises: The fluorescent dye and polyethylene glycol-polypropylene glycol-polyethylene glycol copolymer or phospholipid-polyethylene glycol surfactant are first dissolved in a water-soluble organic solvent, and the amount of the surfactant used is 4 to 20 times the mass of the fluorescent dye. Fluorescent nanoparticles are prepared by a reprecipitation method, and the organic solvent is removed under reduced pressure to obtain a nanoparticle aqueous solution.

9. A composition for use in biological imaging, fluorescent probes, fluorescent sensors, fluorescent labels, and tumor treatment, comprising the fluorescent dye according to claim 1 or 2.

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