Near-infrared two-region cyanine dye, preparation method and application thereof

Abstract

The application belongs to the technical field of near-infrared organic dye synthesis, and particularly discloses a near-infrared two-region merocyanine dye, a preparation method and application thereof; the dye is 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indol-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indol salt; the preparation method is simple, and raw materials are cheap and easy to obtain; the prepared near-infrared organic dye has high light stability, has strong fluorescence emission in the near-infrared two-region, and can be used as a contrast agent and applied to the field of near-infrared two-region biofluorescence imaging.

Description

Technical Field

[0001] This invention belongs to the field of near-infrared organic dye synthesis technology, and specifically discloses a near-infrared II region anthocyanin dye and its preparation method and application. Background Technology

[0002] When dyes that absorb and / or emit in the visible and near-infrared I (NIR) windows (400-900 nm) are used as plant contrast agents, the absorption and autofluorescence of various pigments in plants, such as chlorophyll, xanthophyll, and carotene, can severely interfere with imaging depth and sensitivity. Dyes that absorb and / or emit in the near-infrared II (1000-1700 nm) have attracted considerable attention because they can avoid the influence of various pigments in plants.

[0003] Current near-infrared II fluorescent dyes are mainly divided into two categories: one is benzothiadiazole (BBTD) fluorophores with a donor-acceptor-donor (DAD) structure, and the other is cyanine dyes with a large π-conjugated system. Cyanine fluorescent dyes are typical organic fluorophores, characterized by high extinction coefficients, simple synthetic routes, and long absorption and emission wavelengths. Compared with DAD-type fluorophores, they have a higher molar extinction coefficient (ε>10). 5 M -1 cm -1 With its narrow emission range, cyanine fluorophores require only small amounts to maintain their excellent brightness. Furthermore, the simple synthesis process makes cyanine dyes more modifiable, giving them an advantage over DAD-type fluorophores due to these properties. However, cyanine fluorescent dyes (such as ICG) are easily photobleached, and their conjugated chains are susceptible to oxidation and reduction by oxides and reducing agents such as ONOO. - Its photostability is poor due to the attack characteristics of cysteine ​​and other amino acids.

[0004] Chinese patent CN113713123A describes a fluorescent conjugated polymer nanoprobe for imaging the brain's lymphatic system and blood vessels. The conjugated polymer used is a 9,9-dioctylfluorene-2,1,3-benzothiadiazole copolymer, and the imaging is performed using a stereofluorescence microscope in the visible light region. This invention provides a contrast agent for visible light fluorescence imaging, enabling rapid, high-brightness imaging of the brain's lymphatic system and blood vessels using a simple, convenient, and low-cost stereofluorescence microscope. However, because its fluorescence signal is in the visible light region, its imaging resolution is relatively low, and due to the low penetration depth of visible light, its imaging depth is also limited.

[0005] Chinese patent CN112876873A discloses a near-infrared region II heptamethylcyanine dye, QHS-Cy, which exhibits an absorption peak at 983 nm and a fluorescence emission peak at 1018 nm in dichloromethane. It demonstrates good photostability, high photothermal efficiency, and effective avoidance of biological background fluorescence interference. However, the related synthetic steps result in low yields. Its large π-conjugated structure makes the dye prone to aggregation, and its exposed conjugated backbone and mid-position Cl atom are susceptible to attack by reactive species or photobleaching, thus affecting fluorescence intensity and reducing imaging performance.

[0006] In conclusion, the development of near-infrared II dyes with long absorption / emission wavelengths and photostability is of great significance and has great application potential. Summary of the Invention

[0007] In order to overcome the shortcomings and limitations of the above-mentioned technologies, the purpose of this invention is to provide a near-infrared II cyanine dye, its preparation method and application.

[0008] The near-infrared II cyanine dye of this invention employs a benzoindole heptamethine cyanine fluorophore parent structure, introducing a rigid five-membered ring and a strongly electron-withdrawing acetyl group. The long conjugation length allows the dye to emit near-infrared II fluorescence after excitation. The slightly twisted molecular structure reduces aggregation quenching, and the rigid structure and sterically hindered substituted side groups improve the dye's photostability. The near-infrared II cyanine dye of this invention is synthesized through a simple, high-yield process. Its near-infrared II fluorescence signal and high photostability allow it to overcome tissue autofluorescence interference, making it suitable as a high signal-to-noise ratio near-infrared II biofluorescence imaging contrast agent.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A near-infrared II region anthocyanin dye, the molecular formula of which is C2. 50 H 52 N3O + The specific structure is as follows:

[0011]

[0012] This invention also provides a method for preparing near-infrared II cyanine dyes, comprising the following steps:

[0013] Step I: The compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole salt was dissolved in an organic solvent, and then benzylamine was added. The reaction was carried out under stirring. After the reaction was completed, the compound 2-(2-(2-(benzylamine)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt was obtained by separation and purification.

[0014] Step II: Dissolve 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt in dichloromethane, add N,N-diisopropylethylamine, then add acetyl chloride, and then react under stirring. After the reaction is complete, separate and purify to obtain the near-infrared II cyanine dye.

[0015] Preferably, the structural formula of the 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt is shown below.

[0016]

[0017] Preferably, in step I, the molar ratio of the compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole salt to benzylamine is 1:(2-3); and the organic solvent is N,N-dimethylformamide.

[0018] Preferably, the reaction temperature under stirring in step I is 60-80°C.

[0019] Preferably, the stirring time in step I is 5-15 minutes.

[0020] Preferably, the separation and purification step in step I is as follows: after the reaction is completed, the organic solvent is removed by rotary evaporation, and the resulting solid is directly used in the next reaction.

[0021] Preferably, in step II, the molar ratio of 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt to N,N-diisopropylethylamine is 1:(10-11).

[0022] Preferably, in step II, the molar ratio of 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt to acetyl chloride is 1:(7-9).

[0023] Preferably, in step II, the amount of dichloromethane added per mmol of compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt is 40-60 mL.

[0024] Because the near-infrared II cyanine dye is easily decomposed at high temperatures, has good solubility in dichloromethane, and is easily removed after the reaction, dichloromethane is chosen. Dichloromethane is volatile and has a low boiling point, so excessively high temperatures are not required when removing the solvent via rotary evaporation. However, it is still necessary to control the amount of solvent used to shorten the solvent removal time; otherwise, the yield will drop sharply. Solvents such as petroleum ether and ethyl acetate are unsuitable, while solvents such as N,N-dimethylformamide and tetrahydrofuran have high boiling points.

[0025] Preferably, the temperature at which the reaction is carried out under stirring in step II is 0°C-30°C.

[0026] Preferably, the reaction time under stirring in step II is 35-45 minutes.

[0027] Preferably, the separation and purification steps in step II are as follows: after the reaction is completed, the mixture is extracted with saturated ammonium chloride aqueous solution and dichloromethane, the organic phase is taken, dried, and filtered; the organic solvent is removed by rotary evaporation, and the resulting solid is purified by silica gel column chromatography.

[0028] More preferably, the eluent used in step II of the silica gel chromatography is dichloromethane / methanol.

[0029] This invention also provides the application of near-infrared II cyanine dyes in the preparation of fluorescent probes and in near-infrared II biofluorescence imaging.

[0030] Preferably, the near-infrared II biofluorescence imaging is near-infrared II plant fluorescence imaging.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) In the molecular structure of the near-infrared II cyanine dye of the present invention, the rigid five-membered ring on the polymethyl ether chain reduces the planarity of the conjugated system, reduces fluorescence quenching caused by molecular stacking, and makes the polymethyl ether skeleton more stable. In addition, on the one hand, the acetyl group with strong electron-withdrawing ability in the N-benzylacetamide group at the middle position enhances the electron-pulling ability of the dye molecule, which increases the degree of delocalization of the electron cloud of the dye in the entire conjugated plane, resulting in a redshift of absorption / emission; on the other hand, the N-benzylacetamide group at the middle position has large steric hindrance and simultaneously possesses an aromatic ring and a strong electron-withdrawing group, which can effectively prevent the conjugated skeleton from being attacked by oxidizing or reducing species in the environment, thus improving the photostability of the dye. If the middle position of the whole cyanine dye is a halogen atom (such as IR-806, IR-820), then because the halogen atom itself is unstable and easily attacked by electrophilic active species, and the conjugated skeleton is relatively exposed, the dye is easily attacked by active species in the environment, resulting in photobleaching and deterioration.

[0033] The near-infrared II cyanine dye of this invention has ethyl groups substituents on the heterocyclic nitrogen atoms on both sides. On the one hand, the shorter hydrophobic chain of ethyl groups helps ensure good solubility of the dye in various solvents; on the other hand, the electronegativity of ethyl groups avoids adverse effects on the electronic effects of the substituents at the meso position, thus helping to ensure the dye's spectral properties remain unchanged and its photostability remains high. If the substituents on the nitrogen atoms of the heterocyclic atoms on both sides of the cyanine dye are replaced with longer hydrophobic aliphatic chains (such as propyl groups) or aliphatic groups with slightly electron-withdrawing properties (such as hexanoic acid or butyric acid), the dye's solubility will be affected, and the electronic effects of the substituents at the meso position will be adversely affected, thereby worsening the dye's spectral properties and photostability.

[0034] (2) The cyanine dye of the present invention has strong emission in the near-infrared II region, and is therefore suitable as a contrast agent for use in near-infrared II fluorescence bioimaging with high signal-to-noise ratio.

[0035] (3) The synthesis route of the near-infrared II cyanine dye of the present invention is simple, efficient and low in cost, and has good application prospects. Attached Figure Description

[0036] Figure 1 This is a synthetic route diagram for the near-infrared II region anthocyanin dye prepared according to the present invention.

[0037] Figure 2 The image shows the hydrogen nuclear magnetic resonance spectrum of the product obtained in Example 1.

[0038] Figure 3 This is the mass spectrum of the product obtained in Example 1.

[0039] Figure 4 The image shows the absorption spectrum of the product obtained in Example 1.

[0040] Figure 5 The graph shows the change in absorbance of the product obtained in Example 1 under 808nm laser irradiation.

[0041] Figure 6 This is the fluorescence emission spectrum of Example 1 tested in dichloromethane in the presence of crushed leaves.

[0042] Figure 7 The images show near-infrared fluorescence images of the product obtained in Example 1 after continuous excitation for different times. Detailed Implementation

[0043] The specific implementation of the present invention will be further described below with reference to the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that any processes not specifically described in detail below are those that can be implemented or understood by those skilled in the art by referring to the prior art. Reagents or instruments whose manufacturers are not specified are considered to be conventional products that can be purchased commercially.

[0044] The synthetic route of the near-infrared II cyanine dye of the present invention is as follows: Figure 1 As shown.

[0045] Example 1

[0046] Step I: Dissolve 202.7 mg (0.28 mmol) of compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole iodide in 10 mL of N,N-dimethylformamide. With stirring, add 59.9 mg (0.56 mmol) of benzylamine, and then stir the reaction at 60 °C for 5 minutes. After the reaction was complete, N,N-dimethylformamide was removed by rotary evaporation to give 187 mg of the solid compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide. (Yield: 87%)

[0047] Step II: Dissolve 198.8 mg (0.25 mmol) of compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide in 10 mL of dichloromethane. Add 322.5 mg (2.5 mmol) of N,N-diisopropylethylamine and 136.5 mg (1.75 mmol) of acetyl chloride while stirring at 0 °C. React for 35 minutes. After the reaction was completed, the sample was extracted with saturated ammonium chloride aqueous solution and dichloromethane (10 mL). The organic phase was collected, dried, and filtered. The organic solvent was removed by rotary evaporation. The obtained solid was purified by silica gel column chromatography (the eluent was dichloromethane / methanol, V / V = 15:1) to obtain 120 mg of cyanine dye (yield: 57.2%).

[0048] Characterized by proton nuclear magnetic resonance spectroscopy: 1 1H NMR (400MHz, Methanol-d4): δ 8.16 (d, J = 8.6Hz, 2H), 8.01–7.98 (m, 4H), 7.63 (t, J = 7.0Hz, 2H), 7.58 (d, J = 8.8Hz, 2H), 7.47 (t, J = 7.0Hz, 4H), 7.35 (t, J = 6.6Hz, 3H), 7.30–7.24 (m, 2H), 6.12 (d, J = 14.0Hz, 2H), 4.94 (s, 2H), 4.30–4.27 (m, 4H), 3.04 (s, 4H), 2.02 (s, 3H), 1.81 (s, 6H), 1.61 (s, 6H), 1.44 (t, J = 7.2Hz, 6H). The 1H NMR spectrum is shown below. Figure 2 As shown.

[0049] Further verification was performed using mass spectrometry: MS (ESI): m / z 710.41 [M] + Mass spectrum as shown Figure 3 As shown. Analysis using NMR and mass spectrometry confirmed that the synthesized product was the final product.

[0050] Example 2

[0051] Step I: Dissolve 405.4 mg (0.56 mmol) of compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole iodide in 15 mL of N,N-dimethylformamide. With stirring, add 149.8 mg (1.40 mmol) of benzylamine, and then stir the reaction at 70 °C for 10 minutes. After the reaction was complete, the organic solvent was removed by rotary evaporation to give 357 mg of the solid compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide. (Yield: 83%)

[0052] Step II: Dissolve 397.6 mg (0.50 mmol) of compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide in 25 mL of dichloromethane. Add 677.25 mg (5.25 mmol) of N,N-diisopropylethylamine and 311.9 mg (4.0 mmol) of acetyl chloride while stirring at 25 °C. Then react for 40 minutes while stirring at 25 °C. After the reaction was completed, the sample was extracted with saturated ammonium chloride aqueous solution and dichloromethane (25 mL). The organic phase was collected, dried, and filtered. The organic solvent was removed by rotary evaporation. The obtained solid was purified by silica gel column chromatography (the eluent was dichloromethane / methanol, V / V = 15:1) to obtain 213 mg of cyanine dye (yield: 51%).

[0053] The fluorescent probe obtained in this embodiment has the same characterization results as in Example 1.

[0054] Example 3

[0055] Step I: Take 608.1 mg (0.84 mmol) of compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide in a 100 mL single-necked flask, add 20 mL of N,N-dimethylformamide to dissolve it, and add 269.6 mg (2.52 mmol) of benzylamine while stirring. Then, stir the reaction at 80 °C for 15 minutes. After the reaction was complete, the organic solvent was removed by rotary evaporation to give 515 mg of the solid compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide. (Yield: 80%)

[0056] Step II: Dissolve 596.3 mg (0.75 mmol) of compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide in 45.0 mL of dichloromethane. Add 1064.3 mg (8.25 mmol) of N,N-diisopropylethylamine and 526.4 mg (6.75 mmol) of acetyl chloride while stirring at 30 °C. Then react for 45 minutes while stirring at 30 °C. After the reaction was completed, the sample was extracted with saturated ammonium chloride aqueous solution and dichloromethane (45 mL). The organic phase was collected, dried, and filtered. The organic solvent was removed by rotary evaporation. The obtained solid was purified by silica gel column chromatography (the eluent was dichloromethane / methanol, V / V = 15:1) to obtain 301.6 mg of cyanine dye (yield: 48%).

[0057] The fluorescent probe obtained in this embodiment has the same characterization results as in Example 1.

[0058] Example 4

[0059] Near-infrared absorption test

[0060] The absorption spectrum testing procedure for the near-infrared II region cyanine dye 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide was performed as follows:

[0061] 4.187 mg of the solid compound 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide salt prepared in Example 1 was dissolved in 5.0 mL of dimethyl sulfoxide to prepare a 1.0 mM stock solution. For testing, the solution was diluted with dimethyl sulfoxide to a concentration of 10.0 μM, and the total volume of the test system was 3.0 mL. The absorption spectrum test results are as follows: Figure 4 As shown, by Figure 4 It can be seen that the absorption peak of the near-infrared II cyanine dye of the present invention is 860nm, and the dye can efficiently absorb near-infrared light in the range of 800-1000nm.

[0062] Light stability test

[0063] The compound 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide prepared in Example 1 was dissolved in N,N-dimethylformamide to prepare a 1.0 mM stock solution. The stock solution was diluted with N,N-dimethylformamide to a final concentration of 20.0 μM. The total test volume was 3.0 mL, and the test temperature was 25 °C. The excitation wavelength was 808 nm, and the power was 100 mW / cm². 2 The solution was continuously irradiated, and the absorption spectrum was measured at 0, 20, 40, 60, 80, 100, and 120 minutes. The absorbance at 860 nm was plotted. The measured absorbance changes with laser irradiation time as follows: Figure 5 As shown, from Figure 5 It can be seen that under continuous irradiation by an 808nm laser, the absorbance of the cyanine dye at 860nm remains almost unchanged, indicating that the near-infrared II cyanine dye prepared by this invention has high photostability.

[0064] Near-infrared fluorescence test

[0065] The fluorescence spectroscopy testing procedure for near-infrared II cyanine dye 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide was performed as follows:

[0066] 4.187 mg of the solid compound 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide salt prepared in Example 1 was dissolved in 5.0 mL of dichloromethane to prepare a 1.0 mM stock solution. For testing, the solution was diluted to 10.0 μM with dichloromethane, and crushed leaves were added to the solution. The total volume of the test system was 3.0 mL, and the excitation wavelength was 808 nm. The fluorescence spectroscopy results are as follows: Figure 6 As shown, by Figure 6 It can be seen that the maximum emission peak of the near-infrared II cyanine dye of the present invention is 918 nm, and it still has strong fluorescence emission in the 1000-1100 nm range; moreover, even in the presence of leaves, the dye still exhibits a near-infrared II emission spectrum with a sharp peak, indicating that the fluorescence emission of the dye can avoid the influence of pigments in the leaves.

[0067] Near-infrared II fluorescence imaging test

[0068] The compound 2-(2-(2-(N-benzylacetamide)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole iodide prepared in Example 1 was dissolved in dichloromethane. A certain amount was sealed in a 1 mL centrifuge tube and excited with an 808 nm laser. Fluorescence imaging was recorded after continuous excitation for 0, 40, 80, and 120 minutes. Figure 7 As shown, the cyanine dye exhibits considerable fluorescence signal in the 900-1300nm band, and the fluorescence signal does not show significant attenuation after 120 minutes, indicating that the dye has near-infrared II imaging capability and can maintain high photostability during imaging.

[0069] The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A near-infrared II region anthocyanin dye, characterized in that, The molecular formula of the near-infrared II cyanine dye is C2. 50 H 52 N3O + The specific structure is as follows:

2. The method for preparing the near-infrared II cyanine dye according to claim 1, characterized in that, Includes the following steps: Step I: The compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole salt was dissolved in an organic solvent, and then benzylamine was added. The reaction was carried out under stirring. After the reaction was completed, the compound 2-(2-(2-(benzylamine)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt was obtained by separation and purification. Step II: Dissolve 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt in dichloromethane, add N,N-diisopropylethylamine, then add acetyl chloride, and then react under stirring; after the reaction is completed, separate and purify to obtain the near-infrared II cyanine dye; The structural formula of the 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt is shown below.

3. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, In step I, the molar ratio of the compound 2-(2-(2-chloro-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene) cyclopent-1-en-1-yl) vinyl)-3-ethyl-1,-1-dimethyl-1H-benzo[e]indole salt to benzylamine is 1:(2-3); the organic solvent is N,N-dimethylformamide.

4. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, The reaction temperature under stirring in step I is 60-80℃; the stirring time is 5-15 minutes; the separation and purification steps in step I are as follows: after the reaction is completed, the organic solvent is removed by rotary evaporation, and the obtained solid is directly used for the next reaction.

5. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, In step II, the molar ratio of 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt to N,N-diisopropylethylamine is 1:(10-11); the molar ratio of 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ylidene)ethylidene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt to acetyl chloride is 1:(7-9).

6. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, In step II, the amount of dichloromethane added per mmol of compound 2-(2-(2-(benzylamino)-3-(2-(3-ethyl-1,1-dimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethylene)ethylene)cyclopent-1-en-1-yl)vinyl)-3-ethyl-1,1-dimethyl-1H-benzo[e]indole salt is 40-60 mL.

7. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, The reaction temperature under stirring in step II is 0℃-30℃; the reaction time under stirring in step II is 35-45 minutes.

8. The method for preparing near-infrared II cyanine dye according to claim 2, characterized in that, The separation and purification steps described in step II are as follows: After the reaction is completed, the mixture is extracted with saturated ammonium chloride aqueous solution and dichloromethane, the organic phase is collected, dried, and filtered; the organic solvent is removed by rotary evaporation, and the resulting solid is purified by silica gel column chromatography.

9. The method for preparing near-infrared II cyanine dye according to claim 8, characterized in that, The eluent for the silica gel column chromatography is dichloromethane / methanol.

10. The application of the near-infrared II cyanine dye according to claim 1 in the preparation of fluorescent probes and in near-infrared II biofluorescence imaging.

Images